Method for analyzing antibody

Abstract

One aspect of the present invention provides a method for quantifying, determining, or measuring, in a composition, the proportion of an antigen-binding molecule having at least one disulfide bond formed in a region other than a hinge region, with respect to the total of the antigen-binding molecule having at least one disulfide bond formed in a region other than a hinge region and an antigen-binding molecule not having a disulfide bond in a region other than a hinge region, by using protease FabALACTICA (Registered trademark) and an electrophoresis method and / or a chromatography method.

Description

Antibody analysis methods

[0001] The present 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, a method for increasing or concentrating such antigen-binding molecules, and a method for eliminating heterogeneity in disulfide bonds of antigen-binding molecules.

[0002] Antigen-binding molecules (LINC-Ig®) containing artificial disulfide bonds ("LINC") between amino acid residues in regions outside the hinge region are known (Patent Documents 1 to 4). LINC-Ig®, for example, contains 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. However, when such antigen-binding molecules are expressed in cell culture to obtain them, two types of forms coexist in the cell culture: molecules with appropriate disulfide bonds between heavy chains (LINC forms) and molecules without appropriate disulfide bonds, such as mismatched or incompletely formed forms of the molecules (unLINC forms). To prepare LINC-Ig®, molecules in cell culture that do not have appropriate disulfide bonds between heavy chains, such as open-type molecules with capped sulfur atoms, must be converted to molecules with appropriate disulfide bonds.

[0003] The following methods are known 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: Patent Documents 2 and 4 disclose methods for efficiently obtaining antibodies with LINCs by treating a preparation containing molecules with disulfide bonds between heavy chains and molecules in which disulfide bonds are not appropriately formed with a reducing agent, followed by reoxidation by buffer exchange or the like to form disulfide bonds.

[0004] Patent Document 5 discloses the use of a redox buffer solution (e.g., cysteine / cystine) to enable the reduction-oxidation reaction to be carried out in a downstream process in order to prevent antibody fragmentation.

[0005] Patent Document 6 discloses a method for refolding recombinant antibodies by contact with a redox coupling reagent.

[0006] Non-Patent Document 1 discloses that a redox buffer solution was used to suppress lot-to-lot heterogeneity due to cysteinylation in a specific antibody (MAB007).

[0007] Furthermore, because unLINC bodies may be classified as relatively toxic impurities in pharmaceuticals, a method for analyzing and measuring the amount of unLINC bodies is required for quality control. However, until now, no useful method has been found for measuring, determining, or quantifying the amount or proportion of LINC bodies and / or unLINC bodies in cell culture media containing LINC bodies and unLINC bodies or their purified products.

[0008] WO2020 / 027330WO2021 / 157679WO2021 / 200898WO2021 / 201087WO2020 / 037016WO2006 / 047340WO2021 / 201202

[0009] Pharm Sci. 2008 Feb;97(2):775-90, Removal of Cysteinylation from an Unpaired Sulfhydryl in the Variable Region of a Recombinant Monoclonal IgG1 Antibody Improves Homogeneity, Stability, and Biological ActivitySpoerry C et al., (2016) PLoS ONE 11(10): e0164809. doi:10.1371 / journal.pone.0164809

[0010] As described above, a method is known in which a mixture containing antigen-binding molecules with appropriate disulfide bonds between heavy chains and antigen-binding molecules with inappropriate disulfide bonds (e.g., capping molecules) is treated with a reducing agent, followed by reoxidation, to efficiently obtain antigen-binding molecules with LINCs. However, this method has drawbacks in terms of productivity and ease of production, such as low yield and long reaction time.

[0011] To solve the above-mentioned problems, the present disclosure aims to provide an efficient and easy method for producing and purifying antigen-binding molecules having suitable disulfide bonds between antibody heavy chains. The present disclosure relates to a method for increasing the structural uniformity and relative abundance of antigen-binding molecules having one or more disulfide bonds formed between amino acid residues in regions other than the hinge regions of two antibody heavy chains. In other words, the present 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 other than the hinge regions.

[0012] Another object of the present disclosure is to provide a method for measuring, determining, or quantifying the ratio of LINC bodies to the total of LINC bodies and unLINC bodies in a composition containing LINC bodies and unLINC bodies.

[0013] The present inventors conducted studies to solve the above-mentioned problems and found that at least one disulfide bond can be formed between amino acid residues in a region other than the hinge region more efficiently and with high reproducibility by contacting an antigen-binding molecule bound to an affinity column and having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region (these antigen-binding molecules include antigen-binding molecules in which a disulfide bond is appropriately formed between amino acid residues in a region other than the hinge region and antigen-binding molecules in which at least one disulfide bond is not appropriately formed between amino acid residues in a region other than the hinge region) with a reducing agent and then removing the reducing agent. Specifically, the present inventors have developed a method for stably obtaining, in high yield, antigen-binding molecules having at least one disulfide bond between amino acid residues in a region other than the hinge region (LINC-Ig® format) by inserting two steps, namely, passing a solution containing a reducing agent and removing the reducing agent, into the purification process using Protein A affinity chromatography, a method commonly used for primary purification from cell culture fluid (HCCF).

[0014] Furthermore, the present inventors have newly discovered that a commercially available enzyme called FabALACTICA (registered trademark) (IgdE), which digests a specific site in the upper hinge region of human IgG1 to produce intact and homogeneous Fab and Fc fragments, cleaves unLINC forms of antigen-binding molecules having a cysteine ​​residue introduced into their CH1 region at the hinge region, but does not cleave LINC forms of antigen-binding molecules having a cysteine ​​residue introduced into their CH1 region. Based on this finding, the present inventors have discovered a method for separating LINC forms and unLINC forms using equipment such as an electrophoresis device or a chromatography device, and measuring, determining, or quantitating their content ratios.

[0015] The present disclosure is based on these findings and specifically relates to the following: [1] A method for producing a preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, the method comprising a step of subjecting an antigen-binding molecule having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region to chromatography in the presence of a reducing agent. [2] A method for producing a preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, the method comprising the steps of: (a) contacting, in chromatography, an antigen-binding molecule having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region with a solution containing a reducing agent, and (b) removing the reducing agent. [3] A method for producing a preparation containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, the method comprising a step of subjecting a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and mismatched and / or incomplete disulfide bonds of the antigen-binding molecules to chromatography in the presence of a reducing agent. [4] A method for producing a preparation containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, the method comprising the following steps: (a) contacting, during chromatography, a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and mismatched and / or incomplete disulfide bonds of the antigen-binding molecules with a solution containing a reducing agent, and (b) removing the reducing agent. [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 a region other than the hinge region is formed between the polypeptide chains.[6] The method of [5], wherein one or both of the polypeptide chains have a mutated, substituted, or introduced cysteine ​​residue in a region other than the hinge region. [7] The method of any of [1] to [6], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and 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. [8] The method of any of [1] to [7], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and 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. [9] The method of any of [1] to [8], wherein the antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain, and 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.

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

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

[10] , wherein the chromatography comprises an affinity chromatography support, an ion exchange chromatography support, a hydrophobic interaction chromatography support, a multimode chromatography support including both ion exchange chromatography and hydrophobic interaction chromatography, or a hydroxyapatite support.

[12] The method according to

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

[13] The method of

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

[14] The method of

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

[15] The method of

[11] , wherein the multi-mode chromatography support is a support having a combination of a cation exchange ligand and a hydrophobic ligand, or a support having a combination of an anion exchange ligand and a hydrophobic ligand.

[16] The method of

[11] , wherein the hydroxyapatite support is hydroxyapatite or a conversion thereof (such as fluoroapatite).

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

[16] , comprising a step of contacting a chromatography support packed in a column for column chromatography or coated on a membrane for membrane chromatography with a mixture containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and a mismatched and / or incomplete disulfide bond of the antigen-binding molecule.

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

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

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

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

[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 about 0.01 mM to about 100 mM.

[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.

[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 about 0.001 mM to about 10 mM.

[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 about 0.0001 mM to about 1 mM or about 0.001 mM to about 0.01 mM.

[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 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 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.

[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 about 0.0001 mM to about 10 mM, or about 0.001 mM to about 0.1 mM.

[26] The method of 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.

[27] The method of 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.

[28] The method of 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.

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

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

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

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

[31] The method of

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

[32] The method of

[30] or

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

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

[32] , wherein the mixture is contacted with a solution containing a reducing agent for about 6 seconds to about 1,440 minutes, or about 18 seconds to about 300 minutes.

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

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

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

[34] , wherein removing the reducing agent comprises contacting the antigen-binding molecule with a solution not containing a reducing agent.

[36] The method of

[35] , wherein the chromatography is column chromatography or membrane chromatography, and contacting the mixture with a solution not containing a reducing agent comprises passing the solution not containing a reducing agent through a column for column chromatography or a device for membrane chromatography.

[37] The method of

[35] or

[36] , wherein a solution not containing a reducing agent is passed through a column of column chromatography or a device of membrane chromatography for a passage time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes, and optionally, the passage of the solution is temporarily stopped while the column or device is filled with the solution.

[38] The method of any one of

[35] to

[37] , wherein a solution not containing a reducing agent is passed through a column of column chromatography or a device of membrane chromatography for a passage time of about 4 minutes or about 30 seconds, and optionally, the passage of the solution is temporarily stopped while the column or device is filled with the solution.

[39] The method of any one of

[35] to

[38] , wherein the mixture and the solution not containing a reducing agent are contacted for about 6 seconds to about 1,440 minutes, or about 18 seconds to about 300 minutes.

[40] The method according to any one of

[35] to

[39] , wherein the mixture is contacted with a solution containing no reducing agent for about 20 minutes or about 7.5 minutes.

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

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

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

[41] , further comprising, before step (a), the step of contacting a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and disulfide bond mismatches and / or incomplete formations of the antigen-binding molecules with a chromatography support, and packing the antigen-binding molecules into a column for column chromatography or immobilizing them on a membrane for membrane chromatography.

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

[42] , further comprising, before step (a), the step of removing impurities in the chromatography.

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

[43] , further comprising, before or after step (b), the step of recovering antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region.

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

[44] , wherein contacting the antigen-binding molecule with a solution containing a reducing agent cleaves a disulfide bond formed between amino acid residues capable of forming a disulfide bond and / or decapps a sulfur atom in an amino acid residue capable of forming a disulfide bond.

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

[45] , wherein removal of the reducing agent forms a disulfide bond between amino acid residues capable of forming a disulfide bond.

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

[46] , wherein the amino acid residue capable of forming a disulfide bond is an introduced or engineered cysteine ​​residue.

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

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

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

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

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

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

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

[50] , for increasing the ratio (LINC ratio) of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC bodies) to the total of (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies), and (ii) mismatched and / or unformed disulfide bonds of the antigen-binding molecules (unLINC bodies).

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

[51] , for producing antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC bodies), which are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% by molar ratio relative to the total of (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies), and (ii) mismatched and / or unformed disulfide bonds of the antigen-binding molecules (unLINC bodies).

[0016] The present disclosure also relates to the following inventions: [A1] The method of any one of [1] to

[52] , wherein the chromatography is column chromatography. [A2] The method of [A1], comprising a step of contacting a chromatography carrier packed in a column with a mixture containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and a mismatched and / or incomplete disulfide bond of the antigen-binding molecule. [A3] The method of [A1] or [A2], wherein a solution containing a reducing agent is passed through the column for a passage time of about 2 minutes to about 80 minutes, and optionally, the passage of the solution is temporarily stopped while the column is filled with the solution containing the reducing agent. [A4] The method of any one of [A1] to [A3], wherein a solution containing a reducing agent is passed through the column for a passage time of about 4 minutes to about 24 minutes, and optionally, the passage of the solution is temporarily stopped while the column is filled with the solution containing the reducing agent. [A5] The method of any one of [A1] to [A4], wherein a solution containing a reducing agent is passed through the column at a passage time of about 12 minutes, and optionally, the passage of the solution is temporarily stopped while the column is filled with the solution containing the reducing agent. [A6] The method of any one of [A1] to [A5], wherein a solution containing a reducing agent is passed through the column of column chromatography at a rate of about 25 cm / hour to about 500 cm / hour, or about 50 cm / hour to about 400 cm / hour, and optionally, the passage of the solution is temporarily stopped while the column is filled with the solution containing the reducing agent. [A7] The method of any one of [A1] to [A6], wherein a solution containing a reducing agent is passed through the column of column chromatography at a rate of about 100 cm / hour, and optionally, the passage of the solution is temporarily stopped while the column is filled with the solution containing the reducing agent. [A8] The method according to any one of [A1] to [A7], wherein the flow rate of the solution containing the reducing agent is about 0.1 times (about 0.1 CV (column volume)) to about 100 times (about 100 CV) the column volume. [A9] The method according to any one of [A1] to [A8], wherein the flow rate of the solution containing the reducing agent is about 1 time (1 CV) to about 20 times (about 20 CV) the column volume.[A10] The method of any one of [A1] to [A9], wherein the flow rate of the solution containing the reducing agent is about 10.0 times the column volume (about 10.0 CV). [A11] The method of any one of [A1] to [A10], wherein the mixture is contacted with the solution containing the reducing agent for about 4 minutes to about 1,440 minutes. [A12] The method of any one of [A1] to [A11], wherein the mixture is contacted with the solution containing the reducing agent for about 8 minutes to about 300 minutes. [A13] The method of any one of [A1] to [A12], wherein the mixture is contacted with the solution containing the reducing agent for about 120 minutes. [A14] The method of any one of [A1] to [A13], wherein the amount of antigen-binding molecules supported on the carrier 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 carrier. [A15] The method according to any one of [A1] to [A14], wherein a solution not containing a reducing agent is passed through the column for a passage time of about 2 minutes to about 80 minutes, or the column is filled with the solution and then the passage of the solution is temporarily stopped. [A16] The method according to any one of [A1] to [A15], wherein a solution not containing a reducing agent is passed through the column for a passage time of about 4 minutes to about 24 minutes, or the column is filled with the solution and then the passage of the solution is temporarily stopped. [A17] The method according to any one of [A1] to [A16], wherein a solution not containing a reducing agent is passed through the column for a passage time of about 4 minutes, or the column is filled with the solution and then the passage of the solution is temporarily stopped. [A18] The method according to any one of [A1] to [A17], wherein a solution containing no reducing agent is passed through a column of column chromatography at a rate of about 25 cm / hour to about 500 cm / hour, or about 50 cm / hour to about 400 cm / hour, or the flow of the solution is temporarily stopped while the column is filled with the solution. [A19] The method according to any one of [A1] to [A18], wherein a solution containing no reducing agent is passed through a column of column chromatography at a rate of about 300 cm / hour, or the flow of the solution is temporarily stopped while the column is filled with the solution.[A20] The method according to any one of [A1] to [A19], wherein the flow rate of the solution not containing a reducing agent is about 0.1 times (about 0.1 CV) to about 100 times (about 100 CV), or about 1 time (about 1 CV) to about 20 times (about 20 CV) the column volume. [A21] The method according to any one of [A1] to [A20], wherein the flow rate of the solution not containing a reducing agent is about 5.0 times (about 5.0 CV) the column volume. [A22] The method according to any one of [A1] to [A21], wherein the mixture and the solution not containing a reducing agent are contacted for about 4 minutes to about 1,440 minutes. [A23] The method according to any one of [A1] to [A22], wherein the mixture and the solution not containing a reducing agent are contacted for about 8 minutes to about 300 minutes. [A24] The method according to any one of [A1] to [A23], wherein the mixture is contacted with a solution containing no reducing agent for about 20 minutes.

[0017] The present disclosure also relates to the following inventions: [B1] The method of any one of [1] to

[52] , wherein the chromatography is membrane chromatography. [B2] The method of [B1], comprising a step of contacting a chromatography support coated or immobilized on a membrane for membrane chromatography with a mixture containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and a mismatch and / or incomplete disulfide bond of the antigen-binding molecule. [B3] The method of [B1] or [B2], wherein a solution containing a reducing agent is passed through the membrane device for a passage time of about 2 seconds to about 60 minutes, or wherein the passage of the solution is temporarily stopped while the membrane device is filled with the solution containing the reducing agent. [B4] The method of any one of [B1] to [B3], wherein a solution containing a reducing agent is passed through the membrane device for a passage time of about 3 seconds to about 6 minutes, for example, about 30 seconds, or wherein the passage of the solution is temporarily stopped while the membrane device is filled with the solution containing the reducing agent. [B5] The method according to any one of [B1] to [B4], wherein the amount of the solution containing the reducing agent passing through is about 1 time (about 1 MV (Membrane volume)) to about 500 times (about 500 MV) the volume of the membrane device. [B6] The method according to any one of [B1] to [B5], wherein the amount of the solution containing the reducing agent passing through is about 2 times (about 2 MV) to about 100 times (about 100 MV), for example, about 15 times (about 15 MV), the volume of the membrane device. [B7] The method according to any one of [B1] to [B6], wherein the mixture and the solution containing the reducing agent are contacted for about 6 seconds to about 600 minutes. [B8] The method according to any one of [B1] to [B7], wherein the mixture and the solution containing the reducing agent are contacted for about 18 seconds to about 120 minutes, for example, about 7.5 minutes. [B9] The method according to any one of [B1] to [B8], wherein the amount of antigen-binding molecules supported on the carrier is about 5 g to about 100 g, or about 7 g to about 70 g, for example, about 25 g, per 1 L of membrane device volume. [B10] The method according to any one of [B1] to [B9], wherein a solution containing no reducing agent is passed through the membrane device for a passage time of about 2 seconds to about 60 minutes, or wherein the passage of the solution is temporarily stopped while the membrane device is filled with the solution.[B11] The method according to any one of [B1] to [B10], wherein a solution not containing a reducing agent is passed through the membrane device for a passage time of about 3 seconds to about 6 minutes, for example, about 30 seconds, or wherein the passage of the solution is temporarily stopped while the membrane device is filled with the solution. [B12] The method according to any one of [B1] to [B11], wherein the amount of the solution not containing a reducing agent passed through is about 1 time (about 1 MV) to about 500 times (about 500 MV) the volume of the membrane device. [B13] The method according to any one of [B1] to [B12], wherein the amount of the solution not containing a reducing agent passed through is about 2 times (about 2 MV) to about 100 times (about 100 MV), for example, about 15 times (about 15 MV) the volume of the membrane device. [B14] The method according to any one of [B1] to [B13], wherein the mixture and the solution not containing a reducing agent are contacted for about 6 seconds to about 600 minutes. [B15] The method according to any one of [B1] to [B14], wherein the mixture is contacted with a solution not containing a reducing agent for about 18 seconds to about 120 minutes, for example, about 7.5 minutes.

[0018] The present disclosure also relates to the following inventions:

[101] 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 other than the hinge region (LINC bodies) to the total of antigen-binding molecules having at least one disulfide bond formed in a region other than the hinge region (LINC bodies) and antigen-binding molecules having no disulfide bond in a region other than the hinge region (unLINC bodies), the method comprising the steps of: (a) adding a protease to composition (1) containing LINC bodies and unLINC bodies to prepare composition (2), and (b) subjecting composition (2) to electrophoresis or chromatography.

[102] The method according to

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

[103] The method of

[101] or

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

[104] The method of any one of

[101] to

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

[105] The method of any one of

[101] to

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

[106] The method of any one of

[101] to

[105] , wherein the protease is IgdE.

[107] The method of

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

[108] The method of

[106] or

[107] , wherein the IgdE is a protein selected from the group consisting of (a) to (d) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61, (b) a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 62, (c) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61 in which one or more amino acids have been substituted, deleted, added, and / or inserted, said protein being capable of digesting human IgG1 antibody between the T and H amino acid sequences of 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 to the amino acid sequence set forth in SEQ ID NO: 61, said protein being capable of digesting human IgG1 antibody between the T and H amino acid sequences of KSCDKT / HTCPPCP.

[109] The method of any one of

[101] to

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

[110] The method according to any one of

[101] to

[109] , wherein step (a) further comprises obtaining a composition (3) containing a protease and not containing LINC bodies and unLINC bodies, and step (b) comprises subjecting each of compositions (1) to (3) to non-reducing capillary SDS gel electrophoresis (CE-SDS) to obtain electropherograms (1) to (3) for each of compositions (1) to (3), respectively, wherein electropherogram (1) contains peaks derived from LINC bodies and unLINC bodies not digested with a protease, as well as a peak (peak Z) derived from neither LINC bodies nor unLINC bodies not digested with a protease, The method, wherein electropherogram (2) contains a peak derived from LINC bodies (Peak L), a peak derived from unLINC bodies digested with a protease (Peak unL), a peak derived from a protease (Peak E), and a peak derived from none of LINC bodies, unLINC bodies digested with a protease, or a protease (Peak Z), and electropherogram (3) contains a peak derived from a protease (Peak E).

[111] (c) The method according to

[110] , further comprising the step of calculating the percentage (%) using the following formula (I): where A is the area value of peak L in electropherogram (2), B is the sum of the areas of peak unL, peak E, and peak Z in electropherogram (2), C is the area value of peak Z in electropherogram (1), and D is the area value of peak E in electropherogram (3).

[112] The method according to any one of

[101] to

[109] , wherein step (b) is a step of subjecting composition (2) to hydrophobic interaction chromatography (HIC) to obtain a chromatogram.

[113] The method according to

[112] , further comprising (c) a step of calculating the ratio (%) using the following formula (II): Here, A is the area value of the peak derived from the LINC form in the chromatogram, and B is the sum of the area values ​​of all peaks derived from the unLINC form digested with protease in the chromatogram.

[0019] The present disclosure also relates to the following inventions:

[201] The method of 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.

[202] The method of

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

[203] The method of

[201] or

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

[204] The method of any one of

[201] to

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

[205] The method of any one of

[201] to

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

[206] The method of 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.

[207] The method of any one of

[201] to

[205] , wherein the first antigen-binding domain and the second antigen-binding domain each bind to different epitopes on the same antigen.

[208] The method of any one of

[201] to

[204] , wherein the first antigen-binding domain and the second antigen-binding domain each bind to different antigens.

[209] The method of any one of

[201] to

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

[210] The method of any one of

[201] to

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

[211] The method of 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.

[212] The method of 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.

[213] The method of 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.

[214] The method of any one of

[201] to

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

[215] The method of any one of

[201] to

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

[216] The method of 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 cell surface (the first antigen and the second antigen, respectively), and the antigen-binding molecule has the activity of promoting interaction between cells expressing the first antigen and cells expressing the second antigen.

[217] The method of

[216] , wherein the cells expressing the first antigen are cells with cytotoxic activity, the cells expressing the second antigen are their target cells, and the antigen-binding molecule promotes damage to the target cells by the cells with cytotoxic activity.

[218] The method of

[217] , wherein the cells with cytotoxic activity are T cells, NK cells, monocytes, or macrophages.

[219] The method of any one of

[205] to

[218] , wherein the antigen is selected from the group consisting of a receptor belonging to the cytokine receptor superfamily, a G protein-coupled receptor, an ion channel receptor, a tyrosine kinase receptor, an immune checkpoint receptor, an antigen receptor, a CD antigen, a costimulatory molecule, and a cell adhesion molecule.

[220] The method of any one of

[201] to

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

[221] The method of 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.

[222] The method of any one of

[201] to

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

[223] The method of

[221] or

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

[224] The method of any one of

[202] to

[223] , 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.

[225] The method of any one of

[221] to

[224] , wherein each of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is a Fab molecule, and 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.

[226] The method of

[224] or

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

[227] The method of any one of

[221] to

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

[228] The method of any one of

[221] to

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

[229] The method of any one of

[221] to

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

[230] The method of 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.

[231] The method of

[230] , wherein the Fc region is composed of a first Fc region subunit and a second Fc region subunit capable of stable association.

[232] The method of

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

[233] The method of any one of

[230] to

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

[234] The method of any one of

[230] to

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

[235] The method of any one of

[230] to

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

[236] The method of 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 a native IgG Fc region.

[237] The method of any one of

[230] to

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

[238] The method of any one of

[230] to

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

[239] The method of any one of

[230] to

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

[240] The method of any one of

[230] to

[238] , wherein the Fc region comprises, according to EU numbering, one of the following:(a)N434A / Q438R / S440E; (b)N434A / Q438R / S440D; (c)N434A / Q438K / S440E; (d)N434A / Q438K / S440D; (e)N434A / Y436T / Q438R / S440E; (f)N434A / Y436T / Q438R / S440D; (g)N434A / Y436T / Q438K / S440E; (h)N434A / Y436T / Q438K / S440D; (i)N434A / Y436V / Q438R / S440E; (j)N434A / Y436V / Q438R / S440D; (k)N434A / Y436V / Q438K / S440E; (l)N434A / Y436V / Q438K / S440D; (m)N434A / R435H / F436T / Q438R / S440E; (n)N434A / R435H / F436T / Q438R / S440D; (o)N434A / R435H / F436T / Q438K / S440E; (p)N434A / R435H / F436T / Q438K / S440D; (q)N434A / R435H / F436V / Q438R / S440E; (r)N434A / R435H / F436V / Q438R / S440D; (s)N434A / R435H / F436V / Q438K / S440E; (t)N434A / R435H / F436V / Q438K / S440D; (u)M428L / N434A / Q438R / S440E; (v)M428L / N434A / Q438R / S440D; (w)M428L / N434A / Q438K / S440E; (x)M428L / N434A / Q438K / S440D; (y)M428L / N434A / Y436T / Q438R / S440E; (z)M428L / N434A / Y436T / Q438R / S440D; (aa)M428L / N434A / Y436T / Q438K / S440E; (ab)M428L / N434A / Y436T / Q438K / S440D; (ac)M428L / N434A / Y436V / Q438R / S440E; (ad)M428L / N434A / Y436V / Q438R / S440D; (ae)M428L / N434A / Y436V / Q438K / S440E;(af) M428L / N434A / Y436V / Q438K / S440D; (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E.

[241] The method of any one of

[230] to

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

[242] The method of any one of

[230] to

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

[243] The method of

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

[244] The method of

[242] or

[243] , wherein the multimerization is hexamerization.

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

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

[101] to

[113] , and

[201] to

[244] , wherein the antigen-binding molecule has an amino acid residue in its hinge region where at least one of the cysteine ​​residues has been substituted.

[246] The method of

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

[247] The method of 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.

[248] The method of

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

[249] The method of 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.

[250] The method of

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

[251] The method of 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.

[252] The method of 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 with a cysteine ​​residue at one or more positions selected from the group consisting of EU numbering 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.

[0020] The present disclosure also relates to the following inventions:

[301] The method of any one of

[201] to

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

[302] The method of any one of

[201] to

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

[303] The method of 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.

[304] The method of

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

[305] The method of

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

[306] The positions of amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain 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, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 The method of

[304] , wherein each of the amino acids is independently selected from the group consisting of positions 4, 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.

[307] The method of

[304] , wherein the positions of amino acid residues in the CH1 regions of the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 134, 135, 136, 137, 191, 192, 193, 194, 195, 196, and 197.

[308] The method of

[304] , wherein the positions of amino acid residues in the CH1 regions of the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 135, 136, and 191.

[309] The method of

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

[310] The method of

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

[311] The method of

[304] , wherein the positions of amino acid residues in the CH1 regions of the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 148, 149, and 150.

[312] The method of

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

[313] The method of

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

[314] The method of

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

[315] The method of

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

[316] The method of

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

[317] The method of any one of

[304] to

[316] , wherein the difference in positions of amino acid residues in the CH1 region of the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids.

[318] The method of

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

[319] The method of

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

[320] The method of

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

[321] The method of

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

[322] The method according to

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

[323] The method of

[304] , wherein (i) the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 191, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU numbering position 191; (ii) the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 195, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU numbering position 195; or (iii) the amino acid residue in the CH1 region of the first antigen-binding domain is the amino acid residue at EU numbering position 197, and the amino acid residue in the CH1 region of the second antigen-binding domain is the amino acid residue at EU numbering position 197.

[324] A 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.

[325] The method of 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, and the additional disulfide bonds are formed via amino acid residues at the following positions according to EU numbering in the CH1 regions of the first antigen-binding domain and the second antigen-binding domain: (a) between amino acid residues at any of positions 131 to 138, 194, and 195 in each of the two antigen-binding domains; (b) between the amino acid residue at position 131 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (c) between the amino acid residue at position 132 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (d) between the amino acid residue at position 133 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (e) between the amino acid residue at position 134 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (f) between the amino acid residue at position 135 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (g) between the amino acid residue at position 136 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (h) between the amino acid residue at position 137 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (i) between the amino acid residue at position 138 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (j) between the amino acid residue at position 131 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains; (k) between the amino acid residue at position 132 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains;(l) between the amino acid residue at position 133 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains; (m) between the amino acid residue at position 134 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains; (n) between the amino acid residue at position 135 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains; (o) between the amino acid residue at position 136 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains; (p) between the amino acid residue at position 137 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains; and (q) between the amino acid residue at position 138 in each of the two antigen-binding domains and between the amino acid residue at position 195 in each of the two antigen-binding domains.

[326] The method of 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 positions 136 to 138 (EU numbering) in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two or more oppositely charged amino acid residues at positions 193 to 195 (EU numbering) in the CH1 region.

[327] The method of 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 positions 136 to 138 (EU numbering) 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 positions 193 to 195 (EU numbering) in the CH1 region.

[328] The method of 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 positions 136 to 138 (EU numbering) 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 positions 193 to 195 (EU numbering) in the CH1 region.

[329] Either the first antigen-binding domain or the second antigen-binding domain contains one, two, or more of the following amino acid residues in the CH1 region: (a) the amino acid residue at position 136 (EU numbering) is glutamic acid (E) or aspartic acid (D); (b) the amino acid residue at position 137 (EU numbering) is glutamic acid (E) or aspartic acid (D); (c) the amino acid residue at position 138 (EU numbering) is glutamic acid (E) or aspartic acid (D); and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more of the following amino acid residues in the CH1 region: (d) the amino acid residue at position 193 (EU numbering) is lysine (K), arginine (R), or histidine (H); (e) the amino acid residue at position 194 (EU numbering) is lysine (K), arginine (R), or histidine (H); and (f) the amino acid residue at EU numbering position 195 is lysine (K), arginine (R), or histidine (H).

[330] The method of any one of

[201] to

[252] and

[301] to

[324] , comprising one, two, or more of the following amino acid residues in the CH1 region of either the first antigen-binding domain or the second antigen-binding domain: (a) the amino acid residue at EU numbering position 136 is lysine (K), arginine (R), or histidine (H); (b) the amino acid residue at EU numbering position 137 is lysine (K), arginine (R), or histidine (H); (c) the amino acid residue at EU numbering position 138 is lysine (K), arginine (R), or histidine (H).and the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two or more of the following amino acid residues in the CH1 region: (d) the amino acid residue at position 193 (EU numbering) is glutamic acid (E) or aspartic acid (D); (e) the amino acid residue at position 194 (EU numbering) is glutamic acid (E) or aspartic acid (D); and (f) the amino acid residue at position 195 (EU numbering) is glutamic acid (E) or aspartic acid (D).

[331] The method of 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 combination of mutations of specific charged amino acids (according to EU numbering) in the CH1 region described in Table 1, Table 2, or Table 3.

[332] The method of 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 hydrophobic amino acid residues at positions 136 to 138 (EU numbering) in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain comprises one, two, or more hydrophobic amino acid residues at positions 193 to 195 (EU numbering) in the CH1 region.

[333] The method of

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

[334] The method of 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 combination of specific hydrophobic amino acid mutations (according to EU numbering) in the CH1 region listed in Table 4.

[335] The method of 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 "knob" amino acid residue at positions 136 to 138 (EU numbering) in the CH1 region; and the other of the first antigen-binding domain and the second antigen-binding domain contains one, two, or more "hole" amino acid residues at positions 193 to 195 (EU numbering) in the CH1 region.

[336] The method of 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 "hole" amino acid residues in the CH1 region at EU numbering positions 136 to 138; and the other of the first antigen-binding domain or the second antigen-binding domain comprises one "knob" amino acid residue in the CH1 region at EU numbering positions 193 to 195.

[337] The method of

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

[338] The method of 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 positions 136 to 138 (EU numbering) 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 positions 193 to 195 (EU numbering) in the CH1 region.

[339] The method of 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 positions 136 to 138 (EU numbering) 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 positions 193 to 195 (EU numbering) in the CH1 region.

[340] The method of any one of

[201] to

[252] and

[301] to

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

[341] The method of

[303] , wherein 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.

[342] The method of

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

[343] The method of

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

[344] The method of

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

[345] The method of

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

[346] The method of

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

[347] The method of

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

[348] The method of

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

[349] The method of

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

[350] The method of

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

[351] The methods of

[341] to

[350] , wherein the difference in the positions of amino acid residues in the CL regions of the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids.

[352] The method of

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

[353] The method of

[303] , wherein 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.

[354] The method of

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

[355] The method of

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

[356] The method of any one of

[201] to

[252] and

[301] to

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

[357] The method of

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

[358] A method according to any one of

[341] to

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

[359] The method of

[303] , wherein 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.

[360] The method of

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

[361] The method of

[303] , wherein 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.

[362] The method of

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

[363] The method of

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

[364] The method of

[303] , wherein 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.

[365] The method of

[364] , wherein the positions of amino acid residues in the VHH region of the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 according to Kabat numbering.

[0021] The present disclosure also relates to the following inventions:

[401] The method of 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 a first antigen-binding domain and a second antigen-binding domain capable of binding to CD3 and CD137 but not simultaneously to CD3 and CD137, and a third antigen-binding domain capable of binding to DLL3, preferably human DLL3.

[402] The first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, which may be the same or different, and include the following (a1) to (a4): (a1) an antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 27, a heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 28, and a heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 29, and a light chain variable region comprising a light chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 31, and a light chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 32; (a2) a heavy chain variable region comprising a heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 34, and a heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 35, and The method of

[401] , comprising antibody variable regions independently selected from the group consisting of: (a3) ​​an antibody variable region comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32; (a4) an antibody variable region whose antigen binding competes with the antibody variable region of (a1) or (a2).

[403] The method of

[401] or

[402] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region, which may be the same or different, and comprise antibody variable regions independently selected from the group consisting of (a1) to (a4) below: (a1) an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37; and (a2) an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38, and a light chain variable region comprising 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 of (a1) or (a2); and (a4) an antibody variable region whose antigen-binding competes with the antibody variable region of (a1) or (a2).

[404] The third antigen-binding domain is any one of the following (a1) to (a4): (a1) an antibody variable region comprising a heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 47, and a heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising a light chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 49, a light chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 50, and a light chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 51; (a2) an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53; (a3) ​​an antibody variable region that binds to the same epitope as the antibody variable region of (a1) or (a2); and (a4) The method of any one of

[401] to

[403] , comprising an antibody variable region independently selected from the group consisting of antibody variable regions whose antigen binding competes with the antibody variable region of (a1) or (a2).

[405] The method of any one of

[401] to

[404] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region comprising: a heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 27, a heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 28, and a heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 29, and a light chain variable region comprising: a light chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 31, and a light chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 32.

[406] The method of any one of

[401] to

[405] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37.

[407] The method of any one of

[401] to

[406] , wherein the third antigen-binding domain comprises an antibody variable region comprising: a heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 46, a heavy chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 47, and a heavy chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising: a light chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 49, a light chain CDR 2 comprising the amino acid sequence of SEQ ID NO: 50, and a light chain CDR 3 comprising the amino acid sequence of SEQ ID NO: 51.

[408] The method of any one of

[401] to

[407] , wherein the third antigen-binding domain comprises an antibody variable region comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53.

[409] The method of any one of

[401] to

[408] , wherein the first antigen-binding domain and the second antigen-binding domain each comprise an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37, and the third antigen-binding domain comprises an antibody variable region comprising a heavy chain variable region comprising SEQ ID NO: 52, and a light chain variable region comprising SEQ ID NO: 53.

[410] The method of any one of

[401] to

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

[411] The method of any one of

[401] to

[410] , wherein each of the first, second, and third antigen-binding domains is a Fab comprising a heavy chain comprising a VH region and a CH1 region, and a light chain comprising a VL region and a CL region, and 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 antigen-binding domain or the second antigen-binding domain.

[412] The method of

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

[413] The method of any one of

[401] to

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

[414] The method of any one of

[401] to

[413] , wherein the amino acid residues at positions 123 and 124 (Kabat numbering) in the CL regions of each of the first and second antigen-binding domains are arginine and lysine, respectively, and the amino acid residues at positions 147 and 213 (EU numbering) in the CH1 regions of each of the first and second antigen-binding domains are glutamic acid.

[415] The method of any one of

[401] to

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

[416] The Fc region comprises a first Fc region subunit and a second Fc region subunit, wherein the first Fc region subunit is selected from the group consisting of: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 354, and a tryptophan at position 366, and wherein the second Fc region subunit is selected from the group consisting of: an Fc region polypeptide comprising an alanine at each of positions 234 and 235; an Fc region polypeptide comprising an alanine at each of positions 234, 235, and 297; and

[415] The method of

[415] , wherein the Fc region is selected from the group consisting of Fc region polypeptides comprising an alanine at each of positions 234, 235, and 297, a cysteine ​​at position 349, a serine at position 366, an alanine at position 368, and a valine at position 407, all positions being according to EU numbering.

[417] The method of either

[416] or

[417] , wherein the Fc region comprises any one of the following: (a) a first Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 23 and a second Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 24; (b) a first Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 25 and a second Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 26; or (c) a first Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 58 and a second Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 59.

[418] An antigen-binding molecule comprising: (a1) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 39, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 41, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 42; (a2) a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 43, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 44, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 42; and (a3) ​​a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 45, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 44, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 42;

[419] The method of any one of

[401] to

[417] , wherein the antigen-binding molecule comprises five polypeptide chains in any one of the combinations selected from the group consisting of: a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 54, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 55, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 57; preferably, the five polypeptide chains (chain 1 to chain 5) are linked and / or associated with each other according to the orientation shown in Figure 3.

[419] The method of

[401] , wherein the antigen-binding molecule comprises five polypeptide chains: a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 54, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 55, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 56, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence of SEQ ID NO: 57, wherein the five polypeptide chains (chain 1 to chain 5) are linked and / or associated with each other according to the orientation shown in Figure 3.

[420] The method of

[401] , wherein the antigen-binding molecule comprises five polypeptide chains: a polypeptide chain (chain 1) comprising the amino acid sequence of SEQ ID NO: 54, a polypeptide chain (chain 2) comprising the amino acid sequence of SEQ ID NO: 55, a polypeptide chain (chain 3) comprising the amino acid sequence of SEQ ID NO: 60, and two polypeptide chains (chains 4 and 5) each comprising the amino acid sequence of SEQ ID NO: 57, preferably, the five polypeptide chains (chains 1 to 5) are linked and / or associated with each other according to the orientation shown in Figure 3.

[421] The method of

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

[422] The method of

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

[423] The method of

[401] , wherein the antigen-binding molecule comprises five polypeptide chains: a polypeptide chain (A chain) comprising the amino acid sequence of SEQ ID NO: 71, a polypeptide chain (L chain) comprising the amino acid sequence of SEQ ID NO: 72, a polypeptide chain (P chain) comprising the amino acid sequence of SEQ ID NO: 73, and two polypeptide chains (two S chains) each comprising the amino acid sequence of SEQ ID NO: 74, preferably, the five polypeptide chains are linked and / or associated with each other according to the orientation shown in Figure 6.

[424] The method of

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

[425] The method of

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

[426] The method of

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

[0022] The present disclosure also relates to the following inventions:

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

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

[201] to

[252] ,

[301] to

[365] , and

[401] to

[425] , further comprising a step of measuring, determining, or quantifying the ratio (LINC ratio) of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC bodies) to the total of (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies), and (ii) mismatched and / or unformed disulfide bonds of the antigen-binding molecules (unLINC bodies) in the preparation.

[502] The method of

[501] , wherein the measuring, determining, or quantifying step comprises electrophoresis or chromatography.

[503] The method of

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

[504] The method of

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

[505] The method of any one of

[501] to

[504] , comprising a step of adding a protease to the preparation prior to the measuring, determining, or quantitating step.

[506] The method of

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

[507] The method of

[505] or

[506] , wherein the protease is a cysteine ​​protease that can digest human IgG1 antibodies.

[508] The method of any one of

[505] to

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

[509] The method of any one of

[505] to

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

[510] The method of any one of

[505] to

[509] , wherein the protease is IgdE.

[511] The method of

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

[512] The method of

[510] or

[511] , wherein IgdE is a protein selected from the group consisting of the following (a) to (d): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61; (b) a protein encoded by the base sequence set forth in SEQ ID NO: 62; (c) a protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between the T and H amino acid sequences of 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 to the amino acid sequence set forth in SEQ ID NO: 61, wherein the protein is capable of digesting human IgG1 antibody between the T and H amino acid sequences of the amino acid sequence KSCDKT / HTCPPCP.

[513] The method of any one of

[505] to

[512] , wherein the protease is FabALACTICA®.

[514] The method of any one of

[505] to

[513] , wherein the measuring, determining, or quantitating step comprises non-reducing capillary SDS gel electrophoresis (CE-SDS) or hydrophobic interaction chromatography (HIC).

[515] The method of

[514] , wherein the measuring, determining, or quantitating step comprises non-reducing capillary SDS gel electrophoresis (CE-SDS), and further comprises the step of performing non-reducing capillary SDS gel electrophoresis (CE-SDS) on a preparation to which no protease has been added and on a sample containing only protease.

[516] The method of

[515] further comprises the step of obtaining electropherograms for a preparation to which protease has been added, a preparation to which protease has not been added, and a sample containing only protease, wherein the electropherogram of the preparation to which protease has not been added contains peaks derived from LINC bodies and unLINC bodies not digested by protease, as well as a peak (Peak Z) derived from neither LINC bodies nor unLINC bodies not digested by protease; the electropherogram of the preparation to which protease has been added contains a peak derived from LINC bodies (Peak L), a peak derived from unLINC bodies digested by protease (Peak unL), a peak derived from protease (Peak E), and a peak (Peak Z) derived from none of LINC bodies, unLINC bodies digested by protease, or protease; and the electropherogram of the sample containing only protease contains a peak derived from protease (Peak E).

[517] The method of

[516] , wherein the LINC ratio is calculated by the ratio of (the area value of Peak L) to (the sum of the area values ​​of Peak unL, Peak E, and Peak Z) - (the area value of Peak Z) - (the area value of Peak E) + (the area value of Peak L).

[518] The method of

[514] , wherein the measuring, determining, or quantitating step comprises performing hydrophobic interaction chromatography (HIC) and obtaining a chromatogram.

[519] The method of

[518] , wherein the LINC ratio is calculated by 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 the peaks derived from all unLINC forms.

[520] The method of 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.

[521] The method of any one of

[505] to

[520] , further comprising the step of incubating the preparation after adding the protease.

[0023] The present disclosure also relates to the following inventions:

[601] A preparation comprising an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, produced by the method according to 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] .

[602] A pharmaceutical composition comprising a preparation containing an antigen-binding molecule having at least one disulfide bond formed between amino acid residues in a region other than the hinge region, the antigen-binding molecule being produced by the method according to 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] .

[0024] The present disclosure also relates to the following inventions:

[701] The method according to any one of

[101] to

[113] , wherein the composition (1) is a preparation produced by a 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] .

[702] A composition obtained by a method according to any one of

[101] to

[113] and

[701] .

[0025] The present disclosure also relates to the following inventions:

[801] A method for producing a pharmaceutical composition or pharmaceutical preparation comprising an antigen-binding molecule having at least one disulfide bond formed in a region other than the hinge region (LINC body), the method comprising: (a) measuring, determining, or quantifying the ratio of LINC bodies to the total of LINC bodies and unLINC bodies (LINC ratio) in a composition comprising LINC bodies and antigen-binding molecules that do not have a disulfide bond in a region other than the hinge region (unLINC body) using the method of any one of

[101] to

[113] ; (b) selecting a composition 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 (c) preparing a pharmaceutical composition or pharmaceutical preparation comprising the composition selected in step (b) and a pharmaceutically acceptable carrier.

[802] A method for producing a pharmaceutical composition or pharmaceutical preparation comprising an antigen-binding molecule having at least one disulfide bond formed in a region other than the hinge region (LINC body), the method comprising: (a) measuring, determining, or quantifying the ratio of LINC bodies to the total of LINC bodies and unLINC bodies (LINC ratio) in a composition comprising LINC bodies and antigen-binding molecules that do not have a disulfide bond in a region other than the hinge region (unLINC body) using the method according to any one of

[101] to

[113] ; (b) confirming 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%; (c) preparing a pharmaceutical composition or formulation comprising the composition whose LINC rate has been confirmed in step (b) and a pharmaceutically acceptable carrier.

[803] The composition of

[801] or

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

[201] to

[252] ,

[301] to

[365] , and

[401] to

[425] .

[804] The method of any one of

[801] to

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

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

[201] to

[252] ,

[301] to

[365] , and

[401] to

[425] .

[0026] The present disclosure also relates to the following inventions:

[901] A composition comprising an antigen-binding molecule having at least one disulfide bond formed in a region other than the hinge region (LINC body), an antigen-binding molecule having no disulfide bond in a region other than the hinge region (unLINC body), and a protease.

[902] The composition according to

[901] , wherein the protease is an enzyme that does not digest LINC bodies but can digest unLINC bodies.

[903] The composition according to

[901] or

[902] , wherein the protease is a cysteine ​​protease that can digest human IgG1 antibodies.

[904] The composition according to any one of

[901] to

[903] , wherein the protease is an enzyme that can cleave the hinge region of a human IgG1 antibody.

[905] The composition of any one of

[901] to

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

[906] The method of any one of

[901] to

[905] , wherein the protease is IgdE.

[907] The composition of

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

[908] The composition of

[906] or

[907] , wherein the IgdE is a protein selected from the group consisting of (a) to (d) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61, (b) a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 62, (c) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61 in which one or more amino acids have been substituted, deleted, added, and / or inserted, said protein being capable of digesting human IgG1 antibody between the T and H amino acid sequences of 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 to the amino acid sequence set forth in SEQ ID NO: 61, said protein being capable of digesting human IgG1 antibody between the T and H amino acid sequences of the amino acid sequence KSCDKT / HTCPPCP.

[909] The composition of any one of

[901] to

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

[910] The composition of any one of

[901] to

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

[201] to

[252] ,

[301] to

[365] , and

[401] to

[425] .

[911] The composition of any one of

[901] to

[910] , which is a mixture of a preparation produced by a 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] and a protease.

[0027] The present disclosure provides an efficient and easy method for producing and purifying antigen-binding molecules (LINC bodies) that have suitable disulfide bonds in regions other than the hinge region. The method disclosed herein makes it possible to obtain LINC-Ig formats simply, quickly, and with high yields. The method disclosed herein has the advantage of being adaptable to a wide range of conditions, including the type and concentration of reducing agent, pH, and reaction time.

[0028] Furthermore, compared to production and purification of LINC-Ig formats in a tank, the production and purification of LINC-Ig formats in a column as in the method of the present disclosure has advantages such as suppression of aggregation of antigen-binding molecules, short reaction times, easy operation, the ability to automate the reaction using programmed liquid chromatography (LC), the ability to maintain a constant concentration of the reducing agent during the reaction, and ease of reproducibility across scales.

[0029] The present disclosure also provides a method for measuring, determining, or quantifying the proportion of LINC bodies relative to the total of antigen-binding molecules having at least one disulfide bond formed in a region other than the hinge region (LINC bodies) and antigen-binding molecules having no disulfide bond in a region other than the hinge region (unLINC bodies). The method of the present disclosure makes it possible to measure, determine, or quantify the proportion of LINC-Ig formats contained in a composition. The method of the present disclosure has the advantage of being able to measure, determine, or quantify the ratio of LINC bodies to unLINC bodies for antigen-binding molecules that can form disulfide bonds in regions other than the hinge region, specifically, between various sites in the CH1 region of one heavy chain and various sites in the CH1 region of the other heavy chain.

[0030] This figure shows the analytical results of samples reduced with Cys. From the left, the following samples were subjected to affinity chromatography without the reduction / oxidation steps: Ctrl (1 sample), 0.1-100 mmol / L Cys solution, pH 7.0 (4 samples), and 0.1-100 mmol / L Cys solution, pH 8.0 (4 samples). Both pH 7.0 and 8.0 were confirmed to effectively improve the LINC rate, particularly in the 0.1-10 mmol / L concentration range. For the 100 mmol / L sample, no band was visible on the gel, making it impossible to accurately calculate the LINC rate; therefore, a "-" is indicated. This figure shows the analytical results of samples reduced with TCEP. From the left, the following samples were subjected to affinity chromatography without the reduction / oxidation steps: Ctrl (1 sample), 0.001-1 mmol / L TCEP solution, pH 7.0 (4 samples), and 0.001-1 mmol / L TCEP solution, pH 8.0 (4 samples). The results for 0.1-1 mmol / L are marked with "-" because no bands were observed on the gel, making it impossible to accurately calculate the LINC ratio. Figure 1 shows the design and naming rules for trivalent antibodies in the DUAL / LINC (1+2) format. Figure 2 shows electropherograms obtained by CE-SDS analysis. Figure 3 shows electropherograms with peak names obtained by HIS analysis. Figure 4 shows a standard electropherogram (top) and an enlarged electropherogram (bottom). Figure 5 shows a schematic diagram of the ALPS12 molecule. The dotted line indicates the disulfide bond between the CH1 domains of the heavy chains that form LINC bodies. Figure 6 shows the digestion site of FabALACTICA® in the IgG1 sequence. Figure 7 shows changes in LINC bodies and unLINC bodies after FabALACTICA® digestion. Figure 8 shows the raw data from Table 12. The numbers in Table 12 match those in Figure 9. Ori. refers to the undigested sample, and dig. refers to the digested sample. Figure 9 shows the method for calculating the LINC body content using the data from Figure 4. 11-1 is a diagram showing an overall view of an electropherogram, which is a result of HIC analysis. 11-2 is a diagram showing an enlarged view of FIG. 11-1. 11-3 is a diagram showing a calculation formula for LINC body content using the data of FIG. 11-1 and FIG. 11-2.

[0031] DESCRIPTION OF EMBODIMENTS The techniques and procedures described or referenced herein are generally well understood and can be found in, 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 (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 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 (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using These methods are commonly employed by those skilled in the art using conventional methodologies such as those widely used in Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0032] The following definitions and detailed description are provided to facilitate understanding of the disclosure described herein.

[0033] I. Definitions As used herein, the term "and / or" when describing the site of an amino acid modification includes any combination of "and" and "or." Specifically, for example, "the amino acids at positions 33, 55, and / or 96 are substituted" includes the following variations of amino acid modification: (a) positions 33, (b) positions 55, (c) positions 96, (d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96, and (g) positions 33, 55, and 96.

[0034] Unless otherwise indicated, amino acid residues in the light chain constant region are numbered herein according to Kabat et al., and numbering of amino acid residues in the heavy chain constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0035] Amino Acids Amino acids are described herein by one-letter or three-letter codes or both, for example, 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.

[0036] Amino acid modification Amino acid modification (also referred to herein as "amino acid substitution" or "amino acid mutation") in the amino acid sequence of an antigen-binding molecule can be performed using known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR. In addition, several known methods for modifying amino acids to substitute with unnatural amino acids can also be used (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is appropriate to use a cell-free translation system (Clover Direct (Protein Express)) that contains a tRNA in which an unnatural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is a type of stop codon.

[0037] Furthermore, herein, expressions indicating amino acid modifications may be appropriately used, with the one-letter or three-letter code of the amino acid before and after the number representing a specific position, respectively. For example, the modification N100bL or Asn100bLeu used to substitute an amino acid contained in an antibody variable region represents a substitution of Asn at position 100b (according to Kabat numbering) with Leu. That is, the number indicates the amino acid position according to Kabat numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution. Similarly, the modification P238D or Pro238Asp used to substitute an amino acid in the Fc region contained in an antibody constant region represents a substitution of Pro at position 238 (according to EU numbering) with Asp. That is, the numbers indicate the amino acid positions according to EU numbering, the one-letter or three-letter amino acid code written before the number indicates the amino acid before substitution, and the one-letter or three-letter amino acid code written after the number indicates the amino acid after substitution.

[0038] Polypeptide. As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, 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. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not necessarily be translated from a designated nucleic acid. It may be generated by any method, including chemical synthesis. The polypeptides described herein may be about 3 or more amino acids, 5 or more amino acids, 10 or more amino acids, 20 or more amino acids, 25 or more amino acids, 50 or more amino acids, 75 or more amino acids, 100 or more amino acids, 200 or more amino acids, 500 or more amino acids, 1,000 or more amino acids, or 2,000 or more amino acids in size. Polypeptides may have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are said to be folded, while polypeptides that do not have a defined three-dimensional structure but can adopt multiple different conformations are said to be unfolded.

[0039] Percent (%) Amino Acid Sequence Identity: "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, 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 algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will 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 of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0040] Antigen-Binding Molecules As used herein, the term "antigen-binding molecule," in its broadest sense, refers to any molecule that specifically binds to an antigenic determinant (epitope), including an antigen-binding domain, or any molecule that has antigen-binding activity. It can also refer to molecules such as peptides or proteins having a length of about 5 amino acids or more. Peptides and proteins are not limited to those derived from living organisms; for example, they can be polypeptides produced from artificially designed sequences. They can be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Scaffold molecules, which contain a known stable three-dimensional structure such as an α / β barrel as a scaffold and a portion of the molecule becomes the antigen-binding domain, are also an embodiment of the antigen-binding molecules described herein. In one embodiment, an antigen-binding molecule can contain two or more (e.g., 2, 3, 4, 5, or more) polypeptide chains. In one embodiment, the polypeptide chain constituting the antigen-binding molecule can be an antibody heavy chain or an antibody light chain. That is, in one embodiment, the antigen-binding molecule is an antibody, antibody fragment, or antibody derivative. In one embodiment, the antigen-binding molecule is a trivalent antibody comprising chains 1 to 5 as shown in Figure 3 of the present application. In one embodiment, the antigen-binding molecule is a non-antibody protein, or a fragment or derivative thereof.

[0041] Multispecific antigen-binding molecules A "multispecific antigen-binding molecule" refers to an antigen-binding molecule that specifically binds to two or more antigens. The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two different antigenic determinants. The term "trispecific" means that an antigen-binding molecule can specifically bind to at least three different antigenic determinants. In a specific embodiment, the multispecific antigen-binding molecule of the present application is a trispecific antigen-binding molecule that can bind to either CD3 or CD137, but not both antigens simultaneously, and can specifically bind to DLL3.

[0042] Antigen-binding domain: As used herein, the term "antigen-binding domain" refers to a region that specifically binds to and is complementary to a part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When an antigen has a large molecular weight, the antigen-binding domain can bind only to a specific part of the antigen. This specific part is called an epitope. In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "Fab'." In another embodiment, the antigen-binding domain comprises a non-antibody protein or a fragment thereof that binds to a specific antigen. In a specific embodiment, the antigen-binding domain comprises one or two Fabs comprising the CH1 region. In certain embodiments, the antigen binding domain comprises a hinge region.

[0043] As used herein, the term "antigen-binding domain" refers to a domain that can direct the entity to which it binds to a target site, such as a specific type of tumor cell that expresses a cancer antigen (DLL3). The antigen-binding domain can activate signal transduction through its target antigen, such as a T cell receptor complex antigen (particularly CD3) and / or a costimulatory receptor (CD137).

[0044] As used herein, the terms "first," "second," and "third" with respect to antigen-binding domains, etc., are used for the convenience of distinguishing between two or more different types of moieties, etc. The use of these terms is not intended to confer a particular order or orientation of the antigen-binding molecules, unless otherwise specified.

[0045] As used herein, the term "specifically binds" means that one of the molecules involved in specific binding binds without showing any significant binding to molecules other than its one or more binding partner molecules. This expression is also used when an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. When the epitopes bound by an antigen-binding domain are contained in multiple different antigens, an antigen-binding molecule containing the antigen-binding domain can bind to various antigens containing the epitopes.

[0046] In the present disclosure, "binding to the same epitope" means that the epitopes bound by two antigen-binding domains overlap at least partially. The degree of overlap is not limited, but is at least 10% or more, preferably 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, particularly preferably 90% or more, and most preferably 100%.

[0047] Antibodies The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or trispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0048] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, IgG class 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, from N- to C-terminus, a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, each light chain has, from N- to C-terminus, a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0049] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies containing naturally occurring mutations or variant antibodies that arise during the production of a monoclonal antibody preparation, which are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be produced by a variety of techniques, including, but not limited to, hybridoma technology, recombinant DNA technology, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; these and other exemplary methods for making monoclonal antibodies are described herein.

[0050] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0051] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0052] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0053] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR"), and / or forms structurally defined loops (the "hypervariable loops"), and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) 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).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. 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.

[0054] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0055] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0056] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, for example, from about position 216 to about position 230 according to the EU numbering system, or from about position 226 to about position 243 according to the Kabat numbering system. In naturally occurring IgG antibodies, the cysteine ​​residue at EU numbering position 220 in the hinge region is known to form a disulfide bond with the cysteine ​​residue at EU numbering position 214 in the antibody light chain. Furthermore, it is known that the cysteine ​​residues at EU numbering positions 226 and 229 in the hinge region of two antibody heavy chains form disulfide bonds. Generally, the "hinge region" is defined as spanning positions 216 to 238 (EU numbering) or positions 226 to 251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper hinge, the middle hinge, and the lower hinge. In human IgG1 antibodies, these regions are generally defined as follows: upper hinge: positions 216-225 (EU numbering) or 226-238 (Kabat numbering), middle hinge: positions 226-230 (EU numbering) or 239-243 (Kabat numbering), and lower hinge: positions 231-238 (EU numbering) or 244-251 (Kabat numbering). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S-S bonds in the same positions (see, e.g., Brekke et al., 1995, Immunol (Table 1 of Today 16: 85-90)). Hinge regions as used herein include wild-type hinge regions, as well as variants in which amino acid residues in the wild-type hinge region have been altered by substitution, addition, or deletion.

[0057] The terms "capable of being linked" (of amino acid residues) and "capable of being formed" (of disulfide bonds) include cases where the disulfide bond has already formed, and cases where the disulfide bond has not formed but can later be formed under appropriate conditions.

[0058] The term "disulfide bonds formed between amino acids not within the hinge region" (or "disulfide bonds formed between amino acid residues in a region other than the hinge region") refers to disulfide bonds formed, connected, or linked through amino acids located in any region of an antibody other than the "hinge region" as defined above. For example, such disulfide bonds are formed, connected, or linked through amino acids located at any position in an antibody other than the hinge region (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 through amino acids located in the CH1 region, CL region, VL region, VH region, and / or VHH region. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at EU numbering positions 119-123, 131-140, 148-150, 155-167, 174-178, 188-197, and 201-214 in the CH1 region. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at EU numbering positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, and 164 in the CH1 region. , 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214. In some embodiments, such disulfide bonds are formed, connected, or linked through amino acids located at positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering) in the CH1 region.In one preferred embodiment, such a disulfide bond is formed, connected, or linked through the amino acid located at EU numbering position 191 in the CH1 region.

[0059] The term "mismatched molecule" refers to an antigen-binding molecule in which disulfide bonds are formed between amino acid residues different from those desired. When an antigen-binding molecule contains a disulfide bond formed between desired amino acid residues and a disulfide bond formed between amino acid residues different from those desired, such an antigen-binding molecule is included in the mismatched molecule herein.

[0060] The term "incomplete formation" refers to an antigen-binding molecule in which cysteine ​​residues remain free or in which cysteine ​​residues form disulfide bonds with molecules other than the antigen-binding molecule (e.g., impurity molecules). As used herein, "incomplete formation" is not limited as long as it has such characteristics. For example, even if a molecule contains a disulfide bond formed between amino acid residues in a region other than the hinge region (a desired disulfide bond) or a disulfide bond other than the desired disulfide bond (a mismatch portion), it is still included in "incomplete formation" as long as it has the above characteristics.

[0061] The constant region is preferably an antibody constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 antibody constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 antibody constant region. The constant region is also preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. The amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4, multiple allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these sequences can be used in the present invention. The constant region with altered amino acids may also contain other amino acid mutations or modifications, as long as it contains the amino acid mutations of the present invention.

[0062] As used herein, the term "Fc region" or "Fc domain" refers to a region of an antibody molecule comprising a hinge or a portion thereof, and a fragment consisting of the CH2 and CH3 domains. The Fc region of an IgG class refers, for example, but is not limited to, the region from cysteine ​​226 (EU numbering, also referred to herein as the EU index) to the C-terminus, or from proline 230 (EU numbering) to the C-terminus. The Fc region can be obtained, for example, by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, followed by re-elution of the fraction adsorbed to a protein A or protein G column. The protease is not particularly limited, as long as it can digest a full-length antibody to form Fab or F(ab')2 under appropriately selected enzyme reaction conditions (e.g., pH). Examples include pepsin and papain.

[0063] For example, an Fc region derived from a native IgG can be used as the "Fc region." Here, native IgG refers to a polypeptide that contains the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by the immunoglobulin gamma gene. Native human IgG refers to, for example, native human IgG1, native human IgG2, native human IgG3, or native human IgG4. Native IgG also includes naturally occurring variants thereof. Multiple allotype sequences based on genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of these can be used in the present invention. In particular, the human IgG1 sequence may have DEL or EEM as the amino acid sequence at positions 356 to 358 (EU numbering).

[0064] The Fc domain of antigen-binding molecule is composed of a pair of polypeptide chains that comprise the heavy chain domain of immunoglobulin molecule.For example, the Fc domain of immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises CH2 and CH3 IgG heavy chain constant domain.The two subunits of Fc domain can stably associate with each other.In one embodiment, the antigen-binding molecule described herein comprises one or less Fc domain.

[0065] As used herein, the Fc domain of the antigen-binding molecule is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG1 Fc domain. In a further specific embodiment, the Fc domain is a human IgG1 Fc region.

[0066] Chimeric Antibodies The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. 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 is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0067] Humanized Antibodies A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. A "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0068] Antibody Fragments An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); also see WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting increased in vivo half-lives. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0069] Fv (variable fragment) As used herein, the term "Fv (variable fragment)" refers to the minimum unit of an antibody-derived antigen-binding domain consisting of a pair of an antibody light chain variable region (VL (light chain variable region)) and an antibody heavy chain variable region (VH (heavy chain variable region)). In 1988, Skerra and Plückthun discovered that homogeneous, active antibodies could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). In the Fv prepared from the periplasmic fraction, VH and VL were associated in a manner that allowed them to bind to antigens.

[0070] scFv, Single-Chain Antibodies, and sc(Fv)2. As used herein, the terms "scFv," "single-chain antibody," or "sc(Fv)2" all refer to antibody fragments that contain, in a single polypeptide chain, the variable regions from both the heavy and light chains but lack the constant regions. Generally, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains that enables them to form the desired structure that will allow antigen binding. Single-chain antibodies are discussed in detail by Plückthun in *The Pharmacology of Monoclonal Antibodies*, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994). See also International Patent Application Publication No. WO 1988 / 001649; U.S. Pat. Nos. 4,946,778 and 5,260,203. In certain embodiments, single-chain antibodies may be bispecific and / or humanized.

[0071] An scFv is an antigen-binding domain in which the VH and VL constituting the Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85(16), 5879-5883). The VH and VL can be held in close proximity by the peptide linker.

[0072] sc(Fv)2 is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). The two VH and VL may be derived from different monoclonal antibodies. Such sc(Fv)2 preferably includes bispecific sc(Fv)2 that recognizes two different epitopes present in the same 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 scFvs with a linker such as a peptide linker.

[0073] As used herein, examples of the form of the antigen-binding domain that constitutes sc(Fv)2 include antibodies characterized in that two VH units and two VL units are arranged in the following order, starting from the N-terminus of the single-chain polypeptide: VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]). However, the order of the two VH units and two VL units is not limited to the above configuration and may be arranged in any order. For example, the following order configurations are also possible: [VL]-linker-[VH]-linker-[VH]-linker-[VL] [VH]-linker-[VL]-linker-[VL]-linker-[VH] [VH]-linker-[VH]-linker-[VL]-linker-[VL] [VL]-linker-[VL]-linker-[VH]-linker-[VH] [VL]-linker-[VH]-linker-[VL]-linker-[VH]

[0074] The molecular form of sc(Fv)2 is also described in detail in WO2006 / 132352. Those skilled in the art can follow these descriptions to appropriately prepare the desired sc(Fv)2 for producing the polypeptide complexes disclosed herein.

[0075] Furthermore, the antigen-binding molecules or antibodies of the present disclosure may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer drugs. Alternatively, a glycosylation sequence is suitably inserted into the antigen-binding molecules or antibodies so that the sugar chains exert the desired effect.

[0076] Linkers used to link antibody variable regions include any peptide linker that can be introduced by genetic engineering, synthetic linkers, and linkers such as those disclosed in Protein Engineering, 9 (3), 299-305, 1996. However, in the present disclosure, peptide linkers are preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (although not particularly limited, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When sc(Fv)2 contains three peptide linkers, the lengths of these linkers may be the same or different.

[0077] For example, such peptide linkers include: Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO:5), Ser-Gly-Gly-Gly (SEQ ID NO:6), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:7), Ser-Gly-Gly-Gly-Gly (SEQ ID NO:8), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:9), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:10), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:11), Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:12), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO:7))n, and (Ser-Gly-Gly-Gly-Gly (SEQ ID NO:8))n. Here, n is an integer equal to or greater than 1. The length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0078] Synthetic linkers (chemical cross-linkers) are commonly used to cross-link peptides, including N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These cross-linkers are commercially available.

[0079] Three linkers are usually required to link four antibody variable regions, and the linkers used may be of the same type or different types.

[0080] Fab, F(ab')2, and Fab'. A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain ("Fab heavy chain") and the VL and CL domains of a light chain ("Fab light chain"). The heavy chain of a wild-type Fab molecule cannot form disulfide bonds with another heavy chain molecule. The term "Fab molecule" encompasses wild-type Fab molecules as well as Fab variants in which amino acid residues in the wild-type Fab have been altered by substitution, addition, or deletion. In certain embodiments, Fab variants contain mutated amino acid residues (e.g., substituted, added, or inserted cysteine ​​or lysine residues) that can form disulfide bonds with another heavy chain molecule or portion thereof (e.g., a Fab molecule).

[0081] An scFab is an antigen-binding domain in which one light chain and one heavy chain CH1 region and variable region constituting a Fab are linked by a peptide linker, which can maintain the light chain and the heavy chain CH1 region and variable region in close proximity.

[0082] The terms "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulins (monoclonal antibodies) with proteases such as pepsin and papain, digesting the immunoglobulins (monoclonal antibodies) near the disulfide bond between the hinge regions of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bond between the hinge regions of the two heavy chains, producing two homologous antibody fragments in which an light chain containing a VL (light chain variable region) and a CL (light chain constant region) is linked by a disulfide bond at the C-terminal region to an heavy chain fragment containing a VH (heavy chain variable region) and a CHγ1 (the γ1 region of the heavy chain constant region). These two homologous antibody fragments are each referred to as Fab'.

[0083] "F(ab')2" is composed of two light chains and two heavy chains containing constant regions, i.e., portions of the CH1 and CH2 domains, such that disulfide bonds are formed between the two heavy chains. The F(ab')2 disclosed herein can be suitably prepared by partially digesting a full-length monoclonal antibody or the like having a desired antigen-binding domain with a protease such as pepsin, followed by removal of the Fc fragment by adsorption onto a protein A column. Such a protease is not particularly limited, as long as it can cleave a full-length antibody to produce F(ab')2 in a limited manner by appropriately setting the enzyme reaction conditions, such as pH. Examples of such proteases include pepsin and ficin.

[0084] Fused "Fused" means that the components (e.g., a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0085] "Crossover" Fab A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the Fab heavy and Fab light chains have been exchanged; i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable region and a heavy chain constant region, and a peptide chain composed of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable regions of the Fab light and Fab heavy chains have been exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light and Fab heavy chains have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0086] "Conventional" Fab In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1) made up of the variable and constant regions of the heavy chain, and a light chain (VL-CL) made up of the variable and constant regions of the light chain.

[0087] Single Domain Antibodies As used herein, the term "single domain antibody" is not particularly limited in structure, as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing of VH and VL, whereas single domain antibodies are known to be able to exhibit antigen-binding activity solely through the domain structure of the single domain antibody itself, without pairing with any other domain. Single domain antibodies usually have a relatively low molecular weight and exist in the form of a monomer.

[0088] Examples of single domain antibodies include, but are not limited to, camelid VHHs, shark VHHs, and the like. NAR and antibody fragments comprising all or a portion of the VH domain or all or a portion of the VL domain of an antibody. Examples of single-domain antibodies, which are antibody fragments comprising all or a portion of the VH / VL domains of an antibody, include, but are not limited to, single-domain antibodies artificially produced starting from a human antibody VH or VL, such as those described in U.S. Patent No. 6,248,516 B1. A single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0089] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals carrying genes capable of producing single-domain antibodies. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals carrying genes capable of producing single-domain antibodies include, but are not limited to, the transgenic animals described in International Publication No. WO 2015 / 143414 and U.S. Patent Publication No. US 2011 / 0123527 A1. Humanized single-chain antibodies can also be obtained by replacing the framework sequences of single-domain antibodies obtained from animals with human germline sequences or sequences similar thereto. Humanized single-domain antibodies (e.g., humanized VHHs) are one embodiment of single-domain antibodies.

[0090] Alternatively, single domain antibodies can be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, or the like. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), and synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11 (1691-1701) are among the examples.

[0091] As used herein, an "agonist" antigen-binding molecule or an "agonist" antibody is an antigen-binding molecule or antibody that significantly enhances the biological activity of the antigen to which it binds.

[0092] As used herein, a "blocking" antigen-binding molecule or antibody or an "antagonist" antigen-binding molecule or antibody is an antigen-binding molecule or antibody that significantly inhibits (either partially or completely) the biological activity of the antigen to which it binds.

[0093] Antigen As used herein, the term "antigen" refers to the site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a three-dimensional structure composed of a continuous stretch of amino acids or a discrete region of non-contiguous amino acids), 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, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise indicated, the proteins referred to herein as antigens (e.g., CD3, CD137, DLL3) can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is human CD3, human CD137, or human DLL3. When a specific protein is referred to herein, the term encompasses "full-length," unprocessed proteins, as well as any form of protein produced by processing in cells. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0094] Fc Region with Reduced Fcγ Receptor-Binding Activity As used herein, "reduced Fcγ receptor-binding activity" means that the competitive activity of a test antigen-binding molecule or antibody is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, and particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the competitive activity of a control antigen-binding molecule or antibody, based on, for example, the analytical methods described above.

[0095] Antigen-binding molecules or antibodies containing the Fc domain of a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be used as a control antigen-binding molecule or antibody. The Fc domain structures are shown in SEQ ID NO: 1 (RefSeq Accession No. AAC82527.1 with an A added to the N-terminus), SEQ ID NO: 2 (RefSeq Accession No. AAB59393.1 with an A added to the N-terminus), SEQ ID NO: 3 (RefSeq Accession No. CAA27268.1 with an A added to the N-terminus), and SEQ ID NO: 4 (RefSeq Accession No. AAB59394.1 with an A added to the N-terminus). Furthermore, when antigen-binding molecules or antibodies containing Fc domain variants of a specific antibody isotype are used as test substances, the effect of the mutations of the variants on Fcγ receptor binding activity can be evaluated using an antigen-binding molecule or antibody containing an Fc domain of the same isotype as a control. As described above, antigen-binding molecules or antibodies comprising Fc domain mutants whose Fcγ receptor binding activity has been determined to be reduced are suitably prepared.

[0096] Such known mutants include, for example, a mutant having a deletion of amino acids 231A to 238S (EU numbering) (WO 2009 / 011941), as well as the 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).

[0097] Specifically, preferred antigen-binding molecules or antibodies include those comprising an Fc domain having at least one amino acid mutation (e.g., substitution) selected from the following amino acid positions forming the Fc domain of an antibody of a particular isotype: 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, 332, 439, 445, 449, or 451 (EU numbering). The antibody isotype from which the Fc domain is derived is not particularly limited, and an appropriate Fc domain derived from a monoclonal IgG1, IgG2, IgG3, or IgG4 antibody can be used. It is preferable to use an Fc domain derived from an IgG1 antibody.

[0098] Preferred antigen-binding molecules or antibodies include, for example, the following substitutions, whose positions are designated by EU numbering in amino acids forming the Fc domain of an IgG1 antibody (each number represents the position of an amino acid residue in EU numbering; and the one-letter amino acid code preceding the number represents the amino acid residue before substitution, and the one-letter amino acid code following the number represents the amino acid residue after substitution): (a) L234F, L235E, P331S; (b) C226S, C229S, P238S; (c) C226S, C229S; or (d) C226S, C229S, E233P, L234V, L235A; (e) L439M, A445N, R449Q, E451S; (f) M428L, N434A, Q438R, S440E as well as those having an Fc domain with a deletion of the amino acid sequence at positions 231 to 238.

[0099] Furthermore, preferred antigen-binding molecules or antibodies also include those comprising an Fc domain having any one of the following substitutions, whose positions are designated by EU numbering in amino acids forming the Fc domain of an IgG2 antibody: (e) H268Q, V309L, A330S, and P331S; (f) V234A; (g) G237A; (h) V234A and G237A; (i) A235E and G237A; or (j) V234A, A235E, and G237A. Each number represents the position of the amino acid residue according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution, and the single-letter amino acid code following the number represents the amino acid residue after substitution.

[0100] Furthermore, preferred antigen-binding molecules or antibodies also include those comprising an Fc domain having any one of the following substitutions, the positions of which are designated by EU numbering in amino acids forming the Fc domain of an IgG3 antibody: (k) F241A; (l) D265A; or (m) V264A. Each number represents the position of the amino acid residue according to EU numbering; the one-letter amino acid code preceding the number represents the amino acid residue before substitution, and the one-letter amino acid code following the number represents the amino acid residue after substitution.

[0101] Furthermore, preferred antigen-binding molecules or antibodies also include those comprising an Fc domain having any one of the following substitutions, the positions of which are designated by EU numbering in amino acids forming the Fc domain of an IgG4 antibody: (n) L235A, G237A, and E318A; (o) L235E; or (p) F234A and L235A. Each number represents the position of the amino acid residue according to EU numbering; the single-letter amino acid code preceding the number represents the amino acid residue before substitution, and the single-letter amino acid code following the number represents the amino acid residue after substitution.

[0102] Other preferred antigen-binding molecules or antibodies include, for example, those comprising an Fc domain in which any amino acid at position 233, 234, 235, 236, 237, 327, 330, or 331 (EU numbering) forming the Fc domain of an IgG1 antibody has been substituted with an amino acid at the corresponding position (EU numbering) in the corresponding IgG2 or IgG4.

[0103] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which one or more of the amino acids at positions 234, 235, and 297 (EU numbering) forming the Fc domain of an IgG1 antibody have been 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 one or more of the amino acids at positions 234, 235, and 297 have been substituted with alanine are particularly preferred.

[0104] Preferred antigen-binding molecules or antibodies also include, for example, those comprising an Fc domain in which the amino acid at position 265 (EU numbering) of the amino acids forming the Fc domain of an IgG1 antibody has been 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 has been substituted with alanine are particularly preferred.

[0105] (Preferentially) Enriched (or Increased) The term "(preferentially) enriched (or increased)" refers to an increase in the relative abundance of a desired form, or an increase in the relative proportion of a desired form, or an increase in the population of a desired form (structural isoform). In some embodiments, the methods described herein increase the relative abundance of an antibody structural isoform, such as an antibody having at least one disulfide bond formed between amino acid residues outside the hinge region. In one embodiment, the at least one disulfide bond is formed between amino acid residues at position 191 (EU numbering) in the CH1 region of each of the first and second antigen-binding domains. In certain embodiments, the method produces a homogeneous antibody preparation having at least 50%, 60%, 70%, 80%, 90%, and preferably at least 95% molar ratio of the antibody having at least one disulfide bond formed outside the hinge region.

[0106] Homogeneity A "homogeneous" population of antibodies refers to an antibody population that primarily comprises antibodies of a single form, e.g., at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100%, of the antibodies in a solution or composition are in a properly folded form. Similarly, a "homogeneous" population of antibodies having at least one disulfide bond formed outside the hinge region refers to a population of such antibodies that primarily comprises a single properly folded form, e.g., at least 50%, 60%, 70%, 80% or more, preferably at least 90%, 95%, 96%, 97%, 99%, or 100% of the antibodies having at least one disulfide bond formed outside the hinge region. In a preferred embodiment, the "homogeneous" population of antibodies comprises at least one disulfide bond formed between the amino acid residue at position 191 according to EU numbering in the CH1 region of each of the first and second antigen-binding domains (i.e., the "paired cysteines" at position 191 according to EU numbering in the CH1 region).

[0107] Determining whether an antibody population is homogeneous and the relative abundance or proportion of protein / antibody conformations in a mixture can be performed using any of a variety of analytical and / or qualitative techniques. If two conformations are differentially resolved during a separation technique, such as chromatography, electrophoresis, filtration, or other purification technique, the relative proportions of the conformations in the mixture can be determined using such a purification technique. For example, at least two distinct conformations of a recombinant IgG can be separated by hydrophobic interaction chromatography. Furthermore, because far-ultraviolet circular dichroism has been used to predict the secondary structural organization of proteins (Perczel et al., 1991, Protein Eng. 4:669-679), such a technique can determine whether alternative conformations of a protein exist. Yet another technique used to determine conformation is fluorescence spectroscopy, which can be used to identify complementary differences in tertiary structure that can be assigned to tryptophan and tyrosine fluorescence. Other techniques that can be used to determine conformational differences and therefore the relative proportions of conformations are online SEC to measure aggregation state, differential scanning calorimetry to measure melting transitions (Tm) and component enthalpies, and chaotropic unfolding. Yet another technique that can be used to determine conformational differences and therefore the relative proportions of conformations is LC / MS detection to determine protein heterogeneity.

[0108] Alternatively, if differences in activity exist between antibody / protein conformations, determining the relative proportions of conformations in the mixture can be done by activity assays (e.g., ligand binding, enzymatic activity, biological activity, etc.). The biological activity of the protein can also be used. Alternatively, binding assays can be used where activity is expressed as activity units / mg protein.

[0109] IEC chromatography is used to determine antibody / protein heterogeneity. In such cases, the antibody is purified or considered "homogeneous," meaning that no polypeptide peaks or fractions corresponding to other polypeptides are detected upon analysis by IEC chromatography. In certain embodiments, an antibody is purified or considered "homogeneous" when no polypeptide bands corresponding to other polypeptides are detected upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those skilled in the relevant art will recognize that multiple bands corresponding to a polypeptide can be visualized by SDS-PAGE due to differential glycosylation, differential post-translational processing, and the like. Most preferably, the polypeptide is purified to substantial homogeneity, as indicated by a single polypeptide band upon analysis by SDS-PAGE. The polypeptide band can be visualized by silver staining, Coomassie blue staining, and / or autoradiography (if the polypeptide is radiolabeled).

[0110] Herein, examples of conditions for SDS-PAGE analysis are as follows: Non-reducing SDS-PAGE is performed using 4-20% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad) with 1× Tris / glycine / SDS running buffer (Bio-Rad). Monoclonal antibody samples are heated at 70°C for 10 minutes. 0.2 micrograms are loaded, and electrophoresis is performed at 200 V for 90 minutes. Proteins are visualized using a Chemidoc Imaging System (Bio-Rad). The percentage of individual bands is analyzed using Image Lab software version 6.0 (Bio-Rad), and the percent intensity of each band (e.g., fast-migrating (lower band) and slow-migrating (upper band) bands) is calculated by dividing the band intensity by the sum of the two bands. The gel may then be stained with CBB, a gel image may be captured, and the bands may be quantified using an imaging device. In the gel image, multiple bands, for example, two bands, i.e., an "upper band" and a "lower band," may be observed in the antibody variant sample. In this case, the molecular weight of the upper band may correspond to that of the parent antibody (before modification). The cysteine ​​substitution may cause structural changes, such as cross-linking via disulfide bonds in the Fab, which may result in changes in electrophoretic mobility. In this case, the lower band may be considered to correspond to an antibody with one or more engineered disulfide bonds formed between the CH1 regions. An antibody variant sample with additional cysteine ​​substitutions may exhibit a higher ratio of lower band to upper band compared to a control sample. The additional cysteine ​​residue may enhance / promote Fab disulfide bond cross-linking; increase the percentage or structural uniformity of antibody preparations with engineered disulfide bonds formed at the mutated positions; or decrease the percentage of antibody preparations without engineered disulfide bonds formed at the mutated positions. As used herein, the term "ratio of lower band to upper band" refers to the ratio between the amount / intensity of the upper band and the amount / intensity of the lower band, which can be quantified during the SDS-PAGE test described above.

[0111] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0112] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0113] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0114] II. Methods for producing a preparation containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies), and methods for measuring, determining, or quantifying the proportion of LINC bodies relative to the total of LINC bodies and antigen-binding molecules without disulfide bonds formed between amino acid residues in a region other than the hinge region (unLINC bodies) The present disclosure relates to methods for producing a preparation containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. The method of the present disclosure comprises subjecting a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and disulfide bond mismatches and / or incomplete disulfide bonds of the antigen-binding molecules (hereinafter sometimes referred to herein as a "mixture containing antigen-binding molecules," "mixture of antigen-binding molecules," etc.) to chromatography in the presence of a reducing agent. In the method of the present disclosure, the mixture containing antigen-binding molecules can be subjected to one or more cycles of one type of chromatography. Alternatively, a mixture containing antigen-binding molecules can be subjected to one or more (e.g., two, three, or more) treatments of each of multiple (e.g., two, three, or more) types of chromatography. When multiple chromatographic treatments are performed, they may be consecutive or discontinuous.

[0115] In the method of the present disclosure, chromatography known to those skilled in the art can be used, including, but not limited to, column chromatography, membrane chromatography, thin-layer chromatography, and the like.

[0116] In the production method of the present disclosure, the step of subjecting a mixture containing antigen-binding molecules to chromatography is carried out in the presence of a reducing agent. In the production method of the present disclosure, the form of the reducing agent is not important as long as it is present in the step of subjecting a mixture containing antigen-binding molecules to chromatography. For example, the antigen-binding molecules may be supported on a chromatography support in advance, and then the antigen-binding molecules may be contacted with a reducing agent, or a mixture containing the antigen-binding molecules and a reducing agent may be mixed and passed through chromatography.

[0117] In one aspect, when column chromatography is used, the method of the present disclosure can include a step of contacting a chromatography support packed in a column of column chromatography with a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and disulfide bond mismatches and / or incomplete disulfide bonds of the antigen-binding molecules. Furthermore, in one aspect, when membrane chromatography is used, the method of the present disclosure can include a step of contacting a chromatography support coated on a membrane of membrane chromatography with a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and disulfide bond mismatches and / or incomplete disulfide bonds of the antigen-binding molecules. The mixture containing the antigen-binding molecules may have been contacted with a reducing agent in advance.

[0118] In the present disclosure, the step of subjecting a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and mismatched and / or incomplete disulfide bonds of the antigen-binding molecules to chromatography can include the following steps: (a) contacting the mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and mismatched and / or incomplete disulfide bonds of the antigen-binding molecules with a solution containing a reducing agent, and (b) removing the reducing agent.

[0119] In one aspect, the method of the present disclosure produces or purifies a homogeneous population of antigen-binding molecules or a preparation or concentrate of homogeneous antigen-binding molecules by the steps described herein.

[0120] In one aspect, the method of the present disclosure can increase the proportion of antigen-binding molecules that have at least one disulfide bond between amino acid residues in a region other than the hinge region relative to antigen-binding molecules that are supported on a carrier and have amino acid residues that can form at least one disulfide bond between amino acid residues in a region other than the hinge region (such antigen-binding molecules include antigen-binding molecules in which at least one disulfide bond is properly formed between amino acid residues in a region other than the hinge region, and antigen-binding molecules in which at least one disulfide bond is not properly formed between amino acid residues in a region other than the hinge region, i.e., disulfide bond mismatches and / or incomplete formation of the antigen-binding molecules).

[0121] In one aspect, the method of the present disclosure produces antigen-binding molecules having at least one disulfide bond between amino acid residues in a region other than the hinge region in a molar ratio of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% relative to carrier-supported antigen-binding molecules having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region (such antigen-binding molecules include antigen-binding molecules in which at least one disulfide bond is properly formed between amino acid residues in a region other than the hinge region and antigen-binding molecules in which at least one disulfide bond is not properly formed between amino acid residues in a region other than the hinge region, i.e., disulfide bond mismatches and / or incomplete formation of the antigen-binding molecules).

[0122] Thus, in one aspect, the present disclosure relates to a method for producing a preparation comprising an increased concentration of antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. In one aspect, the preparation produced by the method of the present disclosure can be a concentrate of antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region. Also, in one aspect, the present disclosure relates to a method for increasing the concentration of antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region in a mixture of antigen-binding molecules. In a further aspect, the present disclosure relates to a method for purifying a preparation comprising antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region from a mixture containing antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region and mismatched and / or incomplete disulfide bonds of the antigen-binding molecules. In the present disclosure, antigen-binding molecules that do not have disulfide bonds formed between amino acid residues in regions other than the hinge region include those in which cysteine ​​residues in regions other than the hinge region are capped, those in which cysteine ​​residues remain as cysteines, etc.

[0123] In the method of the present disclosure, a mixture of antigen-binding molecules is contacted with a solution containing a reducing agent. In a specific embodiment, the mixture of antigen-binding molecules supported on a carrier is contacted with a solution containing a reducing agent in a chromatography process. In one aspect, chromatography carriers of the present disclosure include, but are not limited to, affinity chromatography carriers, ion exchange chromatography carriers, hydrophobic interaction chromatography carriers, multimode chromatography carriers including both ion exchange chromatography and hydrophobic interaction chromatography, and hydroxyapatite carriers. In the present disclosure, the terms "carrier," "resin," "ligand," and "substrate" are used interchangeably.

[0124] In the present disclosure, the affinity chromatography support is not limited as long as it exhibits the action of affinity chromatography. In one aspect, examples of affinity chromatography supports include, but are not limited to, protein A supports, protein G supports, protein L supports, sequence-selective peptide supports, and supports that selectively bind to antigen-binding molecules.

[0125] In the method of the present disclosure, commercially available affinity chromatography supports can be used. In a specific aspect, examples of affinity chromatography supports include MabSelect SuRe® (Cytiva), MabSelect Xtra® (Cytiva), MabSelect SuRe® LX (Cytiva), MabSelect SuRe® pcc (Cytiva), MabSelect PrismA® (Cytiva), MabSpeed® rP202 (Cytiva), TOYOPEARL® rProtein A HC-650F (TOSOH), Praesto® Jetted A50 (Purolite), Amsphere® A3 (JSR), Amsphere® Protein A JWT203 (JSR), Eshmuno A® (Merck Millipore), Capto L® (Cytiva), and MabSelect® VL (Cytiva). Examples include, but are not limited to, the following:

[0126] In one aspect, the ion exchange chromatography support of the present disclosure includes an anion exchange resin (anion exchange ligand) or a cation exchange resin (cation exchange ligand). The anion exchange resin and the cation exchange resin of the present disclosure are not limited as long as they exhibit anion exchange activity and cation exchange activity, respectively.

[0127] In the method of the present disclosure, commercially available anion exchange chromatography supports can be used.In a specific aspect, examples of anion exchange chromatography supports include YMC-BioPro (YMC Corporation), Q Sepharose® High Performance (Cytiva Corporation), Q Sepharose® Fast Flow (Cytiva Corporation), Q Sepharose® XL (Cytiva Corporation), Q Sepharose® Big Beads (Cytiva Corporation), Capto® Q ImpRes (Cytiva Corporation), Capto® Q (Cytiva Corporation), Capto® Q XP (Cytiva Corporation), Capto® DEAE (Cytiva Corporation), SOURCE® 30Q (Cytiva Corporation), SOURCE® 15Q (Cytiva Corporation), DEAE Sepharose® Fast Flow (Cytiva Corporation), ANX Sepharose® 4 Fast Flow (Cytiva Corporation), POROS® 50 PI (Thermo Fisher), and POROS® Examples of suitable carriers include, but are not limited to, 50 HQ (Thermo Fisher), POROS (registered trademark) HQ (Thermo Fisher), POROS (registered trademark) D (Thermo Fisher), POROS (registered trademark) PI (Thermo Fisher), Eshumuno (registered trademark) Q (Merck Millipore), Fractogel (registered trademark) TMAE (Merck Millipore), Fractogel (registered trademark) DEAE (Merck Millipore), Macro-Prep (registered trademark) Q (Bio-Rad Laboratories), Macro-Prep (registered trademark) DEAE (Bio-Rad Laboratories), Giga Cap (registered trademark) Q-650M (TOSOH), Giga Cap (registered trademark) DEAE-650M (TOSOH), and Q HyperCel (registered trademark) (PALL).

[0128] Furthermore, commercially available cation exchange chromatography supports can also be used. In a specific aspect, examples of cation exchange chromatography supports include, but are not limited to, Capto (registered trademark) S (Cytiva), Capto (registered trademark) SP ImpRes (Cytiva), Capto (registered trademark) S ImpaAct (Cytiva), SP Sepharose (registered trademark) Fast Flow (Cytiva), CM Sepharose (registered trademark) Fast Flow (Cytiva), SP Sepharose (registered trademark) High Performance (Cytiva), CM Sepharose (registered trademark) High Performance (Cytiva), SP Sepharose (registered trademark) XL (Cytiva), SP Sepharose (registered trademark) Big Beads (Cytiva), Eshumuno (registered trademark) CPX (Merck Millipore), Eshumuno (registered trademark) CP-FT (Merck Millipore), POROS (registered trademark) 50HS (Thermo Fisher), and POROS (registered trademark) XS (Thermo Fisher).

[0129] In the present disclosure, the support for hydrophobic interaction chromatography is not limited as long as it exhibits hydrophobic interaction chromatography activity. In one aspect, the hydrophobic interaction chromatography support comprises a hydrophobic ligand.

[0130] In the method of the present disclosure, commercially available hydrophobic interaction chromatography supports can be used. In certain aspects, the hydrophobic interaction chromatography support may be Phenyl Sepharose® High Performance (Cytiva), Butyl Sepharose® High Performance (Cytiva), Phenyl Sepharose® 6 Fast Flow (Cytiva), Butyl-S Sepharose® 6 Fast Flow (Cytiva), Butyl Sepharose® 4 Fast Flow (Cytiva), Octyl Sepharose® 4 Fast Flow (Cytiva), Capto® Phenyl ImpRes (Cytiva), Capto® Phenyl (Cytiva), Capto® Phenyl (High Sub) (Cytiva), Capto® Butyl (Cytiva), Capto® Butyl ImpRes (Cytiva), Capto® Octyl (Cytiva), Phenyl Sepharose® 6 Fast Flow (Low Sub) (Cytiva), Phenyl Sepharose® 6 Fast Flow (High Sub) (Cytiva), POROS® Ethyl (Thermo Fisher), Fractogel® Phenyl (Merck Millipore), Fractogel® Propyl (Merck Millipore), TOYOPEARL® Butyl (TOSOH), TOYOPEARL® Ether (TOSOH), TOYOPEARL® Hexyl (TOSOH), TOYOPEARL® Phenyl (TOSOH), TOYOPEARL® PPG (TOSOH), TOYOPEARL® SuperExamples include, but are not limited to, Butyl (TOSOH Corporation), TOYOPEARL (registered trademark) Butyl-600 (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-650C (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-650M (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-650S (TOSOH Corporation), TOYOPEARL (registered trademark) Phenyl-600M (TOSOH Corporation), and Macro-Prep (registered trademark) HIC (Bio-Rad Laboratories).

[0131] In one aspect, the multimodal chromatography support of the present disclosure can include a support having a combination of a cation exchange ligand and a hydrophobic ligand, or a support having a combination of an anion exchange ligand and a hydrophobic ligand. In the method of the present disclosure, commercially available multimodal chromatography supports can be used. Examples of supports for multimodal chromatography include, but are not limited to, Capto® adhere (Cytiva), Capto® adhere ImpRes (Cytiva), Capto® MMC (Cytiva), Capto® MMC ImpRes (Cytiva), and Eshmuno® CMX (Merck Millipore).

[0132] In one aspect, the hydroxyapatite chromatography support can include hydroxyapatite or a derivative thereof (e.g., fluoroapatite). In the method of the present disclosure, commercially available hydroxyapatite chromatography supports can also be used. In certain embodiments, supports for hydroxyapatite chromatography include, but are not limited to, Ceramic Hydroxyapatite® Type I (Bio-Rad Laboratories), Ceramic Hydroxyapatite® Type II (Bio-Rad Laboratories), Ceramic Fluoroapatite® (Bio-Rad Laboratories), MPC® Ceramic Hydroxyfluoroapatite (Bio-Rad Laboratories), HA Ultrogel® (PALL), and Ca++Pure-HA® (TOSOH).

[0133] In the method of the present disclosure, commercially available membrane chromatography can also be used.In certain embodiments, the product for membrane chromatography can be exemplified but not limited to Mustang (registered trademark) Q (PALL), Mustang (registered trademark) S (PALL), Sartobind (registered trademark) Q (Sartorius), Sartobind (registered trademark) S (Sartorius), Sartobind (registered trademark) STIC (Sartorius), Sartobind (registered trademark) protein A (Sartorius), Fibro (Cytiva), Protein capture (GORE), Natrix (registered trademark) Q (Merck Millipore), Purexa (registered trademark)-MQ (Purilogics), Purexa (registered trademark)-A (Purilogics) and Purexa (registered trademark)-DMAE (Purilogics).

[0134] In the present disclosure, the terms "reducing reagent," "reducing agent," and "solution containing a reducing agent" are used interchangeably. In some embodiments, the reducing agent is a free thiol. In one aspect, the reducing agent in the present disclosure can be a monothiol, a dithiol, a phosphine, or an inorganic reagent. Examples of monothiols in the present disclosure include, but are not limited to, glutathione, cysteine, lipoic acid, 2-mercaptoethanol, 2-MEA, etc. Examples of dithiols in the present disclosure include, but are not limited to, DTT (dithiothreitol), DTE, DTBA, etc. Examples of phosphines in the present disclosure include, but are not limited to, TCEP (tris(2-carboxyethyl)phosphine), THPP, etc. Examples of inorganic reagents in the present disclosure include, but are not limited to, sodium sulfite, sodium pyrosulfite, etc.

[0135] The reducing reagent preferably comprises a compound selected from the group consisting of glutathione (GSH), dithiothreitol (DTT), 2-mercaptoethanol, 2-aminoethanethiol (2-MEA), TCEP (tris(2-carboxyethyl)phosphine), dithionitrobenzoate, cysteine, sodium sulfite, sodium pyrosulfite, and Na2SO3. In some embodiments, TCEP, 2-MEA, DTT, cysteine, GSH, or Na2SO3 can be used. In some preferred embodiments, cysteine ​​can be used. In some preferred embodiments, TCEP can be used. One or more reducing agents (e.g., two, three, or more) can be used in the methods of the present disclosure.

[0136] In one embodiment, the reducing agent can be in the form of a solution, preferably a buffer solution. Examples of buffer solutions include, but are not limited to, buffer solutions used in chromatography, such as phosphate buffer, Tris buffer, acetate buffer, citrate buffer, tartrate buffer, and borate buffer. Examples of base solutions include sodium hydroxide, potassium hydroxide, lithium hydroxide, and Tris.

[0137] In the present disclosure, "contacting" means subjecting a mixture of antigen-binding molecules to, exposing to, or mixing a mixture of antigen-binding molecules with a solution containing a reducing agent. In the method of the present disclosure, the mixture of antigen-binding molecules can be contacted with a solution containing a reducing agent while bound to a solid support (e.g., a column for column chromatography or a membrane for membrane chromatography).

[0138] Without being bound by theory, the existence of unLINC bodies (i.e., bivalent or trivalent antigen-binding molecules without engineered disulfide bonds or "paired cysteines") may be due to unpaired Cys residues, which often form disulfide bonds with molecules containing free thiol groups, such as cysteinylation and glutathionylation, which "cap" unpaired Cys residues and prevent LINC formation (the formation of engineered disulfide bonds in regions other than the hinge region). To remove unpaired cysteine-capped molecules, reducing agents can help decap the surface cysteines, and further reoxidation of the decapped antigen-binding molecules (e.g., by removing the reducing reagent) can promote disulfide bond formation between the decapped cysteines for LINC formation. Therefore, removal of cysteinylation from unpaired sulfhydryls in unLINC bodies by reduction and reoxidation can remove unLINC bodies and improve antibody homogeneity.

[0139] In one aspect, in the method of the present disclosure, disulfide bonds formed between amino acid residues capable of forming disulfide bonds in the antigen-binding molecules are cleaved by contacting a mixture of antigen-binding molecules with a solution containing a reducing agent, and capped cysteine ​​residues in the antigen-binding molecules are uncapped by contacting a mixture of antigen-binding molecules with a solution containing a reducing agent.

[0140] In one aspect, in the method of the present disclosure, removal of the reducing agent results in the formation of disulfide bonds between amino acid residues capable of forming disulfide bonds in the antigen-binding molecule.

[0141] The mixture of antigen-binding molecules is contacted with the solution containing a reducing agent for a time sufficient to increase the relative proportion of the desired conformation. Any relative increase in proportion is desirable, including, for example, conversion of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the antigen-binding molecules having an undesired conformation to antigen-binding molecules having the desired conformation.

[0142] In one aspect, in the method of the present disclosure, a solution containing a reducing agent is passed through a chromatography system containing a carrier on which an antigen-binding molecule is supported. In a specific embodiment, the antigen-binding molecule forms a stationary phase immobilized on the carrier of the chromatography system, and the solution containing a reducing agent is part of the mobile phase. In this case, the contacting can be performed as part of a chromatographic purification method.

[0143] The reducing agent is present at a concentration sufficient to increase the relative proportion of the desired conformation (e.g., an antigen-binding molecule in the "paired cysteine" form, with one or more engineered disulfide bonds between the two Fabs of the antibody, for example, between regions other than the hinge region). The optimal absolute concentration and molar ratio of the reducing agent depend on the concentration of the total antigen-binding molecules and, in some cases, the specific antigen-binding molecules. The concentration of the reducing agent also depends on the number and accessibility of unpaired cysteines in the antigen-binding molecule.

[0144] In one aspect, the concentration of the reducing agent can be exemplified as a range that can be specified by any combination of a lower limit selected from the group consisting of about 0.0001 mM, about 0.0005 mM, about 0.001 mM, about 0.005 mM, about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.5 mM, and about 1.0 mM, and an upper limit selected from the group consisting of about 100.0 mM, about 75.0 mM, about 50.0 mM, about 25.0 mM, about 10.0 mM, about 5.0 mM, about 1.0 mM, about 0.5 mM, about 0.1 mM, and about 0.01 mM. In some embodiments, the concentration of the reducing agent can be in the range of about 0.00001 mM to about 10.0 mM, about 0.00005 mM to about 5.0 mM, about 0.0001 mM to about 1.0 mM, about 0.0005 mM to about 0.5 mM, about 0.001 mM to about 0.1 mM, or about 0.001 mM to about 0.01 mM. In further embodiments, the concentration of the reducing agent can be in the range of about 0.001 mM to about 100.0 mM, about 0.005 mM to about 75.0 mM, about 0.01 mM to about 50.0 mM, about 0.05 mM to about 25.0 mM, or about 0.1 mM to about 10 mM. In further embodiments, the concentration of the reducing agent can be in the range of about 0.0001 mM to about 100.0 mM, about 0.0005 mM to about 50.0 mM, about 0.001 mM to about 10.0 mM, about 0.005 mM to about 5.0 mM, or about 0.01 mM to about 1.0 mM. In certain embodiments, the concentration of the reducing agent can be about 0.001 mM, about 0.01 mM, about 0.1 mM, about 0.15 mM, about 1.0 mM, or about 10.0 mM.

[0145] 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 a 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 a 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.

[0146] In certain 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 certain 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.

[0147] To maximize the yield of antigen-binding molecules having a desired conformation, the pH of the solution containing the reducing agent is selected to protect the stability of the antigen-binding molecule and optimize disulfide exchange. In one aspect, the pH of the solution containing the reducing agent of the present disclosure can be 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. In one non-limiting embodiment of the present invention, the optimal pH was found to be about 7.0, about 7.5, or about 8.0. However, the optimal pH for a particular embodiment of the present invention can be easily determined experimentally by one skilled in the art.

[0148] In the method of the present disclosure, a chromatography carrier, an antigen-binding molecule, and a reducing agent are contacted within a chromatography column. In one aspect, in the method of the present disclosure, the antigen-binding molecule supported on the carrier is contacted with the reducing agent within a chromatography column. In other words, in the method of the present 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 of column chromatography or a membrane chromatography device containing the antigen-binding molecule supported on the carrier.

[0149] In some embodiments, the solution containing the reducing agent is passed through a column of column chromatography or a membrane chromatography device at a passage time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes. In particular embodiments, the solution containing the reducing agent is passed through a column of column chromatography or a membrane chromatography device at a passage time of about 12 minutes or about 30 seconds. In some embodiments, the passage may be temporarily stopped while the column or device is filled with the solution containing the reducing agent.

[0150] In one aspect, when column chromatography is used, the solution containing the reducing agent is passed through the column at a flow rate of about 2 minutes to about 80 minutes, or about 4 minutes to about 24 minutes. In a particular aspect, when column chromatography is used, the solution containing the reducing agent is passed through the column at a flow rate of about 12 minutes. In one aspect, the flow may be temporarily stopped when the column is filled with the solution containing the reducing agent. In one aspect, when column chromatography is used, the solution containing the reducing agent of the present disclosure may be passed through the column at a velocity of about 25 cm / hr to about 500 cm / hr, about 50 cm / hr to about 400 cm / hr, about 75 cm / hr to about 350 cm / hr, or about 100 cm / hr to about 300 cm / hr. In some aspects, the solution containing the reducing agent of the present disclosure may be passed through the column at a velocity of about 100 cm / hr. In one aspect, the flow may be temporarily stopped when the column is filled with the solution containing the reducing agent.

[0151] In one aspect, when membrane chromatography is used, the solution containing the reducing agent is passed through the membrane device at a flow rate 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 when the membrane device is filled with the solution containing the reducing agent. In one aspect, when membrane chromatography is used, the 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 certain embodiments, the solution containing the reducing agent of the present disclosure may be passed through the membrane device at a rate of about 2 MV / min. The flow may be temporarily stopped when the membrane device is filled with the solution.

[0152] The amount of the solution containing the reducing agent passed through the column is not particularly limited. For example, when column chromatography is used, the amount of the solution containing the reducing agent passed through the column can be about 0.1 times (about 0.1 CV (column volume)) to about 100 times (about 100 CV), about 1 time (about 1 CV) to about 20 times (about 20 CV), or about 3 times (about 3 CV) to about 10 times (about 10 CV) the column volume. In some embodiments, the amount of the solution containing the reducing agent passed through the column can be about 10 times (about 10 CV) the column volume. When membrane chromatography is used, the amount of the solution containing the reducing agent passed through the column can be about 1 time (about 1 MV (membrane volume)) to about 500 times (about 500 MV), or about 2 times (about 2 MV) to about 100 times (about 100 MV) the membrane device volume. In some embodiments, the volume of the solution containing the reducing agent passed through the column can be about 15 times the column volume (about 15 MV).

[0153] The reaction time (contact time) between the mixture of antigen-binding molecules and the solution containing a reducing agent is not particularly limited, and examples thereof include about 6 seconds to about 1,440 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 specific embodiment, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 120 minutes or about 7.5 minutes.

[0154] In one aspect, when column chromatography is used, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 4 minutes to about 1440 minutes, about 8 minutes to about 300 minutes, about 30 minutes to about 240 minutes, about 60 minutes to about 180 minutes, or about 120 minutes. In a specific embodiment, the contact time between the mixture of antigen-binding molecules and the solution containing a reducing agent can be about 120 minutes.

[0155] In one aspect, when membrane chromatography is used, the contact time between the mixture of antigen-binding molecules and the solution containing 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.

[0156] In the methods of the present disclosure, the antigen-binding molecule can be contacted with the reducing agent in various amounts, as needed, such as analytical experimental scale (1 to 50 mL), preparative scale (50 mL to 10 L), and manufacturing scale (10 L or more). In one aspect, the amount of the antigen-binding molecule supported on the substrate can be about 5 to 100 g, about 7 to 70 g, about 10 to 40 g, e.g., about 10 g, or about 25 g per 1 L of chromatography support containing the substrate.

[0157] In one aspect, when column chromatography is used, the amount of antigen-binding molecule supported on 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, for example, about 10 g, per liter of chromatography carrier containing the substrate.

[0158] In one aspect, when membrane chromatography is used, the amount of antigen-binding molecules supported on 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 of membrane device volume.

[0159] The method of the present disclosure includes a step of removing the reducing agent. In the present disclosure, removal of the reducing agent includes completely removing the reducing agent, reducing the concentration of the reducing agent, or chemically inactivating the reducing agent. Removal of the reducing agent promotes reoxidation of disulfide bonds in regions other than the hinge region between the two Fabs that constitute the antigen-binding molecule.

[0160] In one aspect, the reducing agent can be removed by contacting the antigen-binding molecule with a solution that does not contain a reducing agent, or by passing the solution that does not contain a reducing agent through a column for column chromatography or a device for membrane chromatography.

[0161] In some embodiments, the solution without the reducing agent is passed through a column of column chromatography or a membrane chromatography device at a flow time of about 2 seconds to about 80 minutes or about 3 seconds to about 24 minutes. In particular embodiments, the solution without the reducing agent is passed through a column of column chromatography or a membrane chromatography device at a flow time of about 4 minutes. In some embodiments, the flow may be temporarily stopped when the solution is loaded into the column or device.

[0162] In some embodiments, when column chromatography is used, the solution without the reducing agent is passed through the column for a passage time of about 2 minutes to about 80 minutes, or about 4 minutes to about 24 minutes. In particular embodiments, when column chromatography is used, the solution without the reducing agent is passed through the column for a passage time of about 4 minutes. In some embodiments, the passage may be temporarily stopped when the solution is packed into the column.

[0163] In some embodiments, when column chromatography is used, the solution without a reducing agent can be passed through the column at a rate of about 25 cm / hr to about 500 cm / hr, about 50 cm / hr to about 450 cm / hr, about 200 cm / hr to about 400 cm / hr, or about 250 cm / hr to about 350 cm / hr. In certain embodiments, the solution without a reducing agent of the present disclosure can be passed through the column at a rate of about 300 cm / hr. The flow of the solution may be temporarily stopped when the column is filled with the solution.

[0164] In some embodiments, when membrane chromatography is used, the solution without the reducing agent is passed through the membrane device with a transit time of about 2 seconds to about 60 minutes, or about 3 seconds to about 6 minutes, e.g., about 30 seconds. In some embodiments, the passage may be temporarily stopped once the solution has filled the device.

[0165] In some embodiments, when membrane chromatography is used, the solution not containing a reducing agent can 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. Also, in certain embodiments, the solution not containing a reducing agent of the present disclosure can be passed through the membrane device at a rate of about 2 MV / min. The flow of the solution may be temporarily stopped when the membrane device is filled with the solution.

[0166] The reducing agent-free solution is not particularly limited, and examples thereof include buffer solutions, more specifically, buffer solutions commonly used in chromatography techniques, such as phosphate buffer, Tris buffer, acetate buffer, citrate buffer, tartrate buffer, and borate buffer. Examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, and Tris. In one aspect, in the method of the present disclosure, the reducing agent-free solution can be the same type of solution as the reducing agent-containing solution used in the contacting step, except that it does not contain a reducing agent.

[0167] In one aspect, when column chromatography is used, the flow rate of the solution without reducing agent can be about 0.1 times (about 0.1 CV) to about 100 times (about 100 CV), about 1 time (about 1 CV) to about 20 times (about 20 CV), or about 3 times (about 3 CV) to about 10 times (about 10 CV) the column volume. In some embodiments, the flow rate of the solution without reducing agent can be about 5.0 times (about 5.0 CV) the column volume.

[0168] In one aspect, when membrane chromatography is used, the flow rate of the solution without reducing agent can be from about 1 fold (1 MV) to about 500 fold (500 MV), or from about 2 fold (2 MV) to about 100 fold (100 MV), for example, about 15 fold (15 MV), of the volume of the membrane device.

[0169] In one aspect, the reaction time (contact time) between the mixture of antigen-binding molecules and the solution not containing a reducing agent can be from about 6 seconds to about 1,440 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 specific embodiment, the reaction time (contact time) between the mixture of antigen-binding molecules and the solution not containing a reducing agent can be about 20 minutes.

[0170] In one aspect, when column chromatography is used, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution not containing a reducing agent can be about 4 minutes to about 1,440 minutes, or about 8 minutes to about 300 minutes. In a particular embodiment, when column chromatography is used, the reaction time (contact time) between the mixture of antigen-binding molecules and a solution not containing a reducing agent can be about 20 minutes.

[0171] In one aspect, when membrane chromatography is used, the reaction time (contact time) of the mixture of antigen-binding molecules with a solution not containing 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.

[0172] The methods of the present invention can be carried out over a wide temperature range. For example, the methods 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 contacting partially or fully purified preparations of antigen-binding molecules are about 4°C to about 25°C (ambient temperature), or preferably 23°C, although lower and higher temperatures can also be used. In some embodiments, the methods of the present disclosure can be carried out at temperatures of about 20°C to 37°C, preferably 23°C, 25°C, or 37°C, and more preferably 23°C.

[0173] Even for solutions that do not contain a reducing agent, the optimal pH can be easily determined experimentally by those skilled in the art. For example, it can be 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. In certain embodiments, the optimal pH can be about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, or about 7.9.

[0174] In one aspect, the method of the present disclosure can include a step of contacting the mixture of antigen-binding molecules with a chromatography support and packing the antigen-binding molecules into a column or immobilizing them on a membrane before the step of contacting the mixture with a solution containing a reducing agent. In a specific embodiment, when the mixture is a harvested cell culture fluid (HCCF) containing antigen-binding molecules, a step of contacting the cell culture fluid with a chromatography support and packing the antigen-binding molecules into a column or immobilizing them on a membrane can be included before the step of contacting the cell culture fluid with a solution containing a reducing agent. In one aspect, the mixture of antigen-binding molecules contains antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (i.e., LINC bodies) and antigen-binding molecules having no disulfide bond in a region other than the hinge region (i.e., unLINC bodies).

[0175] In some aspects, the antigen-binding molecule may be at least partially purified before the step of contacting with a solution containing a reducing agent. In some embodiments, the production method of the present disclosure may comprise the step of subjecting the cell culture solution containing the antigen-binding molecule to affinity chromatography (preferably Protein A chromatography) before the step of contacting.

[0176] In one aspect, the method of the present disclosure can include a step of removing impurities during chromatography prior to the contacting step. Impurities can be removed by methods well known to those skilled in the art, such as passing a buffer solution through a column containing a substrate to which an antigen-binding molecule has been adsorbed, as described in the Examples, for example.

[0177] In one aspect, the method of the present disclosure can further comprise the step of recovering or eluting antigen-binding molecules having at least one disulfide bond in a region other than the hinge region, and / or purifying the antigen-binding molecules, before or after the step of removing the reducing agent. In one embodiment, the recovery and elution of antigen-binding molecules can be carried out by eluting the antigen-binding molecules from the chromatography column using an elution buffer or other methods well known to those skilled in the art. Those skilled in the art can adjust the pH, composition, etc. of the elution buffer as appropriate depending on the properties of the antigen-binding molecules to be purified.

[0178] In one embodiment, the antigen-binding molecule preparations produced as described herein may be further purified by techniques known in the art, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a particular antigen-binding molecule will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. Affinity chromatography purification can use an antibody, ligand, receptor, or antigen to which the antigen-binding molecule binds. For example, affinity chromatography purification of antigen-binding molecules can use a matrix with protein A or protein G. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate 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-pressure liquid chromatography.

[0179] In certain embodiments, antigen-binding molecules having at least one disulfide bond in a region other than the hinge region produced or purified by the methods described herein may be further treated with a separation treatment step utilizing a chaotropic denaturant, such as sodium dodecyl sulfate (SDS), urea, or guanidinium hydrochloride (GuHCl). A significant amount of chaotropic agent is required to observe detectable unfolding. In some embodiments, the treatment step uses between 0.1 M and 2 M of a chaotrope, which produces an effect equivalent to the use of 0.1 M to 2 M of guanidinium hydrochloride. In certain embodiments, oxidative refolding is achieved in the presence of approximately 1.0 M guanidinium hydrochloride, or an amount of another chaotropic agent that produces the same or similar amount of refolding as 1 M guanidinium hydrochloride. In some embodiments, between about 1.5 M and 0.5 M of a chaotrope is used. The amount of chaotropic agent used is based on the structural stability of the antigen-binding molecule in the presence of the chaotrope. The chaotrope should be present enough to disrupt the local tertiary and / or quaternary structure of the domain interactions of the antigen-binding molecule, but less than that required to completely unfold the secondary structure of the molecule and / or individual domains. To determine the point at which the antigen-binding molecule begins to unfold by equilibrium denaturation, those skilled in the art can titrate the chaotrope into a solution containing the antigen-binding molecule and monitor the structure using techniques such as circular dichroism or fluorescence. There are other parameters that can be used instead of chaotropes to unfold or slightly disrupt the structure of the antigen-binding molecule. Temperature and pressure are two basic parameters that have been used to change the structure of antigen-binding molecules and can be used instead of chaotropic agents during contact with oxidizing and / or reducing agents. The inventors contemplate that any parameter shown to denature or disrupt the structure of antigen-binding molecules can be used by those skilled in the art instead of chaotropic agents.

[0180] The method of the present disclosure increases the ratio of antigen-binding molecules in LINC bodies to antigen-binding molecules supported on a carrier (i.e., the total of antigen-binding molecules in LINC bodies that have at least one disulfide bond in a region other than the hinge region and antigen-binding molecules in unLINC bodies), i.e., the "LINC ratio." The LINC ratio can be calculated by separating crosslinked LINC bodies and uncrosslinked unLINC bodies (open type) by non-reducing SDS-PAGE and comparing the abundance ratios of each, as described in WO2021 / 157679, for example.

[0181] In one embodiment, a preparation of antigen-binding molecules produced as described herein may be further subjected to techniques such as electrophoresis, chromatography, etc. to determine the LINC ratio. That is, the production method of the present disclosure can include, following a chromatographic purification step, a step of measuring, determining, or quantifying the LINC ratio in the preparation obtained by the purification step, i.e., the ratio (LINC ratio) of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC bodies) to the total of (i) antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies), and (ii) disulfide bond mismatched and / or unformed antigen-binding molecules (unLINC bodies). In a specific aspect, the production method of the present disclosure can comprise the step of measuring, determining, or quantifying the ratio (LINC ratio) of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC bodies) to the total number of antigen-binding molecules having at least one disulfide bond in a region other than the hinge region (LINC bodies), and (ii) antigen-binding molecules in which no disulfide bond is formed (unLINC bodies).

[0182] That is, the present disclosure also relates to a method for measuring, determining, or quantifying the ratio of LINC bodies to the total of LINC bodies and unLINC bodies (LINC ratio) in a composition comprising antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies) and disulfide bond mismatched and / or incomplete (unLINC bodies) of the antigen-binding molecules. In a specific aspect, the present disclosure also relates to a method for measuring, determining, or quantifying the ratio of LINC bodies to the total of LINC bodies and unLINC bodies (LINC ratio) in a composition comprising antigen-binding molecules having at least one disulfide bond formed between amino acid residues in a region other than the hinge region (LINC bodies) and disulfide bond incomplete (unLINC bodies) of the antigen-binding molecules.

[0183] In a still further aspect, the present disclosure also relates to a method for measuring, determining, or quantifying the ratio of LINC bodies to the total of LINC bodies and unLINC bodies (LINC ratio) in a composition comprising antigen-binding molecules having at least one disulfide bond formed in a region other than the hinge region (LINC bodies) and antigen-binding molecules having no disulfide bond in a region other than the hinge region (unLINC bodies).

[0184] The measuring, determining, or quantitating method disclosed herein comprises the following steps (a) and (b): (a) adding a protease to composition (1) containing LINC bodies and unLINC bodies to prepare composition (2), and (b) subjecting composition (2) to electrophoresis or chromatography.

[0185] Composition (1) comprising LINC bodies and unLINC bodies of the present disclosure is not particularly limited as long as it contains LINC bodies and unLINC bodies. Furthermore, the content ratio of LINC bodies and unLINC bodies and the degree of purification are not particularly limited. In one embodiment, composition (1) comprising LINC bodies and unLINC bodies of the present disclosure can contain the LINC bodies and their unLINC bodies disclosed herein. In another embodiment, composition (1) comprising LINC bodies and unLINC bodies of the present disclosure can be a preparation obtained by the manufacturing method described herein or a preparation described herein. In one embodiment, composition (1) comprising LINC bodies and unLINC bodies of the present disclosure can be in the form of a crude product. In a further embodiment, composition (1) comprising LINC bodies and unLINC bodies of the present disclosure can be in the form of a drug substance or a standard.

[0186] In one embodiment, the step of measuring, determining, or quantifying the LINC ratio includes electrophoresis, chromatography, or the like. In another embodiment, the method of measuring, determining, or quantifying the LINC ratio of the present disclosure includes subjecting composition (1) and / or (2) and / or composition (3) described below to electrophoresis or chromatography. In a specific embodiment, the method of measuring, determining, or quantifying the LINC ratio of the present disclosure includes subjecting composition (1), (2), and composition (3) described below to electrophoresis or chromatography. In one embodiment, examples of electrophoresis include non-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and non-reducing capillary SDS gel electrophoresis (CE-SDS). In another embodiment, examples of chromatography include hydrophobic interaction chromatography (HIC). These techniques can be performed using equipment well known to those skilled in the art. For example, an instrument for non-reducing capillary SDS gel electrophoresis (CE-SDS) is the LabChip. TM(PerkinElmer), and PA800 (SCIEX). Examples of instruments for hydrophobic interaction chromatography include Proteomix (registered trademark) (Sepax Technologies, Inc.), Shim-pack Bio HIC series (Shimadzu Corporation), and MabPAC HIC-10 (Thermo Fisher Scientific).

[0187] In one embodiment, the manufacturing method of the present disclosure may include adding a protease to a preparation of antigen-binding molecules manufactured as described herein before measuring, determining, or quantifying the LINC ratio. In another embodiment, the method of measuring, determining, or quantifying the LINC ratio of the present disclosure includes adding a protease to composition (1) containing LINC bodies and unLINC bodies to prepare composition (2). The protease of the present disclosure is not particularly limited as long as it is an enzyme that can digest unLINC bodies but not LINC bodies. In one embodiment, it can be a cysteine ​​protease that can digest the hinge region of an antibody. Such an enzyme can degrade the antibody to generate Fab and Fc regions. In a specific embodiment, the antibody is preferably human IgG1.

[0188] In another embodiment, the protease of the present disclosure can be IgdE derived from bacteria of the genus Streptococcus, including, but not limited to, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus suis, Streptococcus porcinus, Streptococcus pseudoporcinus, Streptococcus canis, Streptococcus castoreus, and Streptococcus merionis.

[0189] In certain embodiments, the protease of the present disclosure is preferably a protease that digests human IgG1 antibody between the T H amino acid sequences of KSCDKT / HTCPPCP in the hinge region of the human IgG1 antibody. In one aspect, the protease of the present disclosure can be IgdE derived from Streptococcus agalactiae. IgdE derived from Streptococcus agalactiae is an enzyme disclosed in literature such as Spoerry C et al., (2016) PLoS ONE 11(10): e0164809. doi:10.1371 / journal.pone.0164809, and can be easily obtained by those skilled in the art using well-known techniques.

[0190] In a further specific embodiment, the IgdE derived from Streptococcus agalactiae in the present disclosure can include a protein selected from the group consisting of the following (a) to (d): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61, (b) a protein encoded by the nucleotide sequence set forth in SEQ ID NO: 62, (c) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61 in which one or more amino acids have been substituted, deleted, added, and / or inserted, said protein being capable of digesting human IgG1 antibody between the T and H amino acid residues of 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 to the amino acid sequence set forth in SEQ ID NO: 61, said protein being capable of digesting human IgG1 antibody between the T and H amino acid residues of the amino acid sequence KSCDKT / HTCPPCP.

[0191] Amino acid addition, deletion, substitution, and / or insertion can be performed by methods known in the art. For example, site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 488-492 (1985)) or overlap extension PCR can be performed on a nucleic acid encoding the amino acid sequence. These methods may be performed alone or in combination, as appropriate.

[0192] In general, it is known that modifications (e.g., conservative substitutions, deletions, insertions, and / or additions) of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) amino acids in a protein do not affect the function of the protein or even enhance the function of the original protein. Amino acids are classified into hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T) according to the characteristics of their side chains. Amino acid side chains can also be classified into aliphatic side chains (G, A, V, L, I, P), hydroxyl-containing side chains (S, T, Y), sulfur-containing side chains (C, M), carboxylic acid- and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), and aromatic-containing side chains (H, F, Y, W). The IgdE of the present disclosure also includes proteins in which amino acids contained in a protein comprising the amino acid sequence of SEQ ID NO: 61 have been modified with other amino acids classified into a group having the same characteristics. However, the IgdE of the present disclosure may also comprise non-conservative modifications as long as they are functionally equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 61.

[0193] In one embodiment, IgdE of the present disclosure also encompasses proteins comprising an amino acid sequence that has a high sequence identity with the amino acid sequence represented by SEQ ID NO: 61. In the present disclosure, high identity refers to sequence identity of at least 50% or more, more preferably 70% or more, 75% or more, 80% or more, 85% or more, and even more preferably 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) of the entire amino acid sequence or the entire nucleotide sequence.

[0194] IgdE derived from Streptococcus agalactiae is also available from Genovis, for example, under the name FabALACTICA (IgdE)." That is, in certain embodiments, the protease of the present disclosure is FabALACTICA The present inventors have incorporated IgdE from Streptococcus agalactiae, specifically FabALACTICA (registered trademark) (IgdE), into compositions comprising LINC bodies and unLINC bodies, for example, preparations of antigen-binding molecules produced as described herein. It has been found that the LINC rate in a composition or preparation can be determined by adding IgdE (registered trademark) (IgdE) and then subjecting the preparation containing IgdE to electrophoresis, chromatography, or the like.

[0195] Thus, the present disclosure also relates to a composition comprising an antigen-binding molecule having at least one disulfide bond formed in a region other than the hinge region (LINC body), an antigen-binding molecule having no disulfide bond in a region other than the hinge region (unLINC body), and a protease. Such a composition can be used to determine the LINC ratio. In one embodiment, the LINC body of the antigen-binding molecule, its unLINC body, and the protease contained in the composition can be those described herein. Such a composition may also contain other components as long as it can be used to determine the LINC ratio.

[0196] The concentration of protease added to the preparation is not particularly limited, but in one embodiment, it can be 0.6 to 1.8 units of enzyme / μg protein, for example. In a specific embodiment, when the LINC rate is measured, determined, or quantified using CE-SDS, 1.25 units of protease / μg protein can be used. In a specific embodiment, when the LINC rate is measured, determined, or quantified using HIC, 1.2 units of protease / μg protein can be used.

[0197] In certain embodiments, when the production method of the present invention includes a step of adding a protease to a preparation of antigen-binding molecules produced as described herein, non-reducing capillary SDS gel electrophoresis (CE-SDS) or hydrophobic interaction chromatography (HIC) can be used in the step of measuring, determining, or quantifying the LINC rate. Furthermore, in certain embodiments, the chromatography for obtaining the antigen-binding molecule preparation can be membrane chromatography.

[0198] In one embodiment, in the step of measuring, determining, or quantifying the LINC ratio in the manufacturing method of the present disclosure, the LINC ratio can be calculated as follows. For example, when non-reducing capillary SDS gel electrophoresis (CE-SDS) is used in the measuring, determining, or quantifying step, a step of adding protease to the preparation obtained in the purification step to prepare a "protease-added preparation" is included. A step of obtaining a "sample containing only protease" is also included. Then, electropherograms are obtained for each of the "protease-added preparation," the "protease-free preparation," and the "protease-only sample." The electropherogram obtained from the "protease-free preparation" contains peaks derived from LINC bodies and unLINC bodies undigested by protease, as well as a peak (peak Z) derived from neither LINC bodies nor unLINC bodies undigested by protease. The electropherogram obtained from the "preparation to which protease has been added" contains a peak derived from LINC bodies (Peak L), a peak derived from unLINC bodies digested with protease (Peak unL), a peak derived from protease (Peak E), and a peak derived from neither LINC bodies, unLINC bodies digested with protease, nor protease (Peak Z). The electropherogram obtained from the "sample containing only protease" contains a peak derived from protease (Peak E).

[0199] When the LINC rate is measured, determined, or quantified using CE-SDS, the LINC rate is calculated as the ratio of the "area value of Peak L" to the "total area values ​​of Peaks L, E, and Z" minus the "area values ​​of Peak Z" and "area values ​​of Peak E," plus the "area value of Peak L," as shown in formula (I) below. These area values ​​can be calculated by a person skilled in the art according to the analytical manual for the CE-SDS device. Alternatively, the analytical manual may be modified as necessary and the area values ​​may be calculated accordingly.

[0200] The "preparation to which protease has been added" includes LINC bodies, unLINC bodies digested by proteases, proteases, and degradation products derived from the target protein. Therefore, the "peak Z" includes peaks derived from these. The "sample containing only protease" is not particularly limited as long as it does not contain LINC bodies or unLINC bodies and contains the protease of the present disclosure. Examples of the "sample containing only protease" of the present disclosure include, but are not limited to, FabALACTICA (registered trademark) (Genovis). In one embodiment, the "sample containing only protease" can be added, for example, in an equal amount, to a preparation to which the sample has not been added.

[0201] In another embodiment, in the method of the present disclosure, the LINC ratio can also be calculated as follows. For example, when hydrophobic interaction chromatography (HIC) is used in the measuring, determining, or quantitating step, a protease is added to the preparation obtained by the purification step to obtain a "protease-containing preparation." The "protease-containing preparation" is then subjected to HIC to obtain a chromatogram. In this case, the LINC ratio is calculated as the ratio of the area value of the peak derived from the LINC entity to the sum of the area values ​​of the peak derived from the LINC entity and the area values ​​of the peaks derived from all unLINC entities, as shown in formula (II) below. These area values ​​can be calculated by those skilled in the art according to the analysis manual for the HIC instrument. Alternatively, the analysis manual can be modified as necessary and the area values ​​can be calculated accordingly. Mobile phase A and mobile phase B in hydrophobic interaction chromatography (HIC) can be prepared appropriately by those skilled in the art based on common technical knowledge. Furthermore, those skilled in the art can also select a chromatography carrier appropriately, and for example, those described herein can be used.

[0202] Furthermore, when non-reducing capillary SDS gel electrophoresis (CE-SDS) is used to quantify the LINC ratio, the method for measuring, determining, or quantifying the LINC ratio in the present disclosure further comprises, in addition to compositions (1) and (2), preparing composition (3) containing a protease but not LINC bodies or unLINC bodies. Then, the method comprises subjecting each of compositions (1) to (3) to CE-SDS and obtaining electropherograms (1) to (3) for compositions (1) to (3), respectively. Composition (3) is not particularly limited as long as it does not contain LINC bodies or unLINC bodies and contains the protease of the present disclosure. In one embodiment, the aforementioned FabALACTICA (registered trademark) (Genovis) can be used.

[0203] In the present disclosure, electropherogram (1) includes peaks derived from LINC bodies and unLINC bodies undigested by protease, as well as a peak (Peak Z) derived from neither LINC bodies nor unLINC bodies undigested by protease. Electropherogram (2) includes a peak (Peak L) derived from LINC bodies, a peak (Peak unL) derived from unLINC bodies digested by protease, a peak (Peak E) derived from protease, and a peak (Peak Z) derived from none of LINC bodies, unLINC bodies digested by protease, or protease. Electropherogram (3) includes a peak (Peak E) derived from protease.

[0204] When non-reducing capillary SDS gel electrophoresis (CE-SDS) is used, the LINC rate is calculated by the following formula (I): where A is the area value of peak L in electropherogram (2), B is the sum of the areas of peaks unL, E, and Z in electropherogram (2), C is the area value of peak Z in electropherogram (1), and D is the area value of peak E in electropherogram (3).

[0205] In theory, when unLINC bodies are digested with a protease, three fragments are generated as shown on the right side of Figure 8 (i.e., when the antigen-binding molecule is a trivalent antibody consisting of a molecule with five chains as shown in Figure 3, (i) a fusion product of the first or second antigen-binding domain with the third antigen-binding domain, (ii) the first or second antigen-binding domain that does not constitute the fusion product, and (iii) the Fc region). Furthermore, the present inventors have found that when unLINC bodies are digested with a protease, in addition to these three fragments, only one of the two hinge regions contained in the antigen-binding molecule (specifically, when the antigen-binding molecule is a trivalent antibody consisting of a molecule with five chains as shown in Figure 3, the hinge region of chain 3) is digested, and the hinge region of the other chain (i.e., chain 1) is not digested. In the present disclosure, "peaks derived from unLINC bodies digested with a protease" include peaks derived from the three fragments (i) to (iii) above, as well as peaks derived from fragments resulting from digestion of only the hinge region in chain 3 of a trivalent antibody consisting of a molecule composed of five chains as shown in Figure 3, as described above.

[0206] Furthermore, when hydrophobic interaction chromatography (HIC) is used to measure, determine, or quantify the LINC ratio in the present disclosure, the method includes subjecting composition (2) to hydrophobic interaction chromatography (HIC) to obtain a chromatogram. In this case, the LINC ratio can be calculated based on the following formula (II): Here, A is the area value of the peak derived from the LINC body in the chromatogram, and B is the area value of all peaks derived from the unLINC body digested by the protease in the chromatogram.

[0207] Here, all peaks derived from unLINC bodies digested with proteases include peaks derived from all fragments of unLINC bodies that result from digestion of unLINC bodies with proteases, more specifically, peaks derived from the four fragments mentioned above.

[0208] In the present disclosure, the unLINC rate, that is, the ratio of unLINC bodies to the total of LINC bodies and unLINC bodies, can be determined by subtracting the LINC rate (%) from 100%.

[0209] In one embodiment, the method of the present disclosure can further comprise the step of culturing the preparation or composition after adding the protease to the preparation or composition and before subjecting the preparation or composition to electrophoresis or chromatography. The culturing conditions are not particularly limited, but the preparation or composition can be incubated in a phosphate buffer solution at a pH of 6-8, for example, at 37°C, for 16 to 20 hours, for example, 16 hours, 18 hours, or 20 hours.

[0210] In the present disclosure, the antigen-binding molecule subjected to chromatography for purification may contain a signal sequence. In particular, when an antigen-binding molecule having amino acid residues capable of forming at least one disulfide bond between amino acid residues in a region other than the hinge region is recombinantly expressed in cells, the antigen-binding molecule may have a signal sequence to facilitate expression and / or secretion. In one embodiment, when the translation product of a nucleic acid sequence for recombinantly expressing an antigen-binding molecule contains a signal sequence, the method of the present disclosure may comprise a step of cleaving the signal sequence from the antigen-binding molecule. Cleavage of the signal sequence can be performed before or after the step of subjecting the antigen-binding molecule to chromatography for purification, or before or after the step of quantifying the LINC rate in the chromatographically purified product. Cleavage of the signal sequence can be performed by methods well known to those skilled in the art.

[0211] In one aspect, the amino acid residue capable of forming a disulfide bond is a mutated, substituted, introduced or engineered cysteine ​​residue.

[0212] In one aspect, at least one disulfide bond in a region other than the hinge region is formed between polypeptides constituting the antigen-binding molecule. That is, in certain embodiments, at least one disulfide bond in a region other than the hinge region of the present disclosure is an interchain disulfide bond. In some embodiments, at least one disulfide bond in a region other than the hinge region is one, two, three, four, five, or more interchain disulfide bonds.

[0213] In one aspect, at least one disulfide bond in a region other than the hinge region is an engineered disulfide bond that is not present in wild-type IgG.

[0214] III. Antigen-binding molecules of the present invention can form disulfide bonds between a region other than the hinge region of one heavy chain constituting the antigen-binding molecule and a region other than the hinge region of the other heavy chain. In the present disclosure, an antigen-binding molecule having a disulfide bond between a region other than the hinge region of one heavy chain and a region other than the hinge region of the other heavy chain is referred to as a LINC body, and an antigen-binding molecule without such a disulfide bond is referred to as a unLINC body. In the present disclosure, an example of a region other than the hinge region is the CH1 region. In the present disclosure, unLINC bodies include bodies with mismatched disulfide bonds and / or incomplete formation of disulfide bonds. In the present disclosure, LINC bodies can also be referred to as LINC formats. In addition, unLINC bodies can also be referred to as unLINC formats.

[0215] In one aspect, the antigen-binding molecule of the present disclosure comprises a first antigen-binding domain and a second antigen-binding domain that can be linked to each other via at least one disulfide bond. In one aspect, in the antigen-binding molecule produced or purified by the method of the present disclosure, the first antigen-binding domain and the second antigen-binding domain are linked via at least one disulfide bond. In one embodiment of the above aspect, the first antigen-binding domain and the second antigen-binding domain are linked via two, three, four, or more disulfide bonds.

[0216] In one embodiment, at least one of the first and second antigen-binding domains has antigen-binding activity alone (i.e., one antigen-binding domain alone has antigen-binding activity). In a particular embodiment, both the first and second antigen-binding domains have antigen-binding activity alone.

[0217] In one aspect, each of the first antigen-binding domain and the second antigen-binding domain of the present disclosure can have a Fab, Fab', scFab, Fv, scFv, or VHH structure.

[0218] In one aspect, each of the first antigen-binding domain and the second antigen-binding domain is a Fab molecule, and the antigen-binding molecule comprises at least one disulfide bond formed between the first antigen-binding domain and the second antigen-binding domain, and preferably, the at least one disulfide bond is formed between amino acid residues (cysteines) that are not in the hinge region, preferably between amino acid residues (cysteines) in the CH1 region of each antigen-binding domain.

[0219] In one embodiment of the above aspect, both the first and second antigen-binding domains comprise a Fab and hinge region.

[0220] In a specific embodiment, at least one of the amino acid residues forming a disulfide bond between the antigen-binding domains is a mutant amino acid residue not present in the wild-type Fab or hinge region, e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region, which can be introduced into the wild-type Fab or hinge region by, for example, amino acid substitution.

[0221] Alternatively, in another embodiment, in the antigen-binding molecules of the present disclosure, amino acid residues present in the wild-type Fab or hinge region that may participate in disulfide bonds between antigen-binding domains (e.g., cysteine ​​residues) may be substituted with other amino acid residues or deleted, such as those at positions 220, 226, and 229 (EU numbering) in the hinge region and at position 214 in the CL region.

[0222] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains comprises an antibody fragment that binds to a specific antigen. In certain embodiments, the antibody fragment is a Fab, Fab', scFab, Fv, scFv, or single-domain antibody. In certain embodiments, the first and / or second antigen-binding domain comprises a hinge region. Amino acid residues that form disulfide bonds are present in the first and second antigen-binding domains, respectively, and the bond between the antigen-binding domains is formed by linking these amino acid residues. In certain embodiments, at least one of the amino acid residues that form disulfide bonds between the antigen-binding domains is present within the antibody fragment.

[0223] In one aspect, each of the first and second antigen-binding domains of the present disclosure may or may not include a hinge region. In a specific embodiment, each of the first and second antigen-binding domains of the present disclosure may include a Fab and hinge region that form an F(ab')2 structure.

[0224] In one embodiment of the above aspect, the first and second antigen-binding domains both bind to the same antigen. In a specific embodiment, the first and second antigen-binding domains bind to the same epitope on the same antigen. In another specific embodiment, the first and second antigen-binding domains each bind to a different epitope on the same antigen. In a specific embodiment, the antigen-binding molecule of the present disclosure is a biparatopic antigen-binding molecule (e.g., a biparatopic antibody) that targets one specific antigen.

[0225] In another embodiment of the above aspect, each of the first and second antigen-binding domains binds to a different antigen.

[0226] In another embodiment of the above aspect, the antigen-binding molecule of the present disclosure is a clamping antigen-binding molecule (e.g., a clamping antibody). As used herein, a clamping antigen-binding molecule refers to an antigen-binding molecule that specifically binds to an antigen-antigen-binding molecule complex formed between a certain antigen A and an antigen-binding molecule that binds to the antigen A, thereby increasing the binding activity of the antigen-binding molecule that binds to the antigen A (or stabilizing the antigen-antigen-binding molecule complex formed between the antigen A and the antigen-binding molecule that binds to the antigen A). For example, a CD3-clamping antibody specifically binds to an antigen-antibody complex formed between CD3 and an antibody with reduced binding ability to CD3 (a CD3 antibody with reduced binding), thereby increasing the CD3-binding activity of the CD3 antibody with reduced binding ability (or stabilizing the antigen-antibody complex formed between CD3 and the CD3 antibody with reduced binding ability). In a specific embodiment, the first and / or second antigen-binding domain in an antigen-binding molecule of the present disclosure can be an antigen-binding domain derived from a clamping antigen-binding molecule (clamping antigen-binding domain).

[0227] In one embodiment of the above aspects, the first and second antigen-binding domains both have the same amino acid sequence, hi another embodiment, the first and second antigen-binding domains each have a different amino acid sequence.

[0228] In another embodiment of the above aspect, the antigen-binding molecule of the present disclosure has the activity of regulating the interaction between two antigen molecules. Without being bound by any particular theory, it is believed that the activity of regulating the interaction results from the antigen-binding molecule of the present disclosure holding two antigen molecules in close spatial proximity and reducing their mobility. In a particular embodiment, the antigen-binding molecule of the present disclosure can enhance or attenuate the interaction between two antigen molecules compared to a control antigen-binding molecule. The control antigen-binding molecule differs from the antigen-binding molecule of the present disclosure only in that it has one fewer disulfide bond between the two antigen-binding domains. In a further embodiment, the one fewer disulfide bond can be selected from bonds derived from a mutant amino acid residue not present in the wild-type Fab or hinge region (e.g., a cysteine ​​residue not present in the wild-type Fab or hinge region). The mutant amino acid residue can be, for example, an artificially mutated, substituted, introduced, or engineered cysteine ​​residue.

[0229] In one embodiment, the antigenic molecule is selected from the group consisting of receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0230] In a specific embodiment, the two antigen molecules bound by an antigen-binding molecule of the present disclosure can be a ligand and its receptor, respectively. The antigen-binding molecule of the present disclosure has the activity of promoting activation of the receptor by the ligand. In another specific embodiment, the two antigen molecules bound by an antigen-binding molecule of the present disclosure can be an enzyme and its substrate, respectively. The antigen-binding molecule of the present disclosure has the activity of promoting the catalytic reaction of the enzyme on the substrate.

[0231] In another specific embodiment, the two antigen molecules bound by the antigen-binding molecule of the present disclosure can both be antigens (e.g., proteins) present on the cell surface. The antigen-binding molecule of the present disclosure has the activity of promoting interaction between a cell expressing a first antigen and a cell expressing a second antigen. For example, the cell expressing the first antigen and the cell expressing the second antigen can be a cell with cytotoxic activity and its target cell, respectively. The antigen-binding molecule of the present disclosure promotes damage to the target cell by the cell with cytotoxic activity. The cell with cytotoxic activity is, for example, a T cell, a NK cell, a monocyte, or a macrophage.

[0232] In one embodiment of the above aspect, the antigen-binding molecule of the present disclosure is resistant to protease cleavage. In certain embodiments, the antigen-binding molecule of the present disclosure has increased resistance to protease cleavage compared to a control antigen-binding molecule. In certain embodiments, the antigen-binding molecule of the present disclosure has an increased proportion of full-length molecules (e.g., full-length IgG molecules) remaining after protease treatment compared to a control antigen-binding molecule. In certain embodiments, the antigen-binding molecule of the present disclosure has a decreased proportion of specific fragments (e.g., Fab monomers) generated after protease treatment compared to a control antigen-binding molecule.

[0233] In one embodiment of the above aspect, when an antigen-binding molecule of the present 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 excised. In one embodiment, the protease can cleave the hinge region of the antigen-binding molecule.

[0234] In one aspect, at least one of the first and second antigen-binding domains of the present disclosure can bind to a soluble protein, while in another embodiment, at least one of the first and second antigen-binding domains of the present disclosure can bind to a membrane protein.

[0235] The first antigen-binding domain and the second antigen-binding domain of the present disclosure can bind to a first antigen and a second antigen, respectively. In some embodiments, the first antigen and the second antigen are derived from a human, mouse, rat, monkey, rabbit, or dog. In some embodiments, examples of the first antigen and the second antigen include, but are not limited to, 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), antigens expressed not only on tumor cells, tumor blood vessels, and stromal cells 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, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD antigens, costimulatory molecules, and cell adhesion molecules.

[0236] In some embodiments, either the first antigen or the second antigen can be, for example, a molecule specifically expressed on T cells, and the other antigen can be a molecule expressed on the surface of T cells or any other immune cells. In another embodiment of the combination of the first antigen and the second antigen, preferably, either the first antigen or the second antigen is, for example, a molecule specifically expressed on T cells, and the other antigen is a molecule expressed on immune cells, and is different from the preselected antigen.

[0237] 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 CD3 site to which the antigen-binding molecule of the present disclosure binds can be any epitope present in the gamma, delta, or epsilon chain sequences constituting human CD3. In particular, epitopes present in the extracellular region of the epsilon chain in the human CD3 complex are preferred. The polynucleotide sequences of the gamma, delta, and epsilon chain structures constituting 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 accession numbers). Other examples of antigens include Fcγ receptors, TLRs, lectins, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

[0238] In one embodiment, the first antigen can be a molecule specifically expressed on T cells, preferably a T cell receptor complex molecule such as CD3, more preferably human CD3. In another embodiment, the second antigen can be a molecule expressed on T cells or any other immune cell, preferably a cell surface regulator on immune cells, more preferably a costimulatory molecule expressed on T cells, even more preferably a protein of the "TNF superfamily" or "TNF receptor superfamily," including, but not limited to, human CD137 (4-1BB), CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL. In a preferred embodiment, the first antigen can be CD3 and the second antigen can be CD137. Herein, the terms "first antigen" and "second antigen" are defined interchangeably.

[0239] In certain embodiments, the antigen-binding molecule of the present disclosure specifically binds to all or part of a partial peptide of CD3. In certain embodiments, the CD3 is human CD3 or cynomolgus monkey CD3, most typically human CD3. In certain embodiments, the antigen-binding molecule is cross-reactive with (i.e., specifically binds to) human CD3 and cynomolgus monkey CD3. In some embodiments, the antigen-binding molecule can specifically bind to the ε subunit of CD3, particularly the human CD3ε subunit of CD3 set forth in SEQ ID NO: 13 (NP_000724.1) (RefSeq accession number in parentheses). In some embodiments, the antigen-binding molecule can specifically bind to the CD3ε chain expressed on the surface of eukaryotic cells. In some embodiments, the antigen-binding molecule binds to the CD3ε chain expressed on the surface of T cells.

[0240] In certain embodiments, the antigen binding molecule of the present disclosure specifically binds to the whole or a part of a partial peptide of CD137. As used herein, the term "CD137" is also referred to as 4-1BB and is a member of the tumor necrosis factor (TNF) receptor family. Examples of factors belonging to the TNF superfamily or TNF receptor superfamily include CD137, CD137L, CD40, CD40L, OX40, OX40L, CD27, CD70, HVEM, LIGHT, RANK, RANKL, CD30, CD153, GITR, and GITRL.

[0241] In certain embodiments, the CD137 is human CD 137. In some embodiments, suitable examples of antigen-binding molecules in the present disclosure include antigen-binding molecules that bind to the same epitope as the human CD137 epitope bound by an antibody selected from the group consisting of: an antibody that recognizes a region containing the SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC sequence (SEQ ID NO: 14), an antibody that recognizes a region containing the DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC sequence (SEQ ID NO: 15), an antibody that recognizes a region containing the LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC sequence (SEQ ID NO: 16), and an antibody that recognizes a region containing the LQDPCSNCPAGTFCDNNRNQIC sequence (SEQ ID NO: 17) in the human CD137 protein.

[0242] In one embodiment of the above aspect, at least one of the first and second antigen-binding domains comprises a non-antibody protein or a fragment thereof that binds to a specific antigen. In a particular embodiment, the non-antibody protein is either a pair of a ligand and a receptor that specifically bind to each other. Such receptors include, for example, receptors belonging to the cytokine receptor superfamily, G protein-coupled receptors, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD4 antigens, costimulatory molecules, and cell adhesion molecules.

[0243] In one aspect, the antigen-binding molecule of the present disclosure may further comprise a third antigen-binding domain, which may be fused to either the first or second antigen-binding domain.

[0244] In one aspect, the third antigen-binding domain can be a Fab or scFv, in which case the C-terminus of the third antigen-binding domain may be fused to the N-terminus of the Fab heavy chain (VH region) of either the first or second antigen-binding domain, optionally via a peptide linker.

[0245] In certain embodiments, each of the first, second, and third antigen-binding domains of the present disclosure can be a Fab molecule, in which case the third antigen-binding domain may be 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 or second antigen-binding domain, optionally via a peptide linker.

[0246] In one aspect, examples of peptide linkers in the present disclosure include, but are not limited to, those selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0247] In one aspect, the third antigen-binding domain of the present disclosure is a crossover 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 can be a conventional Fab molecule.

[0248] In one aspect, the third antigen-binding domain of the present disclosure can bind to a third antigen different from the first and second antigens described above. The third antigen-binding domain that binds to the third antigen can be an antigen-binding domain that recognizes any antigen. The third antigen-binding domain of the present disclosure can be an antigen-binding domain that recognizes a molecule that is specifically expressed in cancer cells or cancer tissues.

[0249] In some embodiments, the third antigen is derived from a human, mouse, rat, monkey, rabbit, or dog. In some embodiments, the third antigen is a molecule specifically expressed on cells or organs derived from a human, mouse, rat, monkey, rabbit, or dog. The third antigen is preferably a molecule not systemically expressed on cells or organs. The third antigen is preferably, for example, a tumor cell-specific antigen, including antigens expressed in association with the malignant transformation of cells, as well as abnormal sugar chains that appear on cell surfaces or protein molecules during malignant transformation of cells. Specific examples include the ALK receptor (pleiotrophin receptor), pleiotrophin, 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's 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, virally induced tumor antigens (e.g., envelope antigens of DNA tumor viruses and RNA tumor viruses), colon CEA, carcinoembryonic antigen α-fetoprotein (e.g., oncofetal trophoblast glycoprotein 5T4 and oncofetal bladder tumor antigen), differentiation antigens (e.g., human lung cancer antigens L6 and L20), fibrosarcoma antigen, human T-cell leukemia-associated antigen Gp37, neoglycoprotein, sphingolipid, breast cancer antigen (e.g., EGFR (epidermal growth factor receptor)), NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen APO-1, differentiation antigen For example, I antigen found in fetal erythrocytes, early endodermal I antigen found in adult erythrocytes, I (Ma) found in embryos before implantation or gastric cancer, M18, M39 found in mammary epithelium, SSEA-1, VEP8, VEP9, Myl, VIM-D5 found in bone marrow cells, D156-22, 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 carcinoma cells, TL5 (blood type A) ), EGF receptor found in A431 cells, E1 series (blood type B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric cancer antigen, CO-514 (blood type Lea) found in adenocarcinoma, NS-10 and CO-43 (blood type Leb) found in adenocarcinoma, G49 found in the EGF receptor of A431 cells, MH2 (blood type ALeb / Ley) found in colon cancer, 19.9 found in colon cancer, gastric cancer mucin, T5A7 found in bone marrow cells, R24 found in melanoma, 4.2, GD3, and D1 found in embryonal carcinoma cells.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8, SSEA-3 and SSEA-4 found in 4- to 8-cell embryos, cutaneous T-cell lymphoma-associated antigen, MART-1 antigen, sialyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, paraneoplastic-associated brain-testis cancer antigen (tumor neural antigen MA2 and paraneoplastic neural antigen), neuro-oncological ventral antigen 2 (NOVA2), blood cell cancer antigen gene 520, tumor-associated antigen CO-029, tumor-associated antigens MAGE-C1 (cancer / testis antigen CT7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, and MAGE-4b These include MAGE-X2, cancer-testis antigen (NY-EOS-1), YKL-40, and any fragments of these polypeptides, as well as modified structures thereof (such as the aforementioned modified phosphate groups and sugar chains), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA, and CLEC12A.

[0250] In one preferred embodiment, the third antigen is glypican-3 (GPC3). In yet another embodiment, the third antigen is DLL3 (Delta-like 3). In certain embodiments, the third antigen-binding domain can be a "DLL3 antigen-binding domain" that binds to DLL3.

[0251] The term "DLL3" as used herein refers to any native DLL3 (Delta-like 3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed "full-length" DLL3 and any form of DLL3 produced by cellular processing. The term also encompasses naturally occurring variants of DLL3, such as splice variants or allelic variants. The amino acid sequence of an exemplary human DLL3 is known as NCBI Reference Sequence (RefSeq) NM_016941.3, the amino acid sequence of an exemplary cynomolgus monkey DLL3 is known as NCBI Reference Sequence XP_005589253.1, and the amino acid sequence of an exemplary mouse DLL3 is known as NCBI Reference Sequence NM_007866.2.

[0252] The human DLL3 protein contains a C-terminal transmembrane (TM) region and an intracellular domain, and an N-terminal DSL (Notch) domain. In addition, DLL3 contains six EGF domains, EGF1 to EGF6, from the N- to C-terminus.

[0253] 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 a DLL3 protein expressed on the surface of a eukaryotic cell. In some embodiments, the DLL3 antigen binding domain binds to a DLL3 protein expressed on the surface of a cancer cell.

[0254] In some embodiments, the DLL3 antigen-binding domain of the present disclosure binds to an epitope within the extracellular domain (ECD), i.e., the domain from the N-terminus to just before the TM region, but not to the TM region or the C-terminal intracellular domain. The DLL3 antigen-binding domain of the present disclosure can bind to an epitope within any of the above-mentioned domains / regions within the ECD. In preferred embodiments, the DLL3 antigen-binding domain of the present disclosure binds to an epitope within the region from EGF6 to just before the TM region. More specifically, the DLL3 antigen-binding domain of the present disclosure can bind to an epitope within the region defined by SEQ ID NO: 21 in human DLL3. In some embodiments, the DLL3 antigen-binding domain of the present disclosure binds to an epitope within the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region, or the EGF1, EGF2, EGF3, EGF4, EGF5, or EGF6 region of human DLL3, or the region from EGF6 to just before the TM region. In some embodiments, the antigen binding molecule or DLL3 antigen binding domain may be derived from previously reported anti-DLL3 antibodies (e.g., WO2019131988 and WO2011093097) in which the DLL3 epitope to which it binds has been characterized.

[0255] In human DLL3, the above-mentioned domains / regions are represented by the following amino acid residues (see, for example, www.uniprot.org / uniprot / Q9NYJ7 or WO2013 / 126746): extracellular domain (ECD): amino acid residues at positions 1 to 492; DSL domain: amino acid residues at positions 176 to 215; EGF domain: amino acid residues at positions 216 to 465; EGF1 region: amino acid residues at positions 216 to 249; EGF2 region: amino acid residues at positions 274 to 310; EGF3 region: amino acid residues at positions 312 to 351; EGF4 region: amino acid residues at positions 353 to 389; EGF5 region: amino acid residues at positions 391 to 427; EGF6 region: amino acid residues at positions 429 to 465; EGF6 to just before the TM region: amino acid residues at positions 429 to 492; the TM region: amino acid residues at positions 493 to 513; and the C-terminal intracellular domain: amino acid residues at positions 516 to 618 (or 516 to 587 in some isoforms). The amino acid positions described above also refer to the amino acid positions in the amino acid sequence shown in SEQ ID NO: 22.

[0256] Therefore, the antigen-binding molecules of the present disclosure can bind to the above-mentioned regions / domains having amino acid residues at the above-mentioned positions in human DLL3, i.e., the antigen-binding molecules of the present disclosure can bind to epitopes within the above-mentioned regions / domains having amino acid residues at the above-mentioned positions in human DLL3.

[0257] The DLL3 protein used in the present disclosure is not limited by its origin, and is preferably a human or cynomolgus monkey DLL3 protein.

[0258] In some embodiments, DLL3 ECD fragment proteins (or ECD variants) can be used as DLL3 proteins. Depending on the truncation site, the fragment / variant can include, from N-terminal to C-terminal, DSL domain to EGF6, EGF1 to EGF6, EGF2 to EGF6, EGF3 to EGF6, EGF4 to EGF6, EGF5 and EGF6, or EGF6. The fragment / variant can also include a region extending from immediately after the EGF6 region to immediately before the TM region. A Flag tag can be attached to the C-terminus of the fragment / variant using techniques well known in the art.

[0259] The CD3, CD137, or DLL3 protein of the present disclosure may be a protein having the sequence described above, or may be a modified protein having a sequence derived from the sequence described above by modifying one or more amino acids. Examples of modified proteins having a sequence derived from the sequence described above by modifying one or more amino acids include polypeptides having 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more identity to the amino acid sequence described above. Alternatively, partial peptides of these CD3, CD137, or DLL3 proteins may be used.

[0260] In certain embodiments, the antigen-binding molecules described herein bind to epitopes of CD3, CD137, or DLL3 that are conserved among different species of CD3, CD137, or DLL3. In certain embodiments, the antigen-binding molecules of the present disclosure are trispecific antigen-binding molecules, i.e., trispecific antigen-binding molecules that can specifically bind to three different antigens, i.e., can bind to either CD3 or CD137, but not both antigens simultaneously, and can specifically bind to DLL3.

[0261] In one aspect, the third antigen-binding domain is a conventional Fab, and (a) the first polypeptide comprises (from N-terminus to C-terminus) the VH of the third antigen-binding domain, a heavy chain constant region (CH1), and the VH of the first antigen-binding domain, a heavy chain constant region (CH1), and optionally a hinge region and / or Fc region (CH2 and CH3); (b) the second polypeptide comprises (from N-terminus to C-terminus) the VL of the third antigen-binding domain, and a light chain constant region (CL); (c) the third polypeptide comprises (from N-terminus to C-terminus) the VH of the second antigen-binding domain, a heavy chain constant region (CH1), and optionally a hinge region and / or Fc region (CH2 and CH3); (d) the fourth polypeptide comprises (from N-terminus to C-terminus) the VL of the second antigen-binding domain, and a light chain constant region (CL); and (e) the fifth polypeptide comprises (from N-terminus to C-terminus) the VL of the first antigen-binding domain, and a light chain constant region (CL).

[0262] In one aspect, the third antigen-binding domain is a VH / VL crossover Fab (in which the variable regions of the Fab light chain and Fab heavy chain are swapped), and (a) the first polypeptide comprises (from N-terminus to C-terminus) the VL of the third antigen-binding domain, a heavy chain constant region (CH1); and the VH of the first antigen-binding domain, a heavy chain constant region (CH1); and optionally a hinge region and / or Fc region (CH2 and CH3); (b) the second polypeptide comprises (from N-terminus to C-terminus) the VH of the third antigen-binding domain, and a light chain constant region (CL); (c) the third polypeptide comprises (from N-terminus to C-terminus) the VH of the second antigen-binding domain, a heavy chain constant region (CH1); and optionally a hinge region and / or Fc region (CH2 and CH3); (d) a fourth polypeptide comprising (from N-terminus to C-terminus) the VL of the second antigen-binding domain and a light chain constant region (CL); and (e) a fifth polypeptide comprising (from N-terminus to C-terminus) the VL of the first antigen-binding domain and a light chain constant region (CL).

[0263] In one aspect, the antigen-binding molecule of the present disclosure can further comprise an Fc region. In one aspect, the Fc region is composed of a first and a second Fc region subunit capable of stable association.

[0264] In one aspect, in the antigen-binding molecule of the present disclosure, each of the first antigen-binding domain and the second antigen-binding domain is a Fab, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second Fc region subunit, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other Fc region subunit.

[0265] In one aspect, the Fc region of the present disclosure can be of human origin, hi some embodiments, the Fc region of the present disclosure can be an IgG Fc region, preferably a human IgG Fc region, and more preferably a human IgG1 Fc region.

[0266] In certain embodiments, the Fc region of the antigen-binding molecule is composed of a first and a second Fc region subunit capable of stable association, and the Fc region exhibits reduced binding affinity for human Fcγ receptors compared to native human IgG1 Fc regions.

[0267] In certain embodiments, the Fc region of the antigen-binding molecule described herein comprises a modification that promotes the association of the first and second subunits of the Fc region. In certain embodiments, the modification is a so-called "knob-into-hole" modification that comprises a "knob" modification in one of the two subunits of the Fc region and a "hole" modification in the other of the two subunits of the Fc region, as described in more detail below.

[0268] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, so that the protrusion ("knob") can be positioned in the cavity ("hole"), promoting heterodimer formation and preventing homodimer formation. The protrusion is constructed by replacing small amino acid side chains in the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0269] Thus, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the antigen-binding molecule, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in 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 with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned.

[0270] The protrusions and cavities can be made by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis, or by peptide synthesis.

[0271] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0272] In yet a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0273] In a particular embodiment, the Fc region is composed of first and second Fc region subunits capable of stable association and exhibits reduced binding affinity for human Fcγ receptors compared to native human IgG1 Fc region, wherein the first Fc region subunit is selected from the group consisting of: (a1) an Fc region polypeptide comprising the mutations L234A, L235A; (a2) an Fc region polypeptide comprising the mutations L234A, L235A, N297A; and (a3) ​​an Fc region polypeptide comprising the mutations L234A, L235A, N297A, S354C, T366W, and the second Fc region polypeptide is selected from the group consisting of: (a4) an Fc region polypeptide comprising the mutations L234A, L235A; (a5) an Fc region polypeptide comprising the mutations L234A, L235A, N297A; and (a6) an Fc region polypeptide comprising the mutations L234A, L235A, N297A, Y349C, T366S, L368A, Y407V (amino acid positions are numbered using EU index numbering).

[0274] In certain 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 that of the Fc region of a native IgG. Such an Fc region contains, for example, Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440, according to EU numbering.

[0275] In some embodiments, the Fc region comprises Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering.

[0276] In some embodiments, the Fc region further comprises an Ile or Leu at position 428; and / or an Ile, Leu, Val, Thr, or Phe at position 436, according to EU numbering.

[0277] In some embodiments, the Fc region has the following sequence, according to EU numbering: (a) N434A / Q438R / S440E; (b) N434A / Q438R / S440D; (c) N434A / Q438K / S440E; (d) N434A / Q438K / S440D; (e) N434A / Y436T / Q438R / S440E; (f) N434A / Y436T / Q438R / S440D; (g) N434A / Y436T / Q438K / S440E; (h) N434A / Y436T / Q438K / S440D; (i) N434A / Y436V / Q438R / S440E; (j) N434A / Y436V / Q438R / S440D; (k) N434A / Y436V / Q438K / S440E; (l) N434A / Y436V / Q438K / S440D; (m) N434A / R435H / F436T / Q438R / S440E; (n) N434A / R435H / F436T / Q438R / S440D; (o) N434A / R435H / F436T / Q438K / S440E; (p) N434A / R435H / F436T / Q438K / S440D; (q) N434A / R435H / F436V / Q438R / S440E; (r) N434A / R435H / F436V / Q438R / S440D; (s) N434A / R435H / F436V / Q438K / S440E; (t) N434A / R435H / F436V / Q438K / S440D; (u) M428L / N434A / Q438R / S440E; (v) M428L / N434A / Q438R / S440D; (w) M428L / N434A / Q438K / S440E; (x) M428L / N434A / Q438K / S440D; (y) M428L / N434A / Y436T / Q438R / S440E; (z) M428L / N434A / Y436T / Q438R / S440D; (aa) M428L / N434A / Y436T / Q438K / S440E; (ab) M428L / N434A / Y436T / Q438K / S440D; (ac) M428L / N434A / Y436V / Q438R / S440E; (ad) M428L / N434A / Y436V / Q438R / S440D;(ae) M428L / N434A / Y436V / Q438K / S440E; (af) M428L / N434A / Y436V / Q438K / S440D; (ag) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah) L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E;

[0278] In some embodiments, the Fc region of the antigen binding molecule comprises the following combination of amino acid substitutions: M428L / N434A / Q438R / S440E.

[0279] In one aspect, the Fc region of the present disclosure may comprise a combination of one or more amino acid substitutions that promote multimerization of the Fc region. Examples of amino acid substitutions that promote multimerization include substitutions at at least one site selected from the group consisting of EU positions 247, 248, 253, 254, 310, 311, 338, 345, 356, 359, 382, ​​385, 386, 430, 433, 434, 436, 437, 438, 439, 440, and 447 (see, e.g., WO2016 / 164480). In certain embodiments, multimers include, but are not limited to, dimers, trimers, and tetramers.

[0280] In one aspect, an antigen-binding molecule of the present disclosure may have an amino acid residue in its hinge region substituted with at least one cysteine ​​residue, which may be present at EU numbering positions 226 and / or 229 in the hinge region.

[0281] In one aspect, an antigen-binding molecule of the present disclosure can have the amino acid sequence KSCDKTHTCPPCP in both or one of its heavy chain hinge regions. In a specific embodiment, an antigen-binding molecule of the present disclosure preferably has the amino acid sequence KSCDKTHTCPPCP in both of its heavy chain hinge regions. Furthermore, an antigen-binding molecule of the present disclosure preferably contains an amino acid substitution with a cysteine ​​residue at one or more positions selected from the group consisting of EU numbering 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.

[0282] In certain embodiments, the Fc region comprises any of the following: (a) a first Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 23 and a second Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 24; (b) a first Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 25 and a second Fc subunit comprising the amino acid sequence set forth in SEQ ID NO: 26; or (c) a first Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 58 and a second Fc region subunit comprising the amino acid sequence set forth in SEQ ID NO: 59.

[0283] In one aspect, the antigen-binding molecule of the present disclosure is a multispecific antigen-binding molecule. In some embodiments, the multispecific antigen-binding molecule is a bispecific or trispecific antigen-binding molecule.

[0284] In one aspect, the antigen-binding molecule of the present disclosure is an antibody. In a specific embodiment, the antibody of the present disclosure is an IgG antibody, preferably an IgG1, IgG2, IgG3, or IgG4 antibody.

[0285] IV. Disulfide Bonds In the present disclosure, at least one disulfide bond in a region other than the hinge region may be referred to by the abbreviation "LINC." Using this abbreviation, in some embodiments, antigen-binding molecules in the present disclosure may be referred to as, for example, "Dual / LINC," "DLL3-Dual / LINC," or "paired cysteine ​​form." Antigen-binding molecules in which the first and second antigen-binding domains are not / are not yet linked via at least one disulfide bond may be referred to by the abbreviation "unLINC" or "Dual-LINC-Ig® with unpaired cysteine."

[0286] In one aspect of the present invention, each of the first and second antigen-binding domains comprises (via mutation, substitution, or insertion) at least one cysteine ​​residue in a region other than the hinge region, which forms at least one disulfide bond between the first and second antigen-binding domains in the "LINC" antigen-binding molecules of the present disclosure.

[0287] In some embodiments of the above aspects, at least one disulfide bond linking the first and second antigen-binding domains can hold the two antigen-binding domains (i.e., the first and second antigen-binding domains described above) in close spatial proximity. Due to the link between the first and second antigen-binding domains via a disulfide bond, the antigen-binding molecule of the present disclosure can hold the two antigen-binding molecules in closer proximity than a control antigen-binding molecule that differs from the antigen-binding molecule of the present disclosure only in that it does not have an additional bond introduced between the two antigen-binding domains. In some embodiments, the terms "close spatial proximity" or "closer proximity" include the meaning that the first and second antigen-binding domains described above are held at a shorter distance and / or with reduced mobility.

[0288] As a result, the two antigen-binding domains of the antigen-binding molecule of the present disclosure (i.e., the first and second antigen-binding domains described above) can bind to antigens expressed on the same single cell. In other words, the two antigen-binding domains of the antigen-binding molecule of the present disclosure (i.e., the first and second antigen-binding domains described above) do not each bind to antigens expressed on different cells, resulting in cross-linking of the different cells. Such an antigen-binding mode of the antigen-binding molecule of the present disclosure can be referred to as "cis-binding," whereas the antigen-binding mode of the antigen-binding molecule in which each of the two antigen-binding domains of the antigen-binding molecule binds to antigens expressed on different cells, resulting in cross-linking of the different cells, can be referred to as "trans-binding." In some embodiments, the antigen-binding molecule of the present disclosure primarily binds to antigens expressed on the same single cell in a "cis-binding" manner.

[0289] In some embodiments of the above aspects, due to the disulfide bond between the first and second antigen-binding domains via the disulfide bond described above, the antigen-binding molecule of the present disclosure can reduce and / or prevent undesired cross-linking and activation of immune cells (e.g., T cells, NK cells, or DC cells, etc.). That is, in some embodiments, the first antigen-binding domain of the antigen-binding molecule of the present disclosure binds to any signaling molecule (e.g., a first antigen) expressed on immune cells such as T cells, and the second antigen-binding domain of the antigen-binding molecule of the present disclosure also binds to any signaling molecule (e.g., the first antigen or a second antigen different from the first antigen) expressed on immune cells such as T cells. Thus, the first and second antigen-binding domains of the antigen-binding molecule of the present disclosure can bind to either a first or a second signaling molecule expressed on the same single immune cell, e.g., T cells (i.e., in a cis-binding manner) or on different immune cells, e.g., T cells (i.e., in a trans-binding manner). When the first antigen-binding domain and the second antigen-binding domain bind to signaling molecules expressed on different immune cells, e.g., T cells, in a trans-binding manner, the different immune cells, e.g., T cells, are cross-linked, and in certain circumstances, such cross-linking of immune cells, e.g., T cells, may cause undesired activation of immune cells, e.g., T cells.

[0290] On the other hand, in another embodiment of the antigen-binding molecule of the present disclosure, both the first antigen-binding domain and the second antigen-binding domain can bind to a signaling molecule expressed on the same single immune cell, for example, a T cell, in a "cis-binding" manner, thereby reducing cross-linking of different immune cells, for example, T cells, via the antigen-binding molecule, and avoiding undesired activation of the immune cell.

[0291] In one aspect, the at least one disulfide bond linking the first antigen-binding domain and the second antigen-binding domain may be formed by linking amino acid residues located at the same position in the first antigen-binding domain and the second antigen-binding domain, respectively, or may be formed by linking amino acid residues located at different positions in the first antigen-binding domain and the second antigen-binding domain, respectively.

[0292] The positions of amino acid residues in the antigen-binding domains can be indicated according to the Kabat numbering or EU numbering system (also referred to as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991. For example, if amino acid residues involved in disulfide bond formation between the first and second antigen-binding domains are present at the same corresponding positions in each antigen-binding domain, the positions of those amino acid residues can be indicated by the same numbers according to the Kabat numbering or EU numbering system. Alternatively, if amino acid residues involved in disulfide bond formation between the first and second antigen-binding domains are present at different but non-corresponding positions in each antigen-binding domain, the positions of those amino acid residues can be indicated by different numbers according to the Kabat numbering or EU numbering system.

[0293] In one aspect, amino acid residues that serve as initiation sites for disulfide bonds between the antigen-binding domains are present in each of the first and second antigen-binding domains, and the bond between these antigen-binding domains is formed by linking these amino acid residues. In one embodiment of the above aspect, at least one of the amino acid residues that serve as initiation sites for disulfide bonds between the antigen-binding domains is a mutant amino acid residue that has been artificially mutated, substituted, introduced, or engineered, for example, an artificially introduced cysteine ​​residue. Such a mutant amino acid residue can be introduced into a wild-type antigen-binding domain by techniques such as amino acid substitution. In some embodiments, at least one disulfide bond is formed between an amino acid residue in the heavy chain of the first antigen-binding domain and an amino acid residue in the heavy chain of the second antigen-binding domain, or between an amino acid residue in the light chain of the first antigen-binding domain and an amino acid residue in the light chain of the second antigen-binding domain. In a further embodiment, the amino acid sequence is formed between amino acid residues in any combination of the CH1 region, CL region, VH region, VL region, and VHH region of the first antigen-binding domain and the CH1 region, CL region, VH region, VL region, and VHH region of the second antigen-binding domain.

[0294] In one aspect, at least one disulfide bond is formed between an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CH1 region of the second antigen-binding domain. In a specific embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are located at, for example, any of positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, and 218 to 219 (EU numbering) in the CH1 region. In certain embodiments, the amino acid residues are located at positions 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 33 The amino acid residue is located at any one selected from the group consisting of positions 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 218, and 219. In a specific embodiment, the amino acid residue is located at position 134, 135, 136, 137, 191, 192, 193, 194, 195, 196, or 197 (EU numbering) in the CH1 region. In certain embodiments, the amino acid residue is at position 135, 136, 191, 195, or 197 (EU numbering) of the CH1 region.

[0295] In one embodiment of the foregoing aspects, at least one disulfide bond is formed between an amino acid residue in the CH1 region of the first antigen-binding domain and an amino acid residue in the CH1 region of the second antigen-binding domain. In a specific embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 119, 120, 121, 122, and 123. In a specific embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140. In a specific embodiment, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 148, 149, and 150. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167, respectively. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178, respectively. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197, respectively. In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214, respectively.In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 218 and 219, respectively.

[0296] In one embodiment of the above aspect, the difference in the positions of amino acid residues forming disulfide bonds in the first antigen-binding domain and the second antigen-binding domain (i.e., the distance between them) is within 3 amino acids (i.e., 3 amino acids, 2 amino acids, or 1 amino acid). This means that when comparing the positions of amino acid residues forming disulfide bonds in the CH1 region of the first antigen-binding domain and the positions of amino acid residues forming disulfide bonds in the CH1 region of the second antigen-binding domain using EU numbering, the difference (i.e., the distance) is within 3 amino acids. In a specific embodiment, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residue at position 135 (EU numbering) in the CH1 region of the first antigen-binding domain and any amino acid residue at positions 132 to 138 (EU numbering) in the CH1 region of the second antigen-binding domain. In a specific embodiment, at least one disulfide bond between the first antigen-binding domain and the second antigen-binding domain is formed between the amino acid residue at position 136 (EU numbering) in the CH1 region of the first antigen-binding domain and any amino acid residue at positions 133 to 139 (EU numbering) in the CH1 region of the second antigen-binding domain.

[0297] In a specific embodiment, at least one disulfide bond between the first and second antigen-binding domains is formed between the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain and any amino acid residue at EU numbering positions 188 to 194 in the CH1 region of the second antigen-binding domain. In an exemplary embodiment, at least one disulfide bond between the first and second antigen-binding domains is formed between the amino acid residue at EU numbering position 135 in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one disulfide bond between the first and second antigen-binding domains is formed between the amino acid residue at EU numbering position 136 in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one disulfide bond between the first and second antigen-binding domains is formed between the amino acid residue at EU numbering position 191 in the CH1 regions of the two antigen-binding domains. In a further exemplary embodiment, at least one disulfide bond between the first and second antigen-binding domains is formed between the amino acid residue at EU numbering position 195 in the CH1 regions of the two antigen-binding domains. In a further exemplary embodiment, at least one disulfide bond between the first and second antigen-binding domains is formed between the amino acid residue at EU numbering position 197 in the CH1 regions of the two antigen-binding domains.

[0298] In some embodiments, the antigen-binding molecule of the present disclosure comprises one, two or more additional disulfide bonds between the first antigen-binding domain and the second antigen-binding domain. One, two or more additional disulfide bonds are formed between the first antigen-binding domain and the second antigen-binding domain via amino acid residues at the following positions, according to EU numbering, in each of the CH1 regions of the first antigen-binding domain and the second antigen-binding domain: (a) between amino acid residues at any of positions 131 to 138, 194, and 195 in each of the two antigen-binding domains; (b) between the amino acid residue at position 131 in each of the two antigen-binding domains and between the amino acid residue at position 194 in each of the two antigen-binding domains; (c) between the amino ...

Claims

1. A method for measuring, determining, or quantifying the ratio of LINC bodies (LINC rate) to the sum of antigen-binding molecules having at least one disulfide bond formed in a region other than the hinge region (LINC bodies) and antigen-binding molecules having no disulfide bond in a region other than the hinge region (unLINC bodies), the method comprising the steps of: (a) adding a protease to composition (1) containing LINC bodies and unLINC bodies to prepare composition (2); and (b) subjecting composition (2) to electrophoresis or chromatography.

2. The method of claim 1, wherein the protease is an enzyme that does not digest LINC bodies but can digest unLINC bodies.

3. The method of 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 the TH amino acid sequence of 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 the IgdE is derived from Streptococcus agalactiae.

8. The method according to claim 6 or 7, wherein the IgdE is a protein selected from the group consisting of the following (a) to (d): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 61; (b) a protein encoded by the base sequence set forth in SEQ ID NO: 62; (c) a protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 61, said protein being capable of digesting human IgG1 antibody between the T amino acid sequence of 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 to the amino acid sequence set forth in SEQ ID NO: 61, said protein being capable of digesting human IgG1 antibody between the T amino acid sequence of 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, wherein step (a) further comprises obtaining a composition (3) containing a protease and not containing LINC bodies and unLINC bodies, and step (b) comprises subjecting each of compositions (1) to (3) to non-reducing capillary SDS gel electrophoresis (CE-SDS) to obtain electropherograms (1) to (3) for each of compositions (1) to (3), respectively, wherein electropherogram (1) contains peaks containing LINC bodies and unLINC bodies not digested by proteases, and a peak (peak Z) not derived from LINC bodies or unLINC bodies not digested by proteases, The method, wherein electropherogram (2) contains a peak derived from LINC bodies (Peak L), a peak derived from unLINC bodies digested with protease (Peak unL), a peak derived from protease (Peak E), and a peak (Peak Z) derived from none of LINC bodies, unLINC bodies digested with protease, and protease, and electropherogram (3) contains a peak derived from protease (Peak E).

11. The method of claim 10, further comprising the step of: (c) calculating said percentage by the following formula (I): Here, A is the area value of peak L in electropherogram (2), B is the sum of the areas of peak unL, peak E, and peak Z in electropherogram (2), C is the area value of peak Z in electropherogram (1), and D is the area value of peak E in electropherogram (3).

12. The method according to any one of claims 1 to 9, wherein step (b) is a step of subjecting composition (2) to hydrophobic interaction chromatography (HIC) and obtaining a chromatogram.

13. The method of claim 112, further comprising the step of: (c) calculating said percentage by the following formula (II): Here, A is the area value of the peak derived from the LINC bodies in the chromatogram, and B is the sum of the area values ​​of all peaks derived from the unLINC bodies digested with protease in the chromatogram.

14. The method of any one of claims 1 to 13, 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.

15. The method of claim 14, wherein the at least one disulfide bond is formed between a first antigen-binding domain and a second antigen-binding domain.

16. The method of claim 14 or 15, wherein the at least one disulfide bond is formed between a heavy chain of the first antigen-binding domain and a heavy chain of the second antigen-binding domain.

17. The method of 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 one disulfide bond is formed between the amino acid residue at position 191 (EU numbering) of the heavy chain of the first antigen-binding domain and the amino acid residue at position 191 (EU numbering) of the heavy chain of the second antigen-binding domain, between the amino acid residue at position 195 (EU numbering) of the heavy chain of the first antigen-binding domain and the amino acid residue at position 195 (EU numbering) of the heavy chain of the second antigen-binding domain, or between the amino acid residue at position 197 (EU numbering) of the heavy chain of the first antigen-binding domain and the amino acid residue at position 197 (EU numbering) of 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, which are proteins present on a cell surface, and the antigen-binding molecule has an activity of promoting interaction between a cell expressing the first antigen and a cell expressing the second antigen.

20. The method according to claim 19, wherein the cells expressing a first antigen are cells having cytotoxic activity, the cells expressing a second antigen are their target cells, and the antigen-binding molecule promotes damage to the target cells by the cells having cytotoxic activity.

21. The method according to claim 20, wherein the cells having cytotoxic activity are T cells, NK cells, monocytes, or macrophages.

22. The method of 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, ionotropic receptors, tyrosine kinase receptors, immune checkpoint receptors, antigen receptors, CD4 antigens, costimulatory molecules, and cell adhesion molecules.

23. The method of any one of claims 14 to 22, wherein the first antigen-binding domain and the second antigen-binding domain are capable of binding to CD3 and / or CD137, respectively.

24. The method of 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 a third antigen-binding domain is fused to either the first antigen-binding domain or the second antigen-binding domain.

26. The method of claim 24 or 25, wherein the third antigen-binding domain is a Fab or scFv.

27. The method of 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 of any one of claims 24 to 27, wherein each of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is a Fab molecule, and 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 of claim 27 or 28, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:

20.

30. The method of any one of claims 24 to 29, wherein the third antigen-binding domain is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged, and the first antigen-binding domain and the second antigen-binding domain are conventional Fab molecules.

31. The method of any one of claims 24 to 30, wherein the third antigen-binding domain is capable of binding to an antigen expressed on a cancer cell or tissue.

32. The method of 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 of any one of claims 1 to 32, wherein the antigen-binding molecule further comprises an Fc region.

34. An antibody variable region, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region which may be the same or different, and comprises any one of the following (a1) to (a4): (a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a heavy chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a heavy chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region comprising a light chain CDR 1 comprising the amino acid sequence set forth in SEQ ID NO: 30, a light chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a light chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 32; (a2) a heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 33, a heavy chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and a heavy chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and The method of any one of claims 14 to 33, comprising an antibody variable region independently selected from the group consisting of: (a3) ​​an antibody variable region that binds to the same epitope as the antibody variable region of (a1) or (a2); and (a4) an antibody variable region that competes with the antibody variable region of (a1) or (a2) for antigen binding. The method of any one of claims 14 to 33, comprising an antibody variable region independently selected from the group consisting of: an antibody variable region comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 30, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; 35. The method of any one of claims 14 to 34, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region, which may be the same or different, and comprises an antibody variable region independently selected from the group consisting of (a1) to (a4) below: (a1) an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 36, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37; (a2) an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 38, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 37; (a3) ​​an antibody variable region that binds to the same epitope as the antibody variable region set forth in (a1) or (a2); and (a4) an antibody variable region whose binding to an antigen competes with the antibody variable region set forth in (a1) or (a2).

36. The third antigen-binding domain is selected from the group consisting of the following (a1) to (a4): (a1) an antibody variable region comprising a heavy chain complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 46, a heavy chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 47, and a heavy chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 48, and a light chain variable region comprising a light chain CDR 1 comprising the amino acid sequence set forth in SEQ ID NO: 49, a light chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 51; (a2) an antibody variable region comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 52, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 53; (a3) ​​an antibody variable region that binds to the same epitope as the epitope bound by the antibody variable region set forth in (a1) or (a2); and (a4) an antibody variable region that competes with the antibody variable region of (a1) or (a2) for antigen binding. The method of any one of claims 24 to 35, comprising an antibody variable region independently selected from the group consisting of:

37. The method of any one of claims 14 to 36, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region comprising: a heavy chain complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 27, a heavy chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 28, and a heavy chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable region comprising a light chain CDR 1 comprising the amino acid sequence set forth in SEQ ID NO: 30, a light chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a light chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO:

32.

38. The method of any one of claims 14 to 37, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 36; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

37.

39. The method of any one of claims 24 to 38, wherein the third antigen-binding domain comprises an antibody variable region comprising: a heavy chain complementarity determining region (CDR) 1 comprising the amino acid sequence set forth in SEQ ID NO: 46, a heavy chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 47, and a heavy chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO: 48, and a light chain variable region comprising a light chain CDR 1 comprising the amino acid sequence set forth in SEQ ID NO: 49, a light chain CDR 2 comprising the amino acid sequence set forth in SEQ ID NO: 50, and a light chain CDR 3 comprising the amino acid sequence set forth in SEQ ID NO:

51.

40. The method of any one of claims 24 to 39, wherein the third antigen-binding domain comprises an antibody variable region comprising: a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:52; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

53.

41. The method of any one of claims 24 to 40, wherein each of the first antigen-binding domain and the second antigen-binding domain comprises an antibody variable region comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37; and the third antigen-binding domain comprises an antibody variable region comprising: a heavy chain variable region comprising SEQ ID NO: 52; and a light chain variable region comprising SEQ ID NO:

53.

42. The method of any one of claims 14 to 41, wherein each of the first antigen-binding domain and the second antigen-binding domain is a Fab having a cysteine ​​residue at EU numbering position 191, 195, or 197 of the heavy chain, and having a disulfide bond formed by the two cysteine ​​residues.

43. An antigen-binding molecule comprising: (a1) a polypeptide chain (chain 1) comprising the amino acid sequence set forth in SEQ ID NO: 39, a polypeptide chain (chain 2) comprising the amino acid sequence set forth in SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence set forth in SEQ ID NO: 41, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence set forth in SEQ ID NO: 42; (a2) a polypeptide chain (chain 1) comprising the amino acid sequence set forth in SEQ ID NO: 43, a polypeptide chain (chain 2) comprising the amino acid sequence set forth in SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence set forth in SEQ ID NO: 44, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence set forth in SEQ ID NO: 42; and (a3) ​​a polypeptide chain (chain 1) comprising the amino acid sequence set forth in SEQ ID NO: 45, a polypeptide chain (chain 2) comprising the amino acid sequence set forth in SEQ ID NO: 40, a polypeptide chain (chain 3) comprising the amino acid sequence set forth in SEQ ID NO: 44, and two polypeptide chains (chain 4 and chain 5) each comprising the amino acid sequence set forth in SEQ ID NO: 42; 43. The method of any one of claims 1 to 42, comprising five polypeptide chains in any one of the combinations selected from the group consisting of:

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