Method for producing protein using environmentally responsive peptide

Abstract

The present invention addresses the problem of providing a new method for producing a protein. The problem is solved by a method for producing a molecule 1 comprising an [a] part, a [b] part, a [c] part, and optionally an [e] part. The method includes a step of bringing a starting sample containing one or more molecules 1 and one or more other molecules into contact with a carrier and separating the molecules 1 from other molecules, the number of the [c] parts contained in the other molecule being greater than the number of the [c] parts contained in the molecule 1 by one or more. The [a] part is a part 1 that binds to a target antigen; the [b] part is a first peptide that recognizes a target antigen binding site 1 contained in the [a] part; the [c] part is a second peptide comprising an amino acid sequence containing an amino acid side chain in which the charge changes from no charge to positive charge in response to the pH changing from a neutral condition to an acidic condition; and [e] part is an Fc region of the antibody.

Description

Protein production method using environmentally responsive peptides

[0001] The present invention relates to a method for producing a protein using an environmentally responsive peptide.

[0002] Antibodies have extremely high recognition specificity and binding activity for antigens, i.e., target molecules, and a variety of molecules, including small molecules, peptides, and proteins, can serve as antigens. Furthermore, antibody drugs are highly productive and stable in vivo, and are being developed to target a variety of diseases, including cancer, immune disorders, and infectious diseases. Among these drugs are bispecific (or multispecific) antibodies, which can simultaneously recognize two (or multiple) antigens. Research and development is underway on T-cell engaging (TCE), which can attack target cells by bridging them with cytotoxic T cells (Non-Patent Document 1).

[0003] For example, bispecific antibodies can be produced by coexpressing the heavy and light chains of two types of antibodies. In this process, the coexpressed heavy and light chains randomly associate, resulting in the production of a mixture of multiple types of antibodies in addition to the desired bispecific antibody. Therefore, to improve the production efficiency of a desired bispecific antibody, it is necessary to efficiently associate the desired polypeptide chains and efficiently purify the desired aggregate (the bispecific antibody).

[0004] Techniques for promoting heterodimer formation of antibody heavy chains have been reported as a method for efficiently associating target polypeptide chains (Patent Documents 1 and 2, Non-Patent Document 2). These techniques involve introducing an amino acid mutation into one heavy chain Fc region, and utilizing the bulkiness and charge of the mutated amino acid side chain to promote association with the other heavy chain Fc region, i.e., heterodimer formation. Furthermore, methods for efficiently purifying target bispecific (multispecific) antibodies include a method of adding an affinity tag sequence (Non-Patent Document 3) and a method of purifying by introducing a mutation that reduces affinity to Protein A (Non-Patent Document 4 and Patent Document 3) (Non-Patent Document 5). These methods allow for efficient separation of heterodimers by introducing a tag or mutation into one of the heavy chains.

[0005] Masked antibodies are also known as engineered antibodies that utilize the properties of the tumor microenvironment to specifically act in tumor tissue. Because masked antibodies have suppressed binding activity in normal tissues, they are less toxic to normal tissues via target molecules and are expected to be highly safe antibody drugs (Non-Patent Document 6). Masked antibodies are composed of the antibody itself, a masking domain that inhibits antibody binding, and a linker that connects the antibody and the masking domain and activates the masked antibody specifically in the tumor microenvironment. Known characteristics of the tumor microenvironment include enhanced protease activity and acidified extracellular pH (pHe). Examples of linkers that utilize enhanced protease activity include those equipped with substrates for extracellular proteases, such as matrix metalloproteinases (MMPs) and urokinase-type plasminogen activators (uPA), whose activity is enhanced in tumor tissue, enabling tumor tissue-selective antibody activation (Patent Documents 4 and 5). As an example of utilizing the latter acidic extracellular pH condition, there is a known example in which an amino acid sequence containing basic amino acids is used to impart a charge under acidic conditions, thereby enabling pH-dependent antibody activation (Patent Document 6).

[0006] International Publication WO1996 / 027011 International Publication WO2007 / 114325 International Publication WO2021 / 136256 International Publication WO2010 / 081173 International Publication WO2023 / 100829 International Publication WO2023 / 153442

[0007] Current Opinion in Immunology; 11(5):558-562 (1999). NatBiotechnol. ; 16(7); 677-81 (1998). Nat Med 9, 47-52 (2003) Molecular Immunology; 33(6): 521-530 (1996) MABS; 8 (4); 828-838 (2016) Expert Opin Biol Ther. ;14(8):1049-53(2014).

[0008] A new method for producing proteins is provided.

[0009] In order to solve the above-mentioned problems, the present inventors have found that a target protein having n peptides (pH-responsive linkers) consisting of an amino acid sequence containing amino acid side chains whose charge changes from uncharged to positive in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or less can be separated from other proteins by contacting the two proteins with a support for immobilized metal affinity chromatography (IMAC) in a state in which the two proteins coexist with each other. Based on this finding, the present inventors conducted further intensive research and completed the present invention.

[0010] That is, the present invention provides: [1] a method for producing a molecule (molecule 1) that binds to target antigen 1, comprising the following moieties [a], [b], [c], and optionally [e]: the method comprises the step of contacting a starting sample containing molecule 1 and one or more molecules other than molecule 1 (other molecules) with a carrier for immobilized metal affinity chromatography (IMAC) to separate molecule 1 from other molecules, wherein the number of [c] moieties contained in the other molecules is one or more more or one less than the number of [c] moieties contained in molecule 1: the [a] moiety: moiety 1 that binds to target antigen 1; the [b] moiety: a first peptide that recognizes target antigen-binding site 1 contained in the [a] moiety; the [c] moiety a second peptide consisting of an amino acid sequence containing an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or less; [e] a portion: an Fc region of an antibody; [2] molecule 1 is characterized in that it has a higher binding affinity to target antigen 1 under acidic conditions than under neutral conditions, and preferably EC ５０[3] The method according to [1], wherein the ratio is 3 or more, more preferably 10 or more, and even more preferably 30 or more; [3] The method according to [1] or [2], wherein molecule 1 comprises one or more [a] moieties, one or two or more [c] moieties, and preferably the numbers of [a] moieties, [b] moieties, and [c] moieties are the same, more preferably one or two of each, and even more preferably one of each; [4] The method according to any one of [1] to [3], wherein the second peptide comprises four or more amino acids whose charge changes from uncharged to positive in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or less, and the second peptide consists of an amino acid sequence whose pI value is greater than 6.4, preferably 6.8 or more, more preferably 7.2 or more, and even more preferably 7.6 or more; [5] [6] The method according to any one of [1] to [5], wherein the second peptide consists of an amino acid sequence that does not contain any amino acid that has a negative charge under acidic pH conditions, preferably an amino acid sequence that does not contain aspartic acid and / or glutamic acid; [7] The method according to any one of [1] to [5], wherein the second peptide consists of an amino acid sequence having the following structures (1) and (2): [7] (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which one arginine or lysine is inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a) (the amino acid sequence of (a) or (b) is referred to as a basic amino acid cluster), preferably (b') one or more amino acid sequences in which one arginine is inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), more preferably (b'') one amino acid sequence in which one arginine is inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), and (2) a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains, The method according to any one of [1] to [6], wherein the second peptide comprises an amino acid sequence having the following structure (1) or (2): (1) comprising one amino acid sequence represented by RHHH; and(2) The second peptide comprises a total of four or more amino acids having a basic functional group with a pKa of 7 or less in their side chains. [8] The method according to any one of [1] to [6], wherein the second peptide comprises one or more amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, and a total of four or more amino acids having the basic functional groups with a pKa of 7 or less in their side chains. [9] The method according to any one of [1] to [5], wherein the second peptide comprises a structure represented by an amino acid sequence comprising an amino acid sequence having every other amino acid having a basic functional group with a pKa of 7 or less in its side chain in its side chain.

[10] The method according to any one of [6] to [9], wherein the amino acid is a natural amino acid, preferably histidine.

[11] The method according to any one of [6] to [9], wherein the amino acid is an unnatural amino acid.

[12]

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

[12] , wherein the second peptide consists of an amino acid sequence containing 1 to 4 basic amino acid clusters;

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

[10] ,

[12] and

[13] , wherein the second peptide consists of an amino acid sequence shown in any one of SEQ ID NOs: 49 to 53, 55, 59 to 61, 65, 66, 75 to 81 (Figures 54 and 64), preferably the amino acid sequence shown in SEQ ID NO: 53 (Figure 54);

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

[14] , wherein the second peptide consists of an amino acid sequence containing 10 or less amino acids having basic functional groups with a pKa of 7 or less in their side chains;

[16]

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

[15] , wherein the amino acid sequence of the second peptide comprises two or more basic amino acid clusters, one basic amino acid cluster being located at the amino terminus or the carboxyl terminus of the amino acid sequence, preferably at the amino terminus, and (i) none of the other basic amino acid clusters are located at the amino terminus or the carboxyl terminus of the amino acid sequence, (ii) one other basic amino acid cluster is located at the carboxyl terminus, or (iii) both of the other basic amino acid clusters are located at the amino terminus and the carboxyl terminus of the amino acid sequence.

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

[16] , wherein the amino acid sequence of the second peptide comprises an amino acid sequence that is cleaved by an intracellular and / or extracellular protease and an amino acid sequence that comprises a basic amino acid cluster, wherein the amino acid sequence is: (1) from the amino terminus to the carboxyl terminus, the amino acid sequence that comprises a basic amino acid cluster and the amino acid sequence that is cleaved by an intracellular and / or extracellular protease, linked in this order, or (2) from the amino terminus to the carboxyl terminus, the amino acid sequence that is cleaved by an intracellular and / or extracellular protease and the amino acid sequence that comprises a basic amino acid cluster, linked in this order.

[18] The method according to any one of

[12] to

[17] , wherein the number of amino acids having a basic functional group with a pKa of 7 or less in a side chain contained in the amino acid sequence of the second peptide is 5% or more and 30% or less of the total number of amino acids contained in the amino acid sequence,

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

[18] , wherein molecule 1 is formed by linking the first peptide, the second peptide, and the moiety that binds to target antigen 1 in this order,

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

[19] , wherein the moiety that binds to target antigen 1 is a polypeptide consisting of an amino acid sequence that is not contained in the first peptide or the second peptide,

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

[20] , wherein molecule 1 is formed by a polypeptide,

[22] The method according to

[21] , wherein molecule 1 is linked to the first peptide, the second peptide, and the moiety that binds to target antigen 1 in the order from the amino terminus to the carboxyl terminus,

[23]

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

[23] , wherein the acidic condition is pH 6.5 or less, preferably pH 6.0 or less, more preferably pH 5.5 or less,

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

[24] , wherein the target antigen 1 is an antigen present in tumor tissue, endosome or lysosome,

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

[25] , wherein another moiety, moiety [d], is bound to the moiety that binds to the target antigen 1, and moiety [d] does not comprise the first peptide or the second peptide,

[27] The [d] moiety is one or more selected from the group consisting of an antibody or antigen-binding fragment thereof that is not the [a] moiety, a peptide including an amino acid sequence not contained in the first peptide and the second peptide, a cytokine, a toxin, a radioisotope, a labeling molecule, a photosensitizer, an immunostimulatory substance, an antitumor compound, a drug, a payload, and a polymer, and preferably, the antitumor compound is a camptothecin derivative or a pyrrolobenzodiazepine derivative, and the immunostimulatory substance is a cyclic The method according to

[26] , wherein the camptothecin derivative is a dinucleotide derivative, more preferably N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide;

[28] The method according to

[26] or

[27] , wherein molecule 1 consists of a polypeptide, or consists of a portion [d] and a polypeptide;

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

[28] , wherein the first peptide is obtained by a method comprising the following steps (i) to (iii): (i) contacting a peptide library containing a repeat sequence of an aromatic amino acid and Pro with the moiety that binds to target antigen 1 according to [1]; (ii) recovering peptides that bind to the moiety that binds to target antigen 1; and(iii) preparing the obtained peptide by recombination, chemical synthesis or peptide synthesis,

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

[29] , which comprises a step of preparing a peptide comprising an amino acid sequence contained in the first peptide and / or an amino acid sequence contained in the second peptide by recombination, in vitro translation, chemical synthesis or peptide synthesis,

[31] the method according to any one of [1] to

[30] , which comprises a step of culturing a cell into which a polynucleotide comprising an amino acid sequence contained in a portion that binds to target antigen 1 and, optionally, a nucleotide sequence encoding the amino acid sequence contained in the first peptide and / or the amino acid sequence contained in the second peptide has been introduced, and recovering a polypeptide that binds to the target antigen from the culture,

[32] the method according to any one of [1] to

[31] , wherein molecule 1 comprises n [c] moieties, where n is a positive integer,

[33] the method according to

[32] , wherein another molecule comprises n+1 or more [c] moieties or n-1 or less [c] moieties,

[34]

[35] The method according to any one of

[32] to

[34] , wherein the other molecules comprise a molecule (other molecule 1) comprising n+1 or more [c] moieties and a molecule (other molecule 2) comprising n-1 or fewer [c] moieties;

[36] The method according to any one of

[32] to

[35] , wherein n is 1;

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

[36] , wherein molecule 1 is a multispecific antibody, preferably a bispecific antibody, and optionally the multispecific antibody or bispecific antibody comprises the [d] moiety according to

[27] .

[38] The method according to

[37] , wherein molecule 1 comprises a moiety [a] (anti-target antigen 1 antibody) that is a monovalent or more antibody or antigen-binding fragment thereof that binds to target antigen 1, and a monovalent or more antibody or antigen-binding fragment thereof that binds to target antigen 2 (anti-target antigen 2 antibody), and preferably, the anti-target antigen 1 antibody and the anti-target antigen 2 antibody are monovalent antibodies or antigen-binding fragments thereof;

[39] The method according to

[38] , wherein molecule 1 is a heteromultimer, preferably a heterodimer;

[40]

[41] The method according to any one of

[37] to

[39] , wherein other molecule 1 comprises an anti-target antigen 1 antibody which is an [x × 2]-valent monospecific antibody or an antigen-binding fragment thereof, where x is an integer, and preferably x is 1;

[42] The method according to any one of

[37] to

[41] , wherein other molecule 2 comprises an anti-target antigen 2 antibody which is a [y × 2]-valent monospecific antibody or an antigen-binding fragment thereof, where y is 0 or a positive integer, and preferably y is 0 or 1;

[43] The method according to any one of

[37] to

[42] , wherein other molecule 1 does not comprise an anti-target antigen 2 antibody, and other molecule 2 does not comprise an anti-target antigen 1 antibody;

[44] The method according to any one of

[37] to

[42] , wherein other molecule 1 and other molecule 2 are homomultimers, and preferably homodimers;

[46] The method according to

[44] or

[45] , wherein other molecule 1 contains an anti-target antigen 1 antibody and an anti-target antigen 2 antibody, and other molecule 2 does not contain an anti-target antigen 1 antibody or an anti-target antigen 2 antibody;

[47] The method according to any one of

[44] to

[46] , wherein other molecule 1 and other molecule 2 are homomultimers, preferably homodimers;

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

[36] , wherein the [a] portion is a monospecific antibody or an antigen-binding fragment thereof, and molecule 1 does not contain any antibody or antigen-binding fragment thereof other than the [a] portion;

[49]

[50] The method according to

[48] , wherein the [a] moiety is a monovalent antibody or an antigen-binding fragment thereof;

[51] The method according to

[48] or

[49] , wherein other molecule 1 comprises a bivalent monospecific antibody or an antigen-binding fragment thereof that binds to target antigen 1 (anti-target antigen 1 monospecific antibody), and other molecule 2 does not comprise an anti-target antigen 1 monospecific antibody;

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

[50] , wherein the IMAC is column chromatography;

[53] The method according to

[51] , wherein the mobile phase in IMAC may contain imidazole or histidine;

[54]

[54] The method according to

[53] , wherein molecules containing more [c] moieties are eluted later than molecules containing fewer [c] moieties.

[0011] The present invention also provides: [A1] a method for producing a protein (protein 1) comprising n peptides (second peptides) consisting of an amino acid sequence comprising an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions within a pH range of 7.5 or less, wherein n is a positive integer; the method comprises a step (separation step) of contacting a starting sample comprising protein 1 and one or more proteins other than protein 1 (other proteins) with a support for immobilized metal affinity chromatography (IMAC) to separate protein 1 from at least one other protein (other protein to be separated), wherein the other protein to be separated comprises n+1 or more or n-1 or less second peptides, and n-1 or less is 0 or a positive integer; and [A2] [A1] The method according to [A1], wherein the second peptide comprises four or more amino acids whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or less, and the second peptide comprises an amino acid sequence having a pI value of more than 6.4, preferably 6.8 or more, more preferably 7.2 or more, and even more preferably 7.6 or more; [A3] The method according to [A1] or [A2], wherein the second peptide comprises an amino acid sequence that does not contain any amino acid that is negatively charged in acidic pH conditions, preferably an amino acid sequence that does not contain aspartic acid and / or glutamic acid; [A4] The method according to any one of [A1] to [A3], wherein the second peptide comprises an amino acid sequence having the following structures (1) and (2):[A5] (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which one arginine or lysine is inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a) (the amino acid sequence of (a) or (b) is referred to as a basic amino acid cluster), preferably (b') one or more amino acid sequences in which one arginine is inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), more preferably (b'') one amino acid sequence in which one arginine is inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), and (2) a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains, [A6] The method according to any one of [A1] to [A5], wherein the second peptide consists of an amino acid sequence having the following structure (1) or (2): (1) comprising one amino acid sequence represented by RHHH, and (2) comprising a total of four or more amino acids having a basic functional group with a pKa of 7 or less in their side chains. [A7] The method according to any one of [A1] to [A3], wherein the second peptide consists of a structure represented by an amino acid sequence comprising an amino acid sequence having every other amino acid having a basic functional group with a pKa of 7 or less in their side chains. [A8] The method according to [A7], wherein the amino acid is a natural amino acid, preferably histidine; [A9] The method according to [A7], wherein the amino acid is an unnatural amino acid; [A10] The method according to any one of [A1] to [A9], wherein the second peptide consists of an amino acid sequence containing 1 to 4 basic amino acid clusters; [A11][A12] The method according to any one of [A1] to [A8], [A10] and [A11], wherein the second peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 49 to 53, 55, 59 to 61, 65, 66, 75 to 81 (Figures 54 and 64), preferably the amino acid sequence shown in SEQ ID NO: 53 (Figure 54); [A13] The method according to any one of [A1] to [A12], wherein the second peptide comprises an amino acid sequence containing 10 or less amino acids having basic functional groups with a pKa of 7 or less in their side chains; [A14] The present invention also encompasses the method of any one of [A1] to [A13], wherein the amino acid sequence of the second peptide contains two or more basic amino acid clusters, one basic amino acid cluster being located at the amino terminus or the carboxyl terminus of the amino acid sequence, preferably the amino terminus, and wherein (i) no other basic amino acid cluster is located at the amino terminus or the carboxyl terminus of the amino acid sequence, (ii) one other basic amino acid cluster is located at the carboxyl terminus, or (iii) both other basic amino acid clusters are located at the amino terminus and the carboxyl terminus of the amino acid sequence; [A15] the method of any one of [A1] to [A14], which further comprises a step of removing all or a part of the second peptide after the separation step; and [A16] the method of any one of [A1] to [A15], wherein the other protein to be separated contains one or more second peptides.

[0012] [B1] A method for producing a molecule (molecule 1) that binds to target antigen 1, comprising the following moieties [a], [b], [c], and optionally [e], the method comprising the step of contacting a starting sample containing molecule 1 with a support for immobilized metal affinity chromatography (IMAC): moiety [a]: moiety 1 that binds to target antigen 1; moiety [b]: a first peptide that recognizes target antigen-binding site 1 contained in moiety [a]; moiety [c]: a second peptide consisting of an amino acid sequence including an amino acid side chain that changes charge from uncharged to positively charged in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or less; moiety [e]: an Fc region of an antibody; [B2] [B3] The method according to [B2], wherein the other molecule is an other molecule defined in any one of

[33] to

[35] ,

[40] to

[42] ,

[46] ,

[47] , or

[50] ; [B4] A method for producing a protein (protein 1) comprising n peptides (second peptides) consisting of an amino acid sequence containing an amino acid whose charge changes from uncharged to positive in response to a change in pH from neutral to acidic in the range of pH 7.5 or less, wherein n is a positive integer, and wherein the starting sample comprising protein 1 is subjected to immobilized metal affinity chromatography (IMA). [B5] the method according to [B4], wherein the starting sample contains one or more proteins other than Protein 1 (other proteins), and the number of second peptides contained in the other proteins is one or more more or one less than the number of second peptides contained in Protein 1; [B6] the method according to any one of [1] to

[53] and [B1] to [B3], wherein Molecule 1 contains an [e] moiety, and the [e] moiety contains one or more amino acid mutations that reduce the affinity of Protein A for the carrier compared to a wild-type Fc region;[B7] The method according to [B6], wherein the portion [e] has only one amino acid mutation that reduces the affinity of Protein A for a carrier compared to a wild-type Fc region; [B8] The method according to [B6] or [B7], wherein the one amino acid mutation is a substitution of Ile at position 253 (EU numbering) with Asn; [B9] The method according to any one of [B6] to [B8], further comprising a chromatography step or fractionation step other than IMAC; [B10] The method according to any one of [A1] to [A15], [B4] and [B5], wherein Protein 1 comprises an Fc region of an antibody, and the Fc region comprises one or more amino acid mutations that reduce the affinity of Protein A for a carrier compared to a wild-type Fc region; [B11] The method according to [B10], wherein the Fc region has only one amino acid mutation that reduces the affinity of Protein A for a carrier compared to a wild-type Fc region; [B12] [B13] The method according to any one of [B10] to [B12], further comprising a fractionation step or a chromatography step other than IMAC; [B14] The method according to [B9] or [B13], wherein the chromatography step or the fractionation step other than IMAC is a Protein A chromatography step; [B15] The method according to [B14], wherein the Protein A chromatography step comprises contacting the starting sample with an IMAC carrier and contacting a fraction that passes through the carrier, or a fraction obtained by contacting the starting sample with an IMAC carrier and eluting components adsorbed to the carrier, with a Protein A carrier; [B16] The method according to any one of

[51] ,

[52] , [A1] to [A16] and [B1] to [B15], wherein in IMAC, elution of components adsorbed to the IMAC carrier is elution using a single mobile phase; [B17] the method according to [B14], wherein the starting sample is a fraction obtained by contacting a sample containing molecule 1 or protein 1 with a protein A carrier and eluting the components adsorbed to the carrier; and the like.

[0013] The present invention makes it possible to produce proteins using a new method.

[0014]

[0015] The present invention will be described in detail below. 1. Definitions In the present invention, "DXd" means "N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide".

[0016] In the present invention, "PBD" means "pyrrolobenzodiazepine."

[0017] In the present invention, the term "mimotope" refers to a peptide that binds to the complementarity determining region (CDR) of an antibody. The amino acid sequence of a mimotope is characterized by not necessarily matching the amino acid sequence of the antigen (epitope) recognized by the antibody (Molecular Immunology.; 23(7):709-715 (1986)).

[0018] In the present invention, the term "amino acid" refers to natural amino acids and unnatural amino acids.

[0019] The naturally occurring amino acids are the 20 common amino acids, pyrrolysine and selenocysteine, which are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0020] An unnatural amino acid refers to an amino acid that is not one of the 20 common amino acids, pyrrolysine, or selenocysteine. Other terms that may be used synonymously with unnatural amino acid include "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally occurring amino acid," etc. Unnatural amino acids include amino acids that occur naturally by modification of natural amino acids but are not themselves incorporated into growing polypeptide chains by the translation complex. Additionally, unnatural amino acids include, but are not limited to, amino acids that do not occur in nature and can be obtained synthetically or by modification of natural amino acids.

[0021] In the present invention, the term "gene" refers to a nucleic acid molecule or its complementary strand that contains a nucleotide sequence that encodes an amino acid sequence contained in a protein (the amino acid sequence of a protein), and is single-stranded, double-stranded, triple-stranded or more, and also includes an association of a DNA strand and an RNA strand, a mixture of ribonucleotides and deoxyribonucleotides on a single strand, and a double-stranded, triple-stranded or more nucleic acid molecule containing such a strand.

[0022] In the present invention, "gene," "polynucleotide," and "nucleic acid molecule" are synonymous and interchangeable, and are not limited in any way by the number of their constituent units, such as ribonucleotides, deoxyribonucleotides, nucleotides, and nucleosides, and include, for example, DNA, RNA, mRNA, cDNA, cRNA, probes, oligonucleotides, primers, etc. "Nucleic acid molecule" may be abbreviated to "nucleic acid."

[0023] In the present invention, the terms "polypeptide," "peptide," and "protein (also written as "protein")" are synonymous and interchangeable, and examples thereof include, but are not limited to, enzymes, actin, myosin, cytokines, receptors, ligands, albumin, immunoglobulins, and fragments thereof. They may be either naturally occurring or artificially created, or either isolated from nature or produced by recombinant or synthetic means, without limitation on the organism from which they are derived (if any), and may be either monomers or multimers, or either homomultimers or heteromultimers, which may or may not be modified, and which may or may not be fused, bound, connected, or linked to other moieties.

[0024] In the present invention, the term "antibody" refers to an immunoglobulin having a constant region and a variable region. There is no particular limitation on whether the antibody is a natural or partially or completely synthetic immunoglobulin.

[0025] The basic four-chain antibody structure consists of two identical light chains (L chains) and two identical heavy chains (H chains). The light chains are linked to the heavy chains by a single covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds, depending on the heavy chain isotype. Each light chain and heavy chain has regularly spaced intrachain disulfide bonds. Heavy and light chains have constant regions with very similar amino acid sequences and variable regions with less similar amino acid sequences. Light chains have a variable region (VL) at the amino terminus followed by a constant region (CL). Heavy chains have a variable region (VH) at the amino terminus followed by three constant regions (CH1 / CH2 / CH3). The VL and VH are paired, and the CL is aligned with the first constant region (CH1) of the heavy chain. The paired VL and VH form a single antigen-binding site.

[0026] The constant region of the antibody of the present invention is not particularly limited, but the constant region of a human antibody is preferably used for the antibody of the present invention to treat or prevent a human disease. Examples of the heavy chain constant region of a human antibody include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε. Examples of the light chain constant region of a human antibody include Cκ and Cλ.

[0027] Fab consists of a heavy chain VH followed by CH1 and a light chain VL followed by CL. VH and VL contain complementarity-determining regions (CDRs). Linkers or connecting regions may be present between VH and CH1 and between VL and CL.

[0028] An scFv (single chain Fv) consists of a heavy chain VH, a light chain VL, and a linker that connects these domains. An example of the linker is a repeat sequence of (Gly-Gly-Gly-Gly-Ser).

[0029] VHH (Variable domain of Heavy chain of Heavy chain antibody) is the variable region of an antibody consisting of one domain (also called a single-domain antibody).

[0030] Fc (also referred to as Fc region) is the carboxyl-terminal region of the heavy chain constant region, comprising CH2 and CH3, and is a dimer. The Fc of the present invention may be a native (wild-type) sequence Fc or a mutant Fc in which the native sequence has been mutated (referred to as a "mutant Fc"). In some embodiments of the multispecific and bispecific molecules of the present invention, the Fc region is a mutant Fc, preferably a pair of Fc capable of forming a heterodimer, but is not limited thereto.

[0031] Examples of variant Fc include modified Fc regions (including heterodimeric Fc regions) contained in heteromultimers with improved stability, as disclosed in WO 2013 / 063702; Fc regions containing CH3 regions of immunoglobulins derived from IgG antibodies having "protrusions" and "cavities," as disclosed in WO 96 / 027011; Fc regions containing CH3 domains contained in heterodimers that are electrostatically favored by substituting one or more amino acid residues with charged amino acids, as disclosed in WO 2009 / 089004; heterodimeric Fc regions contained in heterodimers with conformational mutations and / or pI (isoelectric point) mutations, as disclosed in WO 2014 / 110601; and Protein A (Protein A) Fc regions disclosed in WO 2010 / 151792. Examples include, but are not limited to, heterodimeric Fc's comprising a CH3 domain containing a modification that eliminates or reduces binding to IgG1A.

[0032] The variable region consists of highly variable regions called hypervariable regions (HVRs) and relatively invariable regions called framework regions (FRs) separated by the hypervariable regions. Natural heavy and light chain variable regions contain four FRs connected by three hypervariable regions, and the hypervariable regions of each chain are held in close proximity by the FRs together with the hypervariable regions of the other chains, contributing to the formation of the antigen-binding site of antibodies.

[0033] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). The complementarity determining regions, also known as hypervariable regions, are located in the variable regions of the heavy and light chains of an antibody and are regions with particularly high variability in the primary structure. They are usually separated into three regions in the primary structure of the heavy and light chain polypeptide chains. In the present invention, the complementarity determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal end of the heavy chain amino acid sequence, and CDRL1, CDRL2, and CDRL3 from the amino-terminal end of the light chain amino acid sequence. These regions are adjacent to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0034] In the present invention, the positions and lengths of CDRs are determined by the IMGT definition (Developmental and Comparative Immunology 27 (2003) 55-77) unless otherwise specified.

[0035] The FR is the portion of the variable region other than the CDRs. The variable region generally has four FRs: FR1, FR2, FR3, and FR4.

[0036] The CDRs and FRs in the heavy and light chains are arranged from the amino terminus to the carboxyl terminus in the following order: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, respectively.

[0037] The positions of CDR and FR can be determined by various definitions known in the art, such as the definitions of Kabat, Chothia, AbM, contact, etc., in addition to the IMGT.

[0038] In the present invention, the "site" to which an antibody binds, that is, the "site" recognized by an antibody, refers to a partial peptide or partial higher-order structure on an antigen that the antibody binds to or recognizes.

[0039] In the present invention, such a site is also referred to as an epitope or antibody-binding site. In the present invention, a "mutated antibody" refers to a polypeptide that has an amino acid sequence in which amino acids have been substituted, deleted, or added (addition includes insertion) (hereinafter collectively referred to as "mutations") in the amino acid sequence of the original antibody, and that binds to the target antigen of the present invention. The number of mutated amino acids in such a mutant antibody is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50 or less. Such mutant antibodies are also encompassed by the "antibody" of the present invention.

[0040] In the present invention, the term "several" in "one or several" refers to 2 to 10.

[0041] In the present invention, a "molecule" is a molecule comprising an antibody, an antigen-binding fragment of an antibody, as described above, and also includes multispecific molecules formed from antibodies or multiple antigen-binding fragments derived therefrom.

[0042] "A carries B" means "A contains B" or "B is bound, attached, or fused to A." For example, an "antibody carrying a substrate" can be interpreted as an "antibody containing a substrate," an "antibody to which a substrate is bound," an "antibody to which a substrate is attached," or an "antibody to which a substrate is fused."

[0043] In the present invention, "binding fragment" refers to a fragment of a protein that binds to a target molecule and has binding activity to the target molecule. "Binding fragment of a molecule" refers to a fragment of the molecule that has binding activity to the target antigen to which the molecule binds. "Antigen-binding fragment of an antibody" will be described later.

[0044] In the present invention, a "target antigen" refers to a molecule that binds to the target antigen. It is not limited to an antigen that binds to an antibody, but is preferably present in vertebrates, more preferably mammals, even more preferably primates, and most preferably humans. The target antigen may be endogenously or exogenously present, but is preferably present endogenously or in an individual that has been infected or introduced after birth. In a preferred embodiment, the "target antigen" is a disease-related substance or a cell surface antigen, such as, but not limited to, a protein, a nucleic acid molecule, a lipid, a sugar, a fiber, a bone, a membrane, a receptor, a ligand, a cytokine, a hormone, a cell surface antigen, a membrane-bound protein, an enzyme, a metabolite, a pathogen or an endogenous organism, a part thereof or a metabolite thereof, or an artificially introduced substance.

[0045] In the present invention, a "sample" refers to a biological substance containing molecules that bind to a target antigen, and is preferably a liquid such as a solution or suspension, more preferably a solution. In one embodiment, a "sample" may be, for example, but is not limited to, a cell or tissue homogenate or extract, a cell culture supernatant, or a fraction obtained by pretreating the same.

[0046] In the present invention, the term "starting sample" refers to a sample to be subjected to immobilized metal affinity chromatography (IMAC), i.e., a "sample" to be brought into contact with an IMAC carrier. The "starting sample" may or may not have been pretreated. Examples of pretreatment include, but are not limited to, a chromatography step other than IMAC or a fractionation step. In addition, in the present invention, the term "starting sample" is synonymous with "starting raw materials (or starting materials)," "starting fraction," or "starting solution," and these terms are interchangeable.

[0047] 2. Molecules that bind to target antigens The present invention relates to molecules that bind to target antigens, and specifically bind to the target antigen in a specific environment. In the present invention, a molecule that binds to target antigen 1 can be referred to as "molecule 1," and a molecule that binds to target antigen 2 can be referred to as "molecule 2."

[0048] A specific environment refers to the environment in a specific tissue or intracellular organelle, for example, the cancer microenvironment, the environment in an endosome, or the environment in a lysosome.

[0049] The cancer microenvironment refers to the environment within cancer tissue, such as an acidic pH environment or an environment in which proteases are present. While the pH in normal tissue is neutral, e.g., pH 7.0 to 7.5, the pH in cancer tissue is 7.0 or less, preferably 6.5 or less, and more preferably 6.0 or less.

[0050] The molecules that bind to the target antigens of the present invention acquire responsiveness in a specific environment and are therefore sometimes called environmentally responsive molecules, and when the molecules are antibodies, they are called environmentally responsive masked antibodies.

[0051] The target antigen-binding molecule of the present invention comprises a portion that binds to target antigen 1 (portion [a]), a first peptide that recognizes the target antigen-binding site contained in said portion (portion [b]), and a second peptide (portion [c]) that comprises an amino acid sequence including an amino acid side chain that changes charge from uncharged to positive in response to a change in pH from neutral to acidic conditions within a pH range of 7.5 or less, and preferably further comprises an Fc region of an antibody (portion [e]), more preferably in a state in which they are linked directly or indirectly. Here, "indirectly linked" refers to a state in which they are linked via a linker or the like. The portion that binds to target antigen 1 is preferably a polypeptide. The portion binds to target antigen 1, for example, through an antigen-antibody reaction or protein (e.g., receptor)-ligand binding. The portion that binds to target antigen 1 is more preferably an antibody or an antigen-binding fragment of an antibody that binds to target antigen 1 through an antigen-antibody reaction. The first peptide binds to the target antigen-binding site in the portion that binds to target antigen 1, thereby masking the target antigen-binding site. As a result, the target antigen-binding site is unable to bind to target antigen 1 or binds poorly to it.

[0052] The number of [a], [b], [c], and [e] moieties contained in the target antigen-binding molecule of the present invention is not particularly limited, but preferably contains one or more [a], [b], and [c] moieties, respectively. It is more preferable that the [a], [b], and [c] moieties each contain one or more, and it is more preferable that the numbers of [a], [b], and [c] moieties are the same. It is even more preferable that the [a], [b], and [c] moieties each contain one or two. It is even more preferable that the numbers of [a], [b], and [c] moieties are the same. It is even more preferable that the [a], [b], and [c] moieties each contain one. The number of [e] moieties contained in the target antigen-binding molecule of the present invention may usually be a positive even number, but is preferably two or four, and more preferably two. Such [e] moieties form heterodimers or homodimers and may be native, mutant, or a hybrid of native and mutant.

[0053] Figure 1 shows an example of a masked antibody format that is activated in response to a pH change as an example of a molecule that binds to a target antigen of the present invention, but the present invention is not limited thereto. The target antigen-binding portion is a bivalent antibody (IgG), and the second peptide is a peptide containing a cluster of histidine residues (histidine cluster (His cluster), the black square in Figure 1) that exhibits pH responsiveness. The first peptide, shown in a checkered pattern in Figure 1, binds to the antigen-binding site of the antibody and masks said site. The second peptide links the first peptide to the target antigen-binding portion and exhibits pH responsiveness, so is also referred to as a pH-responsive linker. The first peptide can recognize and bind to the target antigen-binding site, but when the second peptide is cleaved and the first peptide is present alone, it binds and dissociates depending on physicochemical conditions, and is therefore not considered to remain permanently bound. For example, in a target antigen-binding molecule of the present invention, the first peptide is bound to the target antigen-binding moiety via the second peptide, and the target antigen-binding molecule assumes a conformation in which the first peptide is positioned near the target antigen-binding site contained in the target antigen-binding moiety, and the first peptide's target antigen-binding site is largely masked under equilibrium conditions unless the conformation of the molecule changes (left side of Figure 1 ). However, this mechanism is not limited to this. Under acidic pH conditions, the histidine residues within the His cluster of the second peptide become positively charged, and the repulsion between the histidine residues induces a conformational change in the linker second peptide, causing the first peptide to dissociate from the target antigen-binding molecule and making it unable to remain bound to the target antigen-binding site (right side of Figure 1 , where "+" indicates a positive charge). As a result, the target antigen-binding site contained in the target antigen-binding moiety becomes capable of binding to the target antigen, and the first peptide is thought to have a higher binding affinity for the target antigen than before the conformational change of the second peptide was induced. That is, the target antigen-binding molecule of the present invention is able to bind to the target antigen with higher affinity under acidic pH conditions than under neutral pH conditions.Such a structural change of the second peptide due to the repulsion between histidine residues is a reversible phenomenon that occurs with a change in pH. Therefore, when a molecule that binds to a target antigen transitions from an acidic pH condition to a neutral pH condition, a structural change opposite to the structural change that occurs when transitioning from a neutral pH condition to an acidic pH condition is induced, and it is thought that binding of the first peptide to the target antigen-binding site reduces the affinity of the molecule that binds to the target antigen for the target antigen. It can be assumed that the target antigen-binding molecule of the present invention exerts its effect primarily through the mechanism described above, but such a mechanism is not limited thereto, and the molecule may exert its effect through other mechanisms or in combination with other mechanisms.

[0054] In the present invention, the expression "binds to a target antigen with higher affinity under acidic pH conditions than under neutral pH conditions" can also be expressed as making the affinity of a molecule that binds to a target antigen for the target antigen under neutral pH conditions weaker than the affinity for the target antigen for the target antigen under acidic pH conditions. In other words, in the present invention, it is sufficient to increase the difference between the affinity of a molecule that binds to a target antigen for the target antigen under acidic pH conditions and the affinity for the target antigen for the target antigen under neutral pH conditions (for example, as described below, EC ５０ (The ratio (neutral pH condition / acidic pH condition) can be increased.) To increase the difference between the affinity of a molecule that binds to a target antigen under acidic pH conditions and the affinity of the molecule that binds to a target antigen under neutral pH conditions, for example, the affinity of the molecule to the target antigen under acidic pH conditions may be increased, the affinity of the molecule to the target antigen under neutral pH conditions may be decreased, or both may be increased.

[0055] In the present invention, the linker portion is made pH-responsive by introducing a basic amino acid cluster into the second peptide. It is also possible to make the mask peptide portion (the first peptide in the present invention) pH-responsive, but in such a configuration, it is necessary to obtain a pH-responsive peptide with a different sequence for each portion that binds to a target antigen. On the other hand, in the configuration of the present invention, the linker portion having a common sequence motif (basic amino acid cluster) of the second peptide can be used universally in portions that bind to different target antigens, which has the advantageous effect of eliminating the need to obtain a pH-responsive peptide with a different sequence for each portion that binds to a target antigen.

[0056] A molecule that binds to a target antigen binds to the target antigen with higher affinity under acidic pH conditions than under neutral pH conditions, thereby preventing the molecule from binding to the antigen in normal tissues and reducing its toxicity. "Reducing toxicity" refers to reducing undesired effects other than the expected efficacy when the molecule that binds to the target antigen is administered to animals such as humans, mice, rats, monkeys, rabbits, and dogs. Examples of toxicity include death or moribundity, weight loss, and organ toxicity (skin, liver, etc.) after drug administration. Whether or not toxicity has been reduced can be determined using methods known to those skilled in the art, such as measuring the subject's weight and checking pathological data after administration of the molecule that binds to the target antigen.

[0057] It is presumed that a molecule that binds to a target antigen binds to the target antigen with higher affinity under acidic pH conditions than under neutral pH conditions, thereby suppressing binding to antigens expressed in normal tissues and improving its blood concentration. "Improving blood concentration" includes increasing the maximum blood concentration (Cmax) and extending the time it takes for the molecule to disappear from the blood when administered to animals such as humans, mice, rats, monkeys, rabbits, and dogs. The target antigen-binding molecule in the blood may or may not have the target antigen-binding moiety masked, as long as it can function as a molecule that binds to the target antigen in a specific environment. Whether or not the "blood concentration has improved" can be determined, for example, by whether or not the concentration (Cmax) at which the blood concentration reaches its maximum after administration of the target antigen-binding molecule has increased, or whether or not the time it takes for the target antigen-binding molecule to disappear from the blood after administration has been extended. These can be determined by measuring the blood drug concentration after administration of the target antigen-binding molecule, or parameters such as the plasma half-life, mean plasma residence time, and plasma clearance of the target antigen-binding molecule, using methods known to those skilled in the art.

[0058] In the present invention, the difference between the affinity to the target antigen at acidic pH and the affinity to the target antigen at neutral pH is not particularly limited, as long as the affinity to the target antigen at acidic pH is stronger than the affinity to the target antigen at neutral pH. ５０ A preferred embodiment has a large ratio (neutral pH condition / acidic pH condition). ５０ EC stands for 50% effective concentration or half effective concentration, and refers to the concentration at which a molecule such as a drug or antibody shows 50% of the maximum response from the minimum value. ５０ The ratio (neutral pH condition / acidic pH condition) is the EC of molecules that bind to the target antigen under neutral pH conditions. ５０ and EC of molecules that bind to target antigens under acidic pH conditions. ５０ Therefore, the EC ５０The larger the ratio (neutral pH condition / acidic pH condition), that is, the higher the EC under neutral pH conditions. ５０ EC under acidic pH conditions ５０ The smaller the value of , the higher the affinity of the molecule for the target antigen at acidic pH compared to neutral pH. ５０ The smaller the ratio (neutral pH condition / acidic pH condition), i.e., the higher the EC under neutral pH conditions. ５０ EC under acidic pH conditions ５０ The larger the value of , the lower the affinity of the molecule for the target antigen at acidic pH compared to neutral pH. ５０ The ratio (neutral pH condition / acidic pH condition) is preferably 3 or more, and more preferably EC ５０ The ratio (neutral pH condition / acidic pH condition) is 10 or more, and more preferably EC ５０ The ratio (neutral pH condition / acidic pH condition) is 30 or more. ５０ The upper limit of the ratio (neutral pH condition / acidic pH condition) is not particularly limited, and may be any value, such as 100, 400, 1000, or 10000, as long as it can be achieved by a person skilled in the art. ５０ The value of the ratio (neutral pH condition / acidic pH condition) is preferably 3 or more and 100 or less, 3 or more and 400 or less, 3 or more and 1,000 or less, or 3 or more and 10,000 or less, more preferably 10 or more and 100 or less, 10 or more and 400 or less, 10 or more and 1,000 or less, or 10 or more and 10,000 or less, and even more preferably 30 or more and 100 or less, 30 or more and 400 or less, 30 or more and 1,000 or less, or 30 or more and 10,000 or less.

[0059] When the antigen is a soluble antigen, the KD (dissociation constant) can be used as a value of affinity to the target antigen, but when the antigen is a membrane-type antigen, the apparent KD (apparent dissociation constant) can be used. The KD (dissociation constant) and the apparent KD (apparent dissociation constant) can be measured by methods known to those skilled in the art, such as Biacore (GE healthcare), ELISA (Enzyme Linked Immunosorbent Assay), FACS, etc.

[0060] Starting Sample In a preferred embodiment, the "starting sample" to be subjected to the method of the present invention comprises not only a molecule (molecule 1) that binds to the target antigen 1 of the present invention, but also one or more molecules other than molecule 1 (other molecules). The number of [c] moieties contained in the "other molecules" is 0 or a positive integer that is one or more more or one or more less than the number of [c] moieties contained in molecule 1, and may be expressed as an average value. Similarly, the number of "other molecules" contained in the "starting sample" is a positive integer, but may be expressed as an average value. It is preferable that the "other molecules" include at least one molecule containing one or more [c] moieties.

[0061] The method for obtaining the starting sample is not particularly limited, but examples include the following: A combination of heavy chains and light chains containing at least two or more of these heavy chains and light chains is appropriately designed, and these heavy chains and light chains are each expressed in a host cell to obtain a mixture containing molecule 1 and "other molecules." This mixture can be used as the starting sample of the present invention, either directly or after further pretreatment as necessary. Such methods are well known as methods for obtaining multispecific antibodies, and starting samples can also be obtained in the present invention using these well-known methods as appropriate. Pretreatments can include chromatography steps or fractionation steps other than IMAC, and preferably include, but are not limited to, protein A chromatography.

[0062] In one aspect of the present invention, molecule 1 contained in the starting sample comprises a moiety [a] (anti-target antigen 1 antibody) which is one or more monovalent antibodies or antigen-binding fragments thereof that bind to target antigen 1, and one or more monovalent antibodies or antigen-binding fragments thereof that bind to target antigen 2 (anti-target antigen 2 antibody), and preferably, the anti-target antigen 1 antibody and the anti-target antigen 2 antibody are monovalent antibodies or antigen-binding fragments thereof. In the above, when molecule 1 comprises an antibody and an antibody, these antibodies bind to each other and form a single molecule as a whole.

[0063] Antibodies of the present invention include antibodies derived from non-human animals (non-human animal antibodies), human antibodies, chimeric antibodies (also called "chimeric antibodies"), humanized antibodies, etc., and preferably human antibodies or humanized antibodies can be used. The scope of the antibodies of the present invention also includes antibody mutants ("mutated antibodies" described below). For example, the scope of human antibodies also includes human mutant antibodies, and the scope of humanized antibodies also includes humanized mutant antibodies.

[0064] Non-human animal antibodies include antibodies derived from vertebrates such as mammals and birds. Mammal-derived antibodies include antibodies derived from rodents such as mouse antibodies and rat antibodies, or camels. Bird-derived antibodies include chicken antibodies.

[0065] Examples of chimeric antibodies include, but are not limited to, antibodies formed by combining a variable region derived from a non-human animal antibody with a constant region of a human antibody (human immunoglobulin).

[0066] Examples of humanized antibodies include, but are not limited to, antibodies in which the CDRs in the variable regions of a non-human animal antibody have been transplanted onto a human antibody (the variable regions of human immunoglobulins), antibodies in which not only the CDRs but also part of the framework region sequence of a non-human animal antibody has been transplanted onto a human antibody, and antibodies in which one or more amino acids derived from any of these non-human animal antibodies have been replaced with human amino acids.

[0067] Antibodies can be produced by various known methods. Known methods include methods using hybridomas, cell-based immunization, and gene recombination techniques. Methods for obtaining human antibodies derived from phage display selected from a human antibody library are also known. For example, a phage display method can be used in which the variable regions of human antibodies are expressed on the surface of phages as scFvs and phages that bind to the antigen are selected. The DNA sequence encoding the variable regions of human antibodies that bind to the antigen can be determined by analyzing the genes of phages selected by binding to the antigen. Once the DNA sequence of an antigen-binding scFv has been elucidated, an expression vector containing that sequence can be constructed and introduced into an appropriate host for expression to obtain a human antibody (WO1992 / 01047, WO1992 / 20791, WO1993 / 06213, WO1993 / 11236, WO1993 / 19172, WO1995 / 01438, WO1995 / 15388, Annu. Rev. Immunol (1994) 12, 433-455). Human antibodies can also be produced by a method using human antibody-producing mice carrying human genomic DNA fragments containing heavy and light chain genes of human antibodies (Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nuc. Acids Res. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.) The constant region of a human therapeutic or prophylactic antibody is preferably that of a human antibody.Examples of the heavy chain constant region of a human antibody include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε. Examples of the light chain constant region of a human antibody include Cκ and Cλ.

[0068] The antigen-binding fragment of an antibody refers to a fragment of the antibody that has binding activity to an antigen to which the antibody binds, and preferably comprises a heavy chain variable region and a light chain variable region. Examples of the antigen-binding fragment of an antibody include Fab, F(ab') ２ Examples of antigen-binding fragments of antibodies include, but are not limited to, single-domain antibodies (sdAbs) such as scFv, Fab', Fv, and VHH. Such antibody antigen-binding fragments may be those obtained by treating the full-length antibody protein molecule with an enzyme such as papain or pepsin, as well as recombinant proteins produced in suitable host cells using recombinant genes, proteins prepared by in vitro translation, and proteins obtained by chemical synthesis or peptide synthesis.

[0069] Mutants of antibodies or antigen-binding fragments thereof Mutants of antibodies or antigen-binding fragments thereof of the present invention (also referred to as "mutated antibodies") can preferably be modified to reduce susceptibility to proteolysis or oxidation, maintain, improve, or inhibit decline or change in biological activity or function, improve or regulate antigen-binding ability, or impart physicochemical or functional properties. It is known that changes in specific amino acid side chains on the surface of proteins can alter the function or activity of the protein; examples of such changes include deamidation of asparagine side chains and isomerization of aspartic acid side chains. Substitutions of other amino acids to prevent such changes in amino acid side chains are also included within the scope of the mutants of the present invention.

[0070] An example of a variant of the present invention is an antibody or antigen-binding fragment thereof having an amino acid sequence in which conservative amino acid substitutions have been made in the amino acid sequence of the antibody or antigen-binding fragment thereof. Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related in their side chains.

[0071] Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and uncharged polar group = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; amide-containing group = asparagine and glutamine; aliphatic group = alanine, valine, leucine, and isoleucine; and aromatic group = phenylalanine, tryptophan, and tyrosine. The amino acid substitutions in such mutant antibodies are preferably made within a range that does not reduce the antigen-binding activity of the original antibody. Among the above amino acids, the negative logarithms of the dissociation constants of groups other than the α-amino group and the α-carboxy group in amino acids having an acidic or basic functional group in their side chains are known to be as follows: arginine: 12.48, aspartic acid: 3.65, cysteine: 8.18, glutamic acid: 4.25, histidine: 6.00, lysine: 10.53, and tyrosine: 10.07 (D. R. Lide, Handbook of Chemistry and Physics, 72nd Edition, CRC Press, Boca Raton, FL, 1991).

[0072] Modified antibodies or antigen-binding fragments thereof and conjugates thereof The present invention provides modified antibodies or antigen-binding fragments thereof. Modified antibodies or antigen-binding fragments thereof of the present invention refer to antibodies or antigen-binding fragments thereof that have been chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, etc. Biological modifications include post-translational modifications (e.g., N-linked or O-linked glycosylation, glycosylation remodeling, processing of the amino- or carboxyl-terminal region, deamidation, aspartic acid isomerization, and methionine oxidation), and modifications in which a methionine residue has been added to the amino terminus by expression in a prokaryotic host cell. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the antibodies or antigens of the present invention, such as enzyme-labeled, fluorescent-labeled, and affinity-labeled antibodies. Such modified antibodies or antigen-binding fragments thereof of the present invention are useful for improving the stability and blood retention of the original antibodies or antigen-binding fragments thereof of the present invention, reducing antigenicity, detecting or isolating such antibodies or antigens, etc.

[0073] Examples of chemical moieties contained in the chemically modified substance include water-soluble polymers such as polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, and polyvinyl alcohol.

[0074] Examples of biologically modified products include those modified by enzymatic treatment or cell treatment, fusion products to which other peptides such as tags have been added by genetic recombination, and products prepared using cells that express endogenous or exogenous glycosylation enzymes as hosts.

[0075] Such modifications may be made at any or desired positions in the antibody or antigen-binding fragment thereof, and the same or two or more different modifications may be made at one or more positions.

[0076] However, these deletions in the heavy chain sequence or modifications of the heavy or light chain sequence do not significantly affect the antigen-binding ability and effector functions (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody, and preferably do not significantly affect them.

[0077] Thus, the present invention also encompasses antibodies that have undergone such deletion or modification. Examples include deletion variants in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain (Journal of Chromatography A; 705; 129-134 (1995)), deletion variants in which two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain and amidation of a new proline residue at the carboxyl terminus (Analytical Biochemistry, 360: 75-83 (2007)), and antibodies in which glutamine or glutamic acid residues at the amino termini of the heavy or light chain of the antibody are modified by pyroglutamylation (WO 2013 / 147153) (collectively referred to as "deletion variants"). However, as long as the antigen-binding ability and effector function are maintained, the deletion variants of the carboxyl termini of the heavy and light chains of the antibody of the present invention are not limited to the above types. When the antibody of the present invention comprises two or more chains (e.g., heavy chains), the two or more chains (e.g., heavy chains) may be any one of heavy chains selected from the group consisting of full-length and the above-mentioned deletion forms, or a combination of any two of these. The quantitative or molecular ratio of each deletion form may be affected by the type of cultured mammalian cells producing the antibody of the present invention and the culture conditions, but the main component of the antibody of the present invention can be one in which one, two, or several amino acid residues are deleted from the carboxyl terminus of at least one, and preferably both, of the two heavy chains.

[0078] Furthermore, even if one to several amino acids derived from an expression vector and / or a signal sequence, etc., are added to the amino and / or carboxyl terminus of an antibody or antigen-binding fragment thereof of the present invention (such as those contained in the molecules, multispecific molecules, bispecific molecules, etc. of the present invention) (and some or all of these amino acids are modified as described above), so long as the desired antigen-binding activity is maintained, the modified antibody or antigen-binding fragment thereof is included in the scope of the molecule of the present invention, and molecules comprising such modified antibodies or antigen-binding fragments are also included in the scope of the molecule of the present invention.

[0079] In the present invention, the term "antibody or antigen-binding fragment thereof" also encompasses "modified antibodies or antigen-binding fragments thereof." Furthermore, the term "antibodies or antigen-binding fragments thereof" included in the molecules, multispecific molecules, bispecific molecules, etc. of the present invention also encompasses such "modified antibodies or antigen-binding fragments thereof."

[0080] Furthermore, antibody-dependent cellular cytotoxicity can be enhanced by modulating the sugar chain modification (glycosylation, defucosylation, etc.) attached to the antibody of the present invention. Techniques for modulating the sugar chain modification of antibodies are known, including, but not limited to, International Patent Publication Nos. WO 1999 / 54342, WO 2000 / 61739, WO 2002 / 31140, and WO 2007 / 133855.

[0081] Moiety that Binds to a Target Antigen: Moiety [a] A target antigen refers to a molecule associated with a specific disease. A molecule associated with a specific disease refers to a molecule that is expressed or whose expression is enhanced in abnormal cells that appear in a patient suffering from the disease associated with the antigen or molecule that causes the disease. The molecule refers to a molecule that can attack the abnormal cells, alleviate the symptoms of the disease, or treat the disease when the target antigen-binding moiety binds to the molecule. The abnormal cells are preferably tumor cells or stromal cells, and the target antigen for tumor cells is a tumor antigen, or for stromal cells, a molecule expressed on stromal cells. Stromal cells interact with tumor cells and play an important role in the growth and progression of cancer. Tumor antigens are antigens expressed on tumor cells or antigens expressed on tumor cells that are cancerous normal cells. Binding of the target antigen-binding moiety to the tumor antigen inhibits tumor cell growth, damages tumor cells, or kills tumor cells (apoptosis or necrosis).

[0082] Examples of tumor antigens include CD98, EGFR, GPRC5D, CD33, CD37, DR5, EPHA2, FGFR2, FGFR4, VEGF, CD20, CD22, CD70, CD166, CD71, CD47, CDH6, A33, CanAg, GPNMB, integrin, tenascin-C, CLDN6, DLL-3, and SLC44A4.

[0083] Furthermore, examples of antibodies against the above-mentioned tumor antigens include anti-CD98 antibodies (described in JP 2017-114763 A, WO 2007 / 114496 A, WO 2008 / 017828 A, WO 2009 / 043922 A, WO 2009 / 090553 A, JP 2012-092068 A, WO 2011 / 118804 A, and WO 2013 / 078377 A, etc.), panitumumab, nimotuzumab, cetuzumab, and the like. Examples of such antibodies include anti-EGFR antibodies such as ximab, ametumumab (SY-101), SYN-004, SCT-200, tomuzotuximab, GC-1118, GR-1401, depatuxizumab (ABT-806), serculutamab, AMG595, and matuzumab, and anti-GPRC5D antibodies (described in WO2018 / 147245, WO2016 / 090329, etc.). The target antigen-binding moiety of the present invention may be derived from or comprise these antibodies.

[0084] Examples of molecules expressed in interstitial cells include FAP (fibroblast activation protein).

[0085] The portion that binds to the target antigen is preferably a polypeptide comprising an amino acid sequence that is not contained in the first peptide or the second peptide, and more preferably consists of said amino acid sequence.

[0086] The target antigen and the portion that binds to the target antigen may be an antigen and an antibody that binds to the antigen or an antigen-binding fragment of the antibody, but are not limited to such combinations, and examples include a ligand and a receptor that binds to the ligand (or vice versa), and a cytokine and a receptor to which the cytokine binds (or vice versa).Further examples of molecules other than antibodies and the like include non-immunoglobulin proteins that bind to the target antigen, nucleic acid molecules such as nucleic acid aptamers, and low-molecular-weight compounds.

[0087] First Peptide: [b] Portion The first peptide, which recognizes the target antigen-binding site contained in the portion that binds to the target antigen (portion [a]), is a peptide that recognizes the target antigen-binding site contained in the portion that binds to the target antigen, thereby masking the site and making it impossible for the portion that binds to the target antigen to bind to the target antigen, making it difficult for the portion to bind to the target antigen, or inhibiting or preventing the binding. When the portion that binds to the target antigen is an antibody or an antigen-binding fragment of an antibody (hereinafter referred to as "antibody, etc."), the first peptide is a peptide that binds to the antigen-binding region of the antibody, etc. Here, "recognizing" the target antigen-binding site is synonymous with "binding" to the target antigen-binding site, "having affinity" for the target antigen-binding site, etc. The antigen-binding region of an antibody, etc., is located in the variable region of the antibody, etc., particularly in the complementarity-determining region (CDR). Because antibodies, etc., bind to an epitope (antigenic determinant) of an antigen, the first peptide is a peptide that mimics the epitope. A peptide that mimics an epitope of an antigen is called a mimotope. In the present invention, the first peptide is preferably a mimotope that binds to a CDR. A mimotope is a peptide consisting of 6 to 30, preferably 10 to 20, more preferably 13 to 17, and particularly preferably 15 amino acids. Mimotopes can be produced, for example, by preparing libraries of various display methods (such as phage display, ribosome display, nucleic acid display, and bacterial display) containing the above-mentioned amino acid numbers, screening them by panning, and selecting phage particles that display peptides that recognize the target antigen-binding site contained in the target antigen-binding portion. DNA encoding the mimotope and its nucleotide sequence can then be obtained from the phage particles. Identifying a peptide as a mimotope (binding to an antibody CDR) can be confirmed by crystallizing the masked antibody and performing X-ray crystallography. Furthermore, if it can be confirmed by SPR or the like that a peptide binds competitively to an antigen of an antibody, this strongly suggests that the peptide is a mimotope that binds to the CDR of the antibody (see Reference Example 4).

[0088] As mentioned above, the moiety that binds to the target antigen may be a molecule other than an antibody or an antigen-binding fragment of an antibody (hereinafter referred to as "antibody, etc."), and examples of the first peptide other than a mimotope include, when the target antigen is a ligand, a peptide that recognizes the ligand-binding site contained in a receptor to which the ligand binds, and when the target antigen is a cytokine, a peptide that recognizes the cytokine-binding site contained in a receptor to which the cytokine binds. When the moiety that binds to the target antigen is a non-immunoglobulin protein, the first peptide is a peptide that recognizes the target antigen site contained in the protein; when the moiety that binds to the target antigen is a nucleic acid molecule, the first peptide is a peptide that recognizes the target antigen site contained in the nucleic acid molecule; and when the moiety that binds to the target antigen is a small molecule compound, the first peptide is a peptide that recognizes the target antigen site contained in the small molecule compound.

[0089] Second Peptide: Portion [c] The second peptide comprises an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions within a pH range of 7.5 or less. Under acidic pH conditions, the amino acid side chain becomes positively charged, inducing a structural change in the linker. Here, "a pH range of 7.5 or less" includes any of the following ranges: pH 1.0 to 7.5, pH 2.0 to 7.5, pH 3.0 to 7.5, pH 4.0 to 7.5, pH 5.0 to 7.5, pH 6.0 to 7.5, and pH 7.0 to 7.5. Furthermore, "a pH range of 7.5 or less" does not necessarily have to be the entire range of pH 7.5 or less (although it may be the entire range), but refers to a change in charge from uncharged to positively charged in response to pH within any range that is entirely within the range of pH 7.5 or less. For example, it goes without saying that a change in the charge of an amino acid side chain in response to a change in pH from neutral to acidic conditions in the pH range of 5.5 to 7.5 falls within the "pH range of 7.5 or less." Furthermore, if a change in the charge of an amino acid in response to a change in pH from neutral to acidic conditions in the pH range of 4.5 to 6.5 falls within the range of 7.5 or less, and therefore falls within the "pH range of 7.5 or less." Here, "neutral pH conditions" refers to conditions where the pH is about 7, for example, conditions where the pH is 7.0 or higher and 7.5 or lower. Furthermore, "acidic pH conditions" refers to conditions where the pH is less than 7, for example, conditions where the pH is less than 7.0, preferably 6.5 or lower, and more preferably 6.0 or lower. The lower limit of the acidic pH conditions is not particularly limited, and may be any low value, such as 1.0, 2.0, or 3.0, as long as it is feasible within the skill of a person skilled in the art. That is, the acidic pH conditions are pH 1.0 or higher and lower than 7.0, pH 2.0 or higher and lower than 7.0, pH 3.0 or higher and lower than 7.0, preferably pH 1.0 or higher and lower than 6.5, pH 2.0 or higher and lower than 6.5, pH 3.0 or higher and lower than 6.5, more preferably pH 1.0 or higher and lower than 6.0, pH 2.0 or higher and lower than 6.0, pH 3.0 or higher and lower than 6.0.Furthermore, because the change in charge of an amino acid side chain is a reversible phenomenon caused by a change in pH, it is naturally believed that an amino acid side chain whose charge changes from uncharged to positive in response to a change in pH "from neutral to acidic" in the pH range of 7.5 or less will also change charge when the pH changes "from acidic to neutral" in the pH range of 7.5 or less. The amino acid side chain whose charge changes from uncharged to positive in response to a change in pH "from neutral to acidic" in the pH range of 7.5 or less may be a side chain of a natural amino acid or a side chain of a non-natural amino acid. In the case of a side chain of an unnatural amino acid, it can be appropriately selected from the unnatural amino acids described in WO 2006 / 132969, WO 2008 / 030612, WO 2008 / 030613, WO 2008 / 030614, WO 2010 / 037062, WO 2018 / 223108, etc., which were published at the time of filing of the present application. The second peptide is also referred to as a pH-responsive linker. The second peptide contains an amino acid having a basic functional group with a pKa of 7 or less in its side chain, the amino acid side chain changing from uncharged to positive in response to a change in pH from neutral to acidic conditions in the pH range of 7.5 or less. pKa is an index used to quantitatively express the acid strength of an acidic compound or acidic functional group, and is expressed as the negative common logarithm (pKa = -log10Ka) of the equilibrium constant Ka in a dissociation reaction in which a hydrogen ion is released. pKa is also called the acid dissociation constant, and is a value measured in water at 25°C. It can be calculated by the method described in Chemistry Handbook, Basic Edition, 5th Revised Edition, II-331 to II-343 (edited by the Chemical Society of Japan, Maruzen Publishing Co., Ltd., 2004). For pKa, values ​​known from literature (e.g., Science Chronology 2013 (Desktop Edition), Maruzen Publishing Co., Ltd., 2012) can also be used. pKa is also used as an index used to quantitatively express the base strength of a basic compound or basic functional group. That is, a proton is added to a basic compound or basic functional group to form a conjugate acid, and the basicity of the original basic compound can be determined based on the acidity of the conjugate acid.In addition, the term "amino acid side chain" in "an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions" and "an amino acid sequence containing an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions" is synonymous with "amino acid residue."

[0090] Since a required property of a pH-responsive linker is that the amino acid side chain contained in the second peptide is positively charged under acidic pH conditions, it is preferable that the second peptide does not contain an amino acid side chain that is negatively charged under acidic pH conditions. This is presumably because the negative charge of the amino acid under acidic pH conditions neutralizes or weakens the positive charge of the amino acid side chain that is positively charged under acidic pH conditions, thereby preventing the structural change of the linker from being induced. In the present invention, the amino acid side chain that is negatively charged under acidic pH conditions is not particularly limited, but when the second peptide is composed of natural amino acids, examples of amino acids that are negatively charged under acidic pH conditions include glutamic acid and aspartic acid. That is, it is preferable that the second peptide does not contain glutamic acid and / or aspartic acid.

[0091] The present invention can also be described using a "pI value" representing the isoelectric point of the second peptide. The term "pI value" of a molecule (also abbreviated as pH(I) or IEP value) as used herein refers to the isoelectric point of a molecule, particularly the isoelectric point of an amino acid, peptide, or protein, which is the pH value at which a particular molecule has no net charge. The higher the pI value of a molecule, i.e., the higher the pH value (basic side) at which a particular molecule has no net charge, the more likely the molecule is to be positively charged under acidic conditions. The lower the pI value of a molecule, i.e., the lower the pH value (acidic side) at which a particular molecule has no net charge, the less likely the molecule is to be positively charged under acidic conditions. Since experimental pI values ​​and calculated pI values ​​may differ slightly, the term "pI value" herein refers to the calculated pI value. pI values ​​can be found, for example, in A. Sillero and J. M. According to Ribeiro, Analytical Biochem., 179(2), 1989, 319-325, the pKa can be calculated from the pKa of each amino acid residue constituting the peptide to be calculated, and the pKa of the amino group at the amino terminal and the carboxyl group at the carboxyl terminal.

[0092] Since a required property of a pH-responsive linker is that the amino acid residues contained in the second peptide are positively charged under acidic pH conditions, the calculated charge under acidic conditions must be positive and greater than the calculated charge under neutral conditions, and the pI value is not particularly limited as long as it satisfies this condition. To induce a structural change in the linker, for example, the calculated charge of the second peptide under acidic conditions of pH 3.0 to 6.5 must be greater than the calculated charge of the second peptide under neutral conditions of pH 7.0 to 7.5. Therefore, the pI value of the second peptide is preferably 6.4 or greater, more preferably 6.8 or greater, even more preferably 7.2 or greater, and most preferably 7.6 or greater. The upper limit of the pI value is not particularly limited, and may be any value, such as 12, 13, or 14, as long as it can be produced within the skills of a person skilled in the art. That is, the pI value of the second peptide is preferably 6.4 or more and 12 or less, 6.4 or more and 13 or less, or 6.4 or more and 14 or less, more preferably 6.8 or more and 12 or less, 6.8 or more and 13 or less, or 6.8 or more and 14 or less, even more preferably 7.2 or more and 12 or less, 7.2 or more and 13 or less, or 7.2 or more and 14 or less, and most preferably 7.6 or more and 12 or less, 7.6 or more and 13 or less, or 7.6 or more and 14 or less.

[0093] For example, the pI value of the second peptide used in the Examples of the present invention calculated according to A. Sillero and J. M. Ribeiro, Analytical Biochem., 179(2), 1989, 319-325 is shown in Table 1. In Table 1, "pH responsiveness" indicates the presence or absence of a structural change under acidic conditions. In Table 1, "pH responsiveness" is expressed in three ways: "yes," "weak," and "no." Changes in binding activity were visually determined by ELISA using the method in Reference Example 5 described below. A clear change in binding activity was recorded as "yes," no change in binding activity was recorded as "no," and a slight change was recorded as "weak."

[0094]

[0095] The second peptide preferably consists of an amino acid sequence having the following structures (1) and (2): (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which one arginine or lysine has been inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), and (2) a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains.

[0096] Here, the amino acid sequence of (a) or (b) is referred to as a "basic amino acid cluster." The amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains is preferably an amino acid sequence consisting of amino acids having 2 to 4 consecutive basic functional groups with a pKa of 7 or less in their side chains, more preferably an amino acid sequence consisting of amino acids having 2 to 3 consecutive basic functional groups with a pKa of 7 or less in their side chains. There is no upper limit to the number of "amino acids having a basic functional group with a pKa of 7 or less in their side chains," but it is preferably 20 or less, more preferably 15 or less, and even more preferably 12 or less.

[0097] As described above, in the present invention, the term "basic amino acid cluster" refers to an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or an amino acid sequence in which one arginine or lysine has been inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence. Furthermore, when a specific amino acid is selected as an amino acid having a basic functional group with a pKa of 7 or less in its side chain, the term "cluster" may be added to the name of the amino acid. For example, when histidine is selected as an amino acid having a basic functional group with a pKa of 7 or less in its side chain, an amino acid sequence consisting of two or more consecutive histidines, or an amino acid sequence in which one arginine or lysine has been inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence, is referred to as a "histidine cluster (His cluster)."

[0098] In the second peptide, the amino acids other than the basic amino acid cluster portion are not particularly limited, as long as they are amino acids other than those having a basic functional group in the side chain with a pKa of 7 or less, arginine, lysine, and amino acids having a negative charge under acidic pH conditions. Examples of such amino acids include naturally occurring amino acids such as glycine, serine, alanine, proline, and threonine. Preferred are amino acid sequences consisting of glycine and / or serine (so-called GS linkers) or amino acid sequences constituting a protease substrate sequence. One to 30, preferably 1 to 20, more preferably 1 to 10 such amino acids may be contained on either or both of the amino-terminal and carboxyl-terminal sides of the basic amino acid cluster.

[0099] Among natural amino acids, histidine is the only amino acid having a basic functional group in its side chain with a pKa of 7 or less, and the number of basic amino acid clusters in a second peptide composed of natural amino acids can be counted by counting the number of histidine clusters. For example, if the second peptide has the amino acid sequence "GGGGSHHGGHHGGHHGGHHGGS" (SEQ ID NO: 42, Figure 52), it can be counted as containing four basic amino acid clusters because it contains four amino acid sequences each consisting of two consecutive histidines. As another example, if the second peptide has the amino acid sequence "GGGGSHHGGHRHGGHHKGGHHGGS" (SEQ ID NO: 43, FIG. 52), it can be counted as containing four basic amino acid clusters because it contains two amino acid sequences consisting of two consecutive histidines, one amino acid sequence in which arginine is inserted between the amino acid sequence in which two consecutive histidines, and one amino acid sequence in which lysine is added to the amino acid sequence in which two consecutive histidines. Examples of amino acid sequences in which one arginine or lysine is inserted or added at any position (including the amino terminus or carboxyl terminus) of an amino acid sequence consisting of two or more consecutive histidines include HRH, RHH, HHR, HRHH, HHRH, RHHH, HHHR, HKH, KHH, HHK, HKHH, HHKH, KHHH, HHHK, etc. This is an example of the case where the amino acid having a basic functional group with a pKa of 7 or less in its side chain is histidine, and the amino acid represented by H may be an amino acid other than histidine having a basic functional group with a pKa of 7 or less in its side chain.

[0100] The basic amino acid cluster in the second peptide preferably includes an amino acid sequence in which one or more arginines are inserted or added at any position (including the amino terminal or carboxyl terminal) of an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, in order to increase the affinity of the molecule that binds to the target antigen for the target antigen at acidic pH; more preferably includes one amino acid sequence in which one arginine is inserted or added at any position (including the amino terminal or carboxyl terminal) of an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains; and even more preferably includes one basic amino acid cluster having an amino acid sequence represented by "RHHH."

[0101] Arginine is an amino acid having a guanidino group, a basic functional group, in its side chain, and its pKa is 12.48. Lysine is an amino acid having an amino group, a basic functional group, in its side chain, and its pKa is 10.53. Therefore, it is believed that the basic functional groups in the side chains of both amino acids are positively charged even under neutral conditions. Therefore, the arginine or lysine can enhance the structural change of the second peptide in response to a change in pH. Due to this effect, it is believed that a second peptide containing a basic amino acid cluster into which arginine or lysine has been inserted or added can induce a structural change even in a mildly acidic environment (e.g., pH 6.5 or higher but less than 7.0).

[0102] In the present invention, an EC of an amino acid sequence in which one or more arginines are not inserted or added at any position (including the amino terminal or carboxyl terminal) of an amino acid sequence consisting of two or more consecutive amino acids having basic functional groups with a pKa of 7 or less in the side chain is used. ５０ The ratio (neutral pH condition / acidic pH condition) and the EC of an amino acid sequence in which one or more arginines are inserted or added at any position (including the amino terminal or carboxyl terminal) of an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in the side chain. ５０By comparing the ratio (neutral pH condition / acidic pH condition), the effect of enhancing pH responsiveness due to the insertion or addition of arginine can be quantified. ５０ The comparison of the ratios is as follows: ５０ Ratio) / (former EC ５０ The ratio can be calculated by the following formula, and it is preferable that the ratio is 4.0 or more.

[0103] The pKa of the basic functional group in the amino acids contained in the basic amino acid cluster and having a basic functional group in the side chain is preferably in the range of 5.0 to 7.0, more preferably in the range of 5.5 to 6.5. The amino acid having a basic functional group in the side chain most preferably includes histidine, whose basic functional group has a pKa of 6.00.

[0104] The number of basic amino acid clusters contained in the second peptide is not particularly limited as long as the clusters are capable of inducing a structural change of the linker by becoming positively charged under acidic pH conditions; however, the second peptide preferably contains 1 to 4 basic amino acid clusters, more preferably 2 to 4 basic amino acid clusters, and even more preferably 3 or 4 basic amino acid clusters.

[0105] The number of amino acids having a basic functional group in a side chain contained in the second peptide is not particularly limited as long as the cluster can be positively charged under acidic pH conditions, thereby inducing a structural change in the linker; however, the number is preferably 10 or less, and more preferably 5% or more and 30% or less of the total number of amino acids contained in the amino acid sequence of the second peptide.

[0106] When the second peptide comprises (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which one arginine or lysine is inserted or added at any position in the amino acid sequence (including the amino terminus or carboxyl terminus), and (2) an amino acid sequence containing a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains, the number of amino acids contained in the second peptide is 10 to 60, preferably 12 to 50, more preferably 15 to 40, and even more preferably 20 to 35.

[0107] The position of the basic amino acid cluster contained in the second peptide is not particularly limited. That is, when the amino acid sequence of the second peptide contains two or more basic amino acid clusters, (1) only one basic amino acid cluster may be located at the amino terminus or carboxyl terminus of the amino acid sequence, (2) one basic amino acid cluster may be located at the amino terminus of the amino acid sequence and another basic amino acid cluster may be located at the carboxyl terminus of the amino acid sequence, or (3) neither basic amino acid cluster may be located at the amino terminus nor the carboxyl terminus of the amino acid sequence. In the present invention, the second peptide may further contain an amino acid sequence that serves as a substrate for an intracellular or extracellular protease and is cleaved by the protease (hereinafter simply referred to as "substrate"). When the amino acid sequence of the second peptide further contains an amino acid sequence that is cleaved by an intracellular or extracellular protease, it is preferable that the amino acid sequence be linked in the following order from the amino terminus to the carboxyl terminus or from the carboxyl terminus to the amino terminus: the basic amino acid cluster, the amino acid sequence that is cleaved by the intracellular or extracellular protease, and the amino acid sequence of the portion that binds to the target antigen (portion [a]).

[0108] Furthermore, the second peptide may include one having a structure represented by an amino acid sequence containing an amino acid sequence having every other amino acid having a basic functional group with a pKa of 7 or less in its side chain. Here, the number of amino acids having a basic functional group with a pKa of 7 or less in its side chain is 4 to 15, preferably 5 to 12, and more preferably 6 to 10. An example of such a sequence is "GGGGSHGHHGHGHGHGHGHGGS" (SEQ ID NO: 44, FIG. 52).

[0109] The second peptide may contain both an amino acid sequence having every other amino acid having a basic functional group with a pKa of 7 or less in its side chain, and (a) an amino acid sequence consisting of two or more consecutive amino acids having basic functional groups with a pKa of 7 or less in their side chains, or (b) an amino acid sequence in which one arginine or lysine has been inserted or added at any position in the amino acid sequence (including the amino terminus or carboxyl terminus).

[0110] The acidic pH condition may be in the range of pH 7.5 or less, as long as the charge of the amino acid having a basic functional group in the side chain of the second peptide is changed. Therefore, the acidic pH condition is preferably in the range of pH 6.5 or less, more preferably in the range of pH 6.0 or less, and even more preferably in the range of pH 5.5 or less.

[0111] The first peptide, the second peptide, and the target antigen-binding moiety may be linked to a linker. That is, any one of the first peptide, the second peptide, and the target antigen-binding moiety may be linked to one or both of the other two via a linker. Here, the linker is a peptide consisting of 2 to 30 amino acids, preferably 2 to 20 amino acids, and more preferably 2 to 10 amino acids.

[0112] The second peptide may contain an amino acid sequence that is cleaved by an intracellular protease or an extracellular protease. When the second peptide contains an amino acid sequence that serves as a substrate for an intracellular and / or extracellular protease and is cleaved by the protease (also simply referred to as a "substrate"; a substrate cleaved by a certain protease is also referred to as a "protease substrate"), the amino acid sequence that serves as a substrate for the intracellular protease can be referred to as the first cleavage amino acid sequence, and the amino acid sequence that serves as a substrate for the extracellular protease can be referred to as the second cleavage amino acid sequence. In the present invention, including the first cleavage amino acid sequence and the second cleavage amino acid sequence in the second peptide is also referred to as combining the first cleavage amino acid sequence and the second cleavage amino acid sequence as a protease cleavage sequence in the second peptide.

[0113] Intracellular proteases, also known as "intracellularly acting proteases," are expressed intracellularly and act intracellularly without being secreted extracellularly, leading to cellular apoptosis. Examples of intracellular proteases include cytoplasmic cysteine ​​proteases such as caspases, calpains (also referred to as "CAPNs"), and tripeptidyl peptidases. Calpains exist in various isoforms, including CAPN1 (μ-calpain), CAPN2 (m-calpain), CAPN3, CAPN4, CAPN5, CAPN6, CAPN7, CAPN8, CAPN9, CAPN10, CAPN11, CAPN12, CAPN13, CAPN14, CAPN15, CAPN16, and CAPN17. Any of these isoforms can be utilized in the present invention. Preferably, CAPN1 (calpain 1) or CAPN2 (calpain 2) is utilized. Tripeptidyl peptidase exists in isoforms such as tripeptidyl peptidase 1 and tripeptidyl peptidase 2, and either isoform can be used in the present invention, preferably tripeptidyl peptidase 2. Accordingly, the first cleavage amino acid sequence in the second peptide is not particularly limited as long as it is an amino acid sequence that serves as a substrate for the above-described intracellular protease, i.e., an amino acid sequence that is recognized and cleaved by the protease, but is preferably an amino acid sequence that is recognized by human CAPN1 and serves as a substrate for it (also referred to as a "CAPN1 substrate" or "CAPN substrate"), and a preferred example of a CAPN substrate is PLFAAP (SEQ ID NO: 67, Figure 55).

[0114] Extracellular proteases, also known as extracellular-acting proteases, are expressed in a state having a signal sequence, secreted extracellularly, and act extracellularly. Examples of extracellular proteases include urokinase-type plasminogen activator (uPA), matrix metalloproteases (MMPs), plasmin, cathepsin, matriptase, and legumain. MMPs include isoforms such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, and any of these isoforms can be used in the present invention. Cathepsin includes isoforms such as cathepsin A, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin F, cathepsin G, cathepsin H, cathepsin K, cathepsin L1, cathepsin L2, cathepsin O, cathepsin S, cathepsin W, and cathepsin X / Z, and any of these isoforms can be used in the present invention. Accordingly, the second cleavage amino acid sequence in the second peptide is not particularly limited as long as it is an amino acid sequence that serves as a substrate for the extracellular protease, i.e., an amino acid sequence that is recognized and cleaved by the protease. Preferably, the second cleavage amino acid sequence is an amino acid sequence that is recognized by human uPA and serves as a substrate for it (also referred to as a "uPA substrate"). Examples of uPA substrates include SGRSANAILE (SEQ ID NO: 36, FIG. 51), SGRSANA (SEQ ID NO: 37, FIG. 51), and SGRSA (SEQ ID NO: 38). Preferred examples of amino acid sequences recognized by human MMP1 and serving as substrates thereof (also referred to as "MMP1 substrates" or "MMP substrates") include VLVPMAMMAS (SEQ ID NO: 39, FIG. 51) and PLGLWA (SEQ ID NO: 40, FIG. 51), and preferred examples of amino acid sequences recognized by human MMP9 and serving as substrates thereof (also referred to as "MMP9 substrates") include PLGLAG (SEQ ID NO: 41, FIG. 51).

[0115] When the second peptide contains both a first cleavage amino acid sequence that is a substrate for an intracellular protease and a second cleavage amino acid sequence that is a substrate for an extracellular protease, the order in which they are linked is not limited, but for example, in a molecule that binds to a target antigen in which the first peptide, the second peptide, and the target antigen-binding moiety are linked in this order, the second cleavage amino acid sequence that is a substrate for an extracellular protease may be included on the first peptide side and the first cleavage amino acid sequence that is a substrate for an intracellular protease may be included on the target-binding moiety; or in a molecule that binds to a target antigen in which the first peptide, the second peptide, and the target antigen-binding moiety are linked in this order, the first peptide side may be included on the first peptide side and the second cleavage amino acid sequence that is a substrate for an extracellular protease may be included on the target-binding moiety. Thus, in a molecule that binds to a target antigen, the first peptide, the second cleavage amino acid sequence, the first cleavage amino acid sequence, and the target antigen-binding moiety may preferably be linked in this order, or the first peptide, the first cleavage amino acid sequence, the second cleavage amino acid sequence, and the target antigen-binding moiety may be linked in this order.

[0116] The preceding paragraphs have exclusively described cases in which the second cleavage amino acid sequence is contained in the second peptide, but the second cleavage amino acid sequence may be contained in a molecule that binds to the target antigen of the present invention, other than the second peptide, for example, in a third peptide inserted at the end of the first peptide, or between the second peptide and the portion that binds to the target antigen.

[0117] The first and second peptides may also partially overlap. For example, in SEQ ID NO: 13, Figure 28 (full-length amino acid sequence of the MHT1806 H chain), the C-terminus (HH) of the mimotope peptide (amino acids 20 to 35) is part of a histidine cluster, and part of the first and second peptides overlap.

[0118] The second peptide includes (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which one arginine or lysine is inserted or added at any position (including the amino or carboxyl terminal) of the amino acid sequence, and (2) an amino acid sequence containing a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains, as well as an amino acid sequence that is cleaved by a protease, which is the first cleavage amino acid sequence and / or the second cleavage amino acid sequence. When both of the above are contained, it is preferable that, when the amino acid sequence is cleaved by a protease, the molecule that binds to the target antigen does not contain (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which a single arginine or lysine has been inserted or added at any position (including the amino terminus or carboxyl terminus) of the amino acid sequence, and (2) an amino acid sequence containing a total of four or more amino acids having the above basic functional groups with a pKa of 7 or less in their side chains.

[0119] When the second peptide contains both (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, or (b) one or more amino acid sequences in which one arginine or lysine has been inserted or added at any position in the amino acid sequence (including the amino or carboxyl terminus), and (2) an amino acid sequence containing a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains, and an amino acid sequence that is cleaved by a protease, which is the first cleavage amino acid sequence and / or the second cleavage amino acid sequence, the number of amino acids contained in the second peptide is 15 to 150, preferably 15 to 120, more preferably 15 to 70, and even more preferably 15 to 50.

[0120] Other Moiety: Moiety [d] The target antigen-binding molecule of the present invention may further comprise another moiety. In the present invention, such another moiety is also referred to as "moiety [d]." Moiety [d] binds to the target antigen-binding portion of the molecule. Moiety [d] consists of one or more compounds selected from the group consisting of an antibody or antigen-binding fragment thereof that is not the target antigen-binding portion, a peptide including an amino acid sequence not contained in the first peptide and the second peptide, a cytokine, a toxin, a radioisotope, a labeled molecule, a photosensitizer (also referred to as a "photosensitizer"), an immunostimulatory substance, an antitumor compound, a drug, a payload, and a polymer.

[0121] Here, examples of peptides containing an amino acid sequence not contained in the first peptide and the second peptide include antibodies, antigen-binding fragments of antibodies, naturally occurring non-immunoglobulin proteins, artificially created proteins, receptor proteins or ligand-binding fragments thereof, ligand proteins, proteins that regulate blood dynamics (e.g., antibody Fc, albumin), etc. Examples of antibodies include antibodies that are not molecules that bind to target antigens, antibodies that bind to sites other than the target binding target site in molecules that bind to target antigens, and antibodies that function as multispecific molecules (e.g., bispecific antibodies) by binding to molecules that bind to target antigens. Examples of cytokines include interleukins, interferons, chemokines, colony-stimulating factors, tumor necrosis factors, growth factors, etc. Examples of toxins include biological toxins such as cyanotoxins, hemotoxins, necrotoxins, neurotoxins, and cytotoxins, as well as environmental toxins. Examples of radioisotopes include １３１ I, ２１１ A.T., ８９Examples of labeling molecules include fluorescent substances such as FITC and PE, enzymes such as HRP and AP, and biotin. Examples of photosensitizers include phthalocyanine derivatives, chlorin derivatives, and bacteriochlorin derivatives. Examples of immunostimulating substances include adjuvants. Examples of polymers include natural or artificial sugar chains, synthetic resins, and polyethylene glycol. Examples of antitumor compounds include topoisomerase inhibitors, mitosis inhibitors, cell division inhibitors, microtubule polymerization / depolymerization inhibitors, glucocorticoid receptor modulators, DNA binders, alkylating agents, radioactive isotopes, siRNA, antibodies, or antigen-binding fragments thereof. Examples of drugs include antitumor agents, the above-mentioned immunostimulating substances and cytokines, and the antitumor compounds, drugs, and payloads contained in the ADCs described below, but are not limited to these.

[0122] When the [d] moiety is a polypeptide, the molecule that binds to the target antigen of the present invention may consist of the polypeptide, and when the [d] moiety is a compound other than a polypeptide, the molecule that binds to the target antigen of the present invention may consist of the [d] moiety and the polypeptide.

[0123] The moiety [d] may be linked to a molecule of the present invention that binds to a target antigen via a linker.

[0124] When the [d] moiety is an antitumor compound, drug, or payload, and the molecule that binds to a target antigen is an antibody or an antigen-binding portion thereof (e.g., an antibody), the molecule that binds to the target antigen and includes the [d] moiety can be referred to as an ADC (Antibody-Drug Conjugate). ADCs are described in, for example, Methods Mol Biol. (2013) 1045:1-27; Nature Biotechnology (2005) 23, pp. 1137-1146. The antitumor compound is not particularly limited as long as it is a substance that can exert a pharmacological effect by binding to an antibody or the like. Examples of antitumor compounds that can be used as antitumor agents include emtansine (4-({3-[(3-{[(1S)-2-{[(1S,2R,3S,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5] hexacosa-10,12,14(26),16,18-pentaen-6-yl]oxy}-1-methyl-2-oxoethyl]methylamino}-3-oxopropyl)sulfanyl]-2,5-dioxopyrrolidin-1-yl}methyl)cyclohexylcarbonyl group) (e.g., WO2001 / 000244, WO2001 / 000245), topoisomerase inhibitors (e.g., Liang, X. et al., Eur. J. Med. Chem. Vol. 171, 2019, pp. 129-168), preferably topoisomerase I inhibitors, for example, camptothecin derivatives, preferably irinotecan and its active metabolite SN-38 (e.g., EP 137145A1, US 4604463A,), exatecan (e.g., EP 495432A1, US 5637770A), exatecan derivatives (e.g., WO 2014 / 057687), etc. Suitable exatecan derivatives include N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide (for example, WO2014 / 057687, WO2014 / 061277, WO2015 / 146132, WO2020 / 100954, WO2015 / 098099) can be exemplified. Other antitumor compounds include pyrrolobenzodiazepine derivatives (e.g., WO2019 / 065964, WO2013 / 173496, WO2014 / 130879, WO2017 / 004330, WO2017 / 004025, WO2017 / 020972, WO2016 / 036804, WO2015 / 095124, WO2015 / 052322, WO2015 / 052534, WO2016 / 115191, WO2015 / 052321, WO201 5 / 031693, WO2011 / 130613) can also be exemplified, and preferred pyrrolobenzodiazepine derivatives include (11a'S)-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-1',11a'-dihydro-5'H-spiro[cyclopropan-1-yl]oxy] propane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5'-one, (11a'S)-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-1',10',11',11a'-tetrahydro-5'H-spiro[cyclopropane-1,2'-pyrrolo[ 2,1-c][1,4]benzodiazepine]-5'-one, (11a'S,11a''''S)-8',8''-[1,5-pentanediylbis(oxy)]bis(7'-methoxy-1',11a'-dihydro-5'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5'-one), and (11a'S)-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,Examples of the drug include (11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-1',11a'-dihydro-5'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-5'-one (WO 2019 / 065964). The drug may also be an immunostimulatory substance such as a STING agonist (e.g., WO 2021 / 202984, WO 2020 / 229982, WO 2020 / 050406, WO 2021 / 177438), a TLR7 / 8 agonist, or a TLR8 agonist (e.g., WO 2018 / 009916, WO 2019 / 084060). Suitable STING agonists include cyclic dinucleotide derivatives, such as (5R,7R,8R,12aR,14R,15R,15aS,16R)-15,16-dihydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6- Tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyne-2,10-dione, (5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16 -Hydroxy-7-[1-(2-hydroxyethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-2,10-bis(sulfanyl)-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione, (5R,7R,8R,12aR,14R,15R,15aR,16R)-7-[1-(2-aminoethyl)-6-oxo-1,6-dihydro-9H-purin-9-yl]-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulene-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione, N-(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16 R)-14-(8,9-dihydro-6-thia-2,3,5-triazabenzo[cd]azulen-2(7H)-yl)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-bis(sulfanyl)octahydro-2H,10H,12H-5,8-methano-2λ5,10λ5-furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,An example of such a glycoprotein is 9-dihydro-1H-purin-1-yl}ethyl)-2-hydroxyacetamide (WO 2021 / 177438). In the present invention, the [d] moiety binds to the target antigen-binding moiety. When the target antigen-binding moiety is an antibody or an antigen-binding fragment thereof, the [d] moiety can be conjugated to the antibody or its antigen-binding fragment by known methods. Techniques for homogenizing glycans have been developed for the production of therapeutic or prophylactic antibodies or glycoprotein molecules containing their Fc regions. Enzymatic transglycosylation is known as a method for homogenizing glycans added to glycoproteins. This is a multistep process consisting of cleavage of glycans (hydrolysis) and condensation of additional glycans (transglycosylation) in an in vitro environment. In particular, when the purpose is to convert N-glycans, a group of enzymes known as endo-β-N-acetylglucosaminidases (ENGases) is used. The enzyme is required to have the following characteristics: 1) substrate specificity, i.e., the ability to hydrolyze complex-type glycans, and 2) the ability to transglycosylate specific structures. Two known transglycosylation methods are known: the oxazoline method, in which a single ENGase is used to transfer a glycan with an activated reducing end, e.g., an oxazolinated glycan, to a GlcNAc (N-acetylglucosamine) acceptor; and the one-pot method, in which two types of ENGase are used to directly transfer a glycan with an inactivated reducing end to a GlcNAc acceptor (WO 2022 / 050300, WO 2018 / 003983). In the present invention, for example, transglycosylation reactions using these methods can be used to conjugate the [d] moiety of an antitumor compound, drug, payload, or the like to an antibody or its antigen-binding fragment, directly or via a linker or the like. When the [d] moiety is a photosensitizer and the target antigen-binding moiety is or contains an antibody or its antigen-binding portion (e.g., an antibody), a molecule that binds to the target antigen and contains the [d] moiety can be used in photodynamic therapy (PDT), and the molecule can be called an ADP (Antibody-Directed Phototherapy) molecule. For details about ADP molecules, see Antibodies (2013) 2,The photosensitizer is not particularly limited as long as it is a substance that can exert a pharmacological effect by irradiating the site where an antibody or the like is bound with light, and examples of the photosensitizer include (2S)-2-[[2-[(2S,3S)-7-carboxy-3-(2-carboxyethyl)-17-ethenyl-12-ethyl-2,8,13,18-tetramethyl-2,3,23,24-tetrahydroporphyrin-5-yl]acetyl]amino]butanedioic acid (e.g., U.S. Pat. No. 5,633,275, U.S. Pat. No. RE37180), IR700 (IRDye (registered trademark) 700DX) (e.g., WO2 013 / 009475, WO2004 / 038378, WO2015 / 187677, WO2017 / 031363, WO2017 / 031367, WO2018 / 156815, WO2019 / 232478, WO2020 / 205623), 2,4-difluoro-N-methyl-3-[10,15,20-tris[2,6-difluoro-3-(methylsulfamoyl)phenyl]-2,3,12,13,22,24-hexahydroporphyrin-5-yl]benzenesulfonamide (e.g., WO2016 / 151458), and the like can be exemplified.

[0125] In or near a tissue containing cells having the target antigen, the cleavable linker (second peptide) contained in the molecule that binds to the target antigen is cleaved, and the active ingredient (e.g., a drug, an antitumor compound, an immunostimulatory substance, etc.) contained in the molecule that binds to the target antigen exerts its effect.

[0126] Fc Region: [e] Portion In the present invention, the Fc region may be referred to as the "[e] portion." The Fc region may be either an Fc region derived from a naturally occurring immunoglobulin or a variant thereof. Examples include an Fc region derived from a vertebrate, preferably a mammal, more preferably a primate or rodent, even more preferably a primate, even more preferably a human or cynomolgus monkey, and most preferably a human immunoglobulin, and a variant thereof. Preferred Fc regions derived from human immunoglobulin are the Fc regions of human IgG1 or human IgG2. In another embodiment of the present invention, the Fc region can interact with protein A, but the interaction with protein A can be regulated. In yet another embodiment of the present invention, the Fc region can regulate effector activity, and may or may not contain a mutation therefor. In another embodiment of the present invention, the Fc region may or may not contain a mutation that promotes heterodimer formation. Furthermore, in certain embodiments of the invention, the Fc region may or may not contain the "ProA affinity reducing mutations" of the present invention (described elsewhere).

[0127] 3. Method for Producing Molecules that Bind to Target Antigens (1) Method for Identifying Mimotopes Contained in First Peptides Peptides that bind to the complementarity-determining regions (CDRs) contained in any antibody or its antigen-binding fragment can be identified using a peptide library. Peptide libraries can be constructed using known methods. For example, a peptide library for various displays, such as ribosomes, composed of completely random amino acids can be constructed, and peptides with high affinity for the CDRs can be selected. Alternatively, a peptide library for various displays (ZPZP lib) can be constructed that contains a repeating motif of aromatic amino acids and Pro (ZPZP motif) near the center, and peptides with high affinity for the CDRs can be selected. Here, Z represents an aromatic amino acid, such as histidine (His), phenylalanine (Phe), tyrosine (Tyr), or tryptophan (Trp), and P represents proline.

[0128] Mimotopes contained in the first peptide can also be identified by the above methods.

[0129] The CDR loop of an antibody contains many aromatic amino acids. Because aromatic amino acids tend to interact with each other, it is preferable that an aromatic amino acid be present near the center of the peptide. (2) Method for producing a molecule that binds to a target antigen Peptides that are molecules that bind to the target antigen of the present invention and that contain the amino acid sequence contained in the first peptide and / or the amino acid sequence contained in the second peptide can be prepared by recombination, in vitro translation, chemical synthesis, peptide synthesis, etc.

[0130] For example, a molecule of the present invention that binds to a target antigen can be produced by introducing a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the portion that binds to the target antigen, and optionally an amino acid sequence contained in the first peptide and / or an amino acid sequence contained in the second peptide, or a vector comprising the polynucleotide, into cells, culturing the cells, and recovering a polypeptide that binds to the target antigen from the culture.

[0131] A polynucleotide comprising a nucleotide sequence encoding the amino acid sequence contained in the target antigen-binding portion and, optionally, the amino acid sequence contained in the first peptide and / or the second peptide may be linked to DNA encoding each peptide and may further be operably linked to elements such as a promoter, enhancer, polyadenylation signal, etc. Here, "operably linked" refers to linking elements so that they perform their functions. These DNAs may be inserted into an expression vector, host cells may be transformed with the vector, and the host cells may be cultured to produce and recover the target antigen-binding molecule. The vector may also contain DNA encoding a signal peptide that promotes secretion of the target antigen-binding molecule from the host cell. In this case, the DNA encoding the signal peptide and the DNA encoding the target antigen-binding molecule are ligated in-frame. After the target antigen-binding molecule is produced, the signal peptide can be removed to obtain the target antigen-binding molecule as a mature protein.

[0132] The expression vector is not particularly limited as long as it can be replicated in a host such as an animal cell, bacteria, or yeast, and examples thereof include known plasmids and phages. Examples of vectors used to construct an expression vector include pcDNA™ (ThermoFisher Scientific), Flexi™ Vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). Prokaryotic cells such as Escherichia coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, can be used as host cells, but eukaryotic cells are preferred. For example, HEK293 cells, a human embryonic kidney cell line, or Chinese hamster ovary (CHO) cells may be used as animal cells. The expression vector can be introduced into host cells by known methods to transform the host cells. Examples of such methods include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The produced antibody can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatographies, filters, ultrafiltration, salting out, dialysis, and the like may be appropriately selected and combined. (3) Method for producing a peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide A peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide of the present invention can be prepared by recombination, in vitro translation, chemical synthesis, peptide synthesis, and the like.

[0133] For example, a peptide consisting of an amino acid sequence including the amino acid sequence of the second peptide of the present invention can be produced by introducing a polynucleotide encoding a peptide consisting of an amino acid sequence including the amino acid sequence of the second peptide into cells, culturing the cells, and recovering the desired peptide from the culture.

[0134] A polynucleotide encoding a peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide may be ligated to DNA encoding each peptide, and may further be operably linked to elements such as a promoter, enhancer, or polyadenylation signal. Here, "operably linked" refers to linking elements so that they perform their functions. These DNAs may be inserted into an expression vector, host cells transformed with the vector, and the host cells cultured to produce and recover the peptide. The vector may also contain DNA encoding a signal peptide that promotes secretion of the peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide from the host cell. In this case, the DNA encoding the signal peptide and the DNA encoding the peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide are ligated in-frame. After the peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide is produced, the signal peptide can be removed to obtain the peptide comprising an amino acid sequence containing the amino acid sequence of the second peptide as a mature protein.

[0135] The expression vector is not particularly limited as long as it can be replicated in a host such as an animal cell, bacteria, or yeast, and examples thereof include known plasmids and phages. Examples of vectors used to construct an expression vector include pcDNA™ (ThermoFisher Scientific), Flexi™ Vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). Prokaryotic cells such as Escherichia coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, can be used as host cells, but eukaryotic cells are preferred. For example, HEK293 cells, a human embryonic kidney cell line, or Chinese hamster ovary (CHO) cells may be used as animal cells. The expression vector can be introduced into host cells by known methods to transform the host cells. Examples of such methods include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis, and the like may be appropriately selected and combined. (4) Immobilized Metal Affinity Chromatography (IMAC) In the present invention, "IMAC" refers to a method of separating multiple molecules with different affinities for a metal using a carrier on which a metal is immobilized.

[0136] In the present invention, IMAC can be performed by methods well known to those skilled in the art. IMAC can be performed in either a column or batch system using a metal-immobilized affinity chromatography support (also referred to as a "resin"), but preferably in a column packed with the support. The mobile phase may be any liquid capable of dissolving a molecule (molecule 1) that binds to the target antigen of the present invention, preferably a liquid capable of dissolving one or more molecules (other molecules) that bind to target antigens other than molecule 1, more preferably an aqueous solution. The mobile phase may contain buffers, salts, surfactants, organic solvents, stabilizers, isotonicity agents, protease inhibitors, etc., but the components that can be contained in the mobile phase are not limited thereto. Examples of buffer-containing liquids include Tris buffer, phosphate buffer, carbonate buffer, etc., and preferably Tris buffer, more preferably Tris buffer at a pH of 7 to 9, even more preferably Tris buffer at a pH of 7.5 to 8.5, and even more preferably Tris buffer at a pH of 8. The Tris concentration in the mobile phase can be, for example, in the range of 0 to 2 M, preferably 0 to 1 M, more preferably 0 to 0.5 M, and even more preferably 0 to 0.02 M. The salt is preferably NaCl, and the NaCl concentration in the mobile phase can be, for example, in the range of 0 to 2 M, more preferably 0 to 1 M, and even more preferably 0 to 0.15 M.

[0137] As the metal-immobilized affinity carrier, those well known to those skilled in the art can be used, and for example, those in which divalent metal ions such as Ni (nickel), Co (cobalt), Cu (copper), Fe (iron), Zn (zinc), etc., preferably Ni, Co and Cu, more preferably Ni, are supported on agarose beads or the like using a chelating agent such as NTA (nitrilotriacetic acid) or IDA (iminodiacetic acid) can be used. Such an IMAC carrier includes, for example, Ni-charged IMAC Sepharose. ＴＭ 6 Fast Flow (cytiva), Ni-charged Profinity ＴＭ Examples of suitable resins include, but are not limited to, IMAC Resin, uncharged (Bio-Rad), HisPur Ni-NTA Agarose Resin (ThermoFisher), and the like.

[0138] In the present invention, IMAC is not particularly limited as long as it includes a step of contacting a starting sample containing a molecule that binds to target antigen 1 (molecule 1) and one or more molecules other than molecule 1 (other molecules) with a carrier for immobilized metal affinity chromatography (IMAC) to separate molecule 1 from other molecules, but examples of such a step include: (i) a step of equilibrating a column packed with the carrier with a buffer (also referred to as an "equilibration buffer"); (ii) a step of adding (also referred to as "loading") the starting sample to the column; (iii) a step of washing the column with one or more buffers (referred to as "washing buffer"); (iv) adding to the column a buffer solution (also referred to as an "elution buffer") containing a linear or stepwise (preferably linear) concentration gradient of imidazole or histidine (preferably a gradient in which the concentration of imidazole or histidine increases); (v) recovering fractions eluted from the column containing molecule 1, preferably fractions containing molecule 1 but not containing one or more other molecules; any of the above steps (i) to (v) can be omitted, multiple steps can be combined, or additional steps can be added, as long as one or more other molecules can be separated from molecule 1. After elution, the column can be washed, regenerated, and stored by methods well known to those skilled in the art.

[0139] The concentration gradient of imidazole or histidine can be prepared, for example, linearly or stepwise in the concentration range of 0 to 2M, preferably 0 to 1M, more preferably 0.02 to 0.5M.

[0140] The temperature at which IMAC is carried out is, for example, 0 to 50°C, but is not limited thereto, and is preferably 0 to 37°C, and more preferably 4 to 25°C.

[0141] 4. Method for Producing a Protein In the method for producing a molecule that binds to a target antigen described in 3 above, "a molecule that binds to a target antigen" may be replaced with "a certain protein," and the present invention also provides a method for producing a protein. For example, the present invention provides a method for producing a protein (protein 1) comprising n peptides (referred to as "second peptides" in the present invention) consisting of an amino acid sequence containing amino acids whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions within a pH range of 7.5 or less, where n is a positive integer, and the method includes a step (separation step) of contacting a starting sample containing protein 1 and one or more proteins other than protein 1 (other proteins) with an IMAC carrier to separate protein 1 from at least one other protein (other protein to be separated), wherein the other protein to be separated contains n+1 or more second peptides or n-1 or less, where n-1 or less is 0 or a positive integer. The other protein preferably contains one or more second peptides. In one aspect, the second peptide comprises an amino acid sequence that contains four or more amino acids whose charge changes from uncharged to positive in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or less, and that has a pI value of greater than 6.4, preferably 6.8 or more, more preferably 7.2 or more, and even more preferably 7.6 or more. In another aspect, the second peptide comprises an amino acid sequence that does not contain any amino acids that are negatively charged under acidic pH conditions, preferably an amino acid sequence that does not contain aspartic acid and / or glutamic acid. In another aspect, the second peptide comprises (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups in their side chains with a pKa of 7 or less, or (b) an amino acid sequence in which one arginine or lysine has been inserted or added at any position (including the amino terminus or carboxyl terminus) of the amino acid sequence of (a) (the amino acid sequence of (a) or the amino acid sequence of (b) is referred to as a basic amino acid cluster).(b') preferably comprises one or more amino acid sequences in which an arginine has been inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), and more preferably comprises (b'') comprises one amino acid sequence in which an arginine has been inserted or added at any position (including the amino or carboxyl terminus) of the amino acid sequence of (a), and (2) comprises an amino acid sequence containing a total of four or more amino acids having a basic functional group with a pKa of 7 or less in their side chains. In yet another aspect, the second peptide comprises an amino acid sequence containing (1) one amino acid sequence represented by RHHH, and (2) comprises an amino acid sequence containing a total of four or more amino acids having a basic functional group with a pKa of 7 or less in their side chains. In another embodiment, the second peptide comprises an amino acid sequence containing at least one amino acid having two or more consecutive basic functional groups with a pKa of 7 or less in their side chains, and a total of four or more amino acids having basic functional groups with a pKa of 7 or less in their side chains. In another embodiment, the second peptide is represented by an amino acid sequence containing every other amino acid having a basic functional group with a pKa of 7 or less in its side chain, the basic functional group having a pKa of 7 or less, preferably 5.0 to 7.0, more preferably 5.5 to 6.5, in its side chain, and the amino acids may be natural amino acids, preferably histidine, or unnatural amino acids. In one embodiment, the second peptide comprises an amino acid sequence containing one to four basic amino acid clusters. The number of amino acids contained in the second peptide is 10 to 60, preferably 12 to 50, more preferably 15 to 40, and even more preferably 20 to 35. For example, the second peptide may comprise any one of the amino acid sequences set forth in SEQ ID NOS: 49 to 53, 55, 59 to 61, 65, 66, and 75 to 81 (Figures 54 and 64), and preferably, but is not limited to, the amino acid sequence set forth in SEQ ID NOS: 53 (Figure 54). In another embodiment, the second peptide consists of an amino acid sequence containing 10 or less amino acids having basic functional groups with a pKa of 7 or less in their side chains.In another embodiment, the amino acid sequence of the second peptide contains two or more basic amino acid clusters, one basic amino acid cluster being located at the amino terminus or carboxyl terminus of the amino acid sequence, preferably the amino terminus, and (i) no other basic amino acid cluster is located at the amino terminus or carboxyl terminus of the amino acid sequence, (ii) another basic amino acid cluster is located at the carboxyl terminus, or (iii) both other basic amino acid clusters are located at the amino terminus and the carboxyl terminus of the amino acid sequence. The protein production method may further comprise a step of removing all or a part of the second peptide after the separation step. The protein to be subjected to such a production method is not particularly limited, and examples thereof include naturally occurring proteins or variants thereof, artificially created proteins, modified proteins, fusion proteins, synthetic proteins, recombinant proteins, proteins containing non-peptide moieties, fragments thereof, and molecules containing any of these and other moieties.

[0142] 5. The Production Method of the Present Invention Including a Chromatography Step or Fractionation Step Other Than IMAC In one embodiment, the method of the present invention may also include a chromatographic step or fractionation step other than IMAC, which may be performed before or after the IMAC step. In other words, the chromatographic step or fractionation step other than IMAC may be a pretreatment of the sample to be subjected to IMAC in the method of the present invention, i.e., the starting sample, or may be a step performed on the sample obtained as a result of the IMAC step.

[0143] Such a chromatography step or fractionation step other than IMAC is preferably a Protein A chromatography step. The Protein A chromatography step is suitable when the molecule 1 that binds to the target antigen 1 of the present invention or another molecule contains an Fc region, or when the protein 1 described in [A1] or another protein contains an Fc region. More preferably, the sample subjected to Protein A chromatography contains, in addition to the molecule 1 that binds to the target antigen 1 of the present invention or the protein 1 described in [A1], another molecule or another protein. Even more preferably, the Fc region contained in the molecule 1 that binds to the target antigen 1 of the present invention or the protein 1 described in [A1], or the other molecule or other protein, contains one or more mutations that reduce the affinity for Protein A (hereinafter referred to as "ProA affinity-reducing mutations").

[0144] In one embodiment, molecule 1 that binds to target antigen 1 is a multispecific, preferably bispecific, more preferably bispecific antibody, comprising one or more pairs of Fc heterodimers, one chain of which comprises a ProA affinity-reducing mutation and the other chain of which does not comprise a ProA affinity-reducing mutation. This results in a difference in affinity for a Protein A chromatography support between molecule 1 that binds to target antigen 1 with multispecificity and other molecules in which the Fc region forms a homodimer or molecules that do not comprise an Fc region (e.g., molecules consisting only of an antibody light chain), making it possible to separate molecule 1 that binds to target antigen 1 with the desired multispecificity from other molecules or to concentrate molecule 1 that binds to target antigen 1 with the desired multispecificity from a mixture of these molecules (see Examples). Other molecules in which the Fc region forms a homodimer either have neither chain containing a mutation that reduces affinity for ProA, or both chains containing a mutation that reduces affinity for ProA. Molecules that do not contain an Fc region do not have affinity for ProA, and therefore these molecules differ from molecule 1 in affinity for the Protein A chromatography carrier.

[0145] Any known mutation can be used as the ProA affinity-reducing mutation. When a molecule of the present invention that binds to a target antigen containing an Fc region or a protein described in [A1] is a component of a therapeutic or prophylactic drug administered to humans, it is preferable that the Fc region be closer to the human wild-type from the standpoint of safety. Therefore, the number of mutations contained in the Fc region is preferably small, and more preferably, there is only one ProA affinity-reducing mutation. When an Fc region consisting of only one ProA affinity-reducing mutation forms a homodimer, there is only one set of ProA affinity-reducing mutations, but this is also included in the meaning of "one" in the present invention. Even more preferred ProA affinity-reducing mutations include the substitution of Ile at position 253 of human IgG1 with Asn (I253N), the substitution of Ile at position 253 (same as above) with Asp (I253D), and the substitution of His at position 435 with Arg (H435R) (all amino acid numbers are according to EU numbering), with I253N being preferred, but not limited thereto. In the present invention, unless otherwise specified, the numbers of amino acids or amino acid residues are based on EU numbering.

[0146] In IMAC, the mobile phase used to elute the components adsorbed to the IMAC carrier is not limited, and examples include a linear concentration gradient, a stepwise concentration gradient, and a single mobile phase. In the present invention, "adsorption" to the carrier is synonymous with "binding" to the carrier and can be used interchangeably.

[0147] Furthermore, one or more other steps may be performed between the IMAC and other chromatography steps or fractionation steps. Examples of such other steps include modification of molecule 1 or protein 1, such as addition of one or more amino acids to the N-terminus or C-terminus, removal of one or more amino acids from the N-terminus or C-terminus, glycosylation, addition of a glycosylation, removal of a glycosylation, and conjugation with a drug. Such other steps may be performed before or after the IMAC and other chromatography steps or fractionation steps. However, the other steps are not limited to these.

[0148] 6. Use of molecules, proteins, etc. that bind to target antigens obtained by the methods of the present invention Molecules, proteins, etc. that bind to target antigens obtained by the methods of the present invention can be used, for example, but not limited to, the prevention and / or treatment, testing, and diagnosis of various diseases such as cancer.

[0149] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.

[0150] Unless otherwise specified, the genetic manipulation procedures in the following examples were performed according to the methods described in "Molecular Cloning" (Sambrook, J., Fritsch, E.F., and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989) or other experimental manuals used by those skilled in the art, or, when commercially available reagents or kits were used, according to the instructions provided with the commercially available products. Primers necessary for constructing genes and vectors were synthesized by outsourcing (FASMAC Corporation and Thermo Fisher Scientific) as needed.

[0151] The following Reference Examples 1 to 14 are the same as Examples 1 to 14 described in WO2023 / 153442 (application number: PCT / JP2023 / 004196), and are also included in the disclosure of this specification.

[0152] Reference Example 1 Preparation of anti-TROP2 antibody HT1-11 and evaluation of antigen-binding activity 1)-1 Expression and purification of anti-TROP2 antibody HT1-11 Same as Example 1)-1 of WO2023 / 153442. The anti-TROP2 antibody HT1-11 (heavy chain amino acid sequence shown in SEQ ID NO: 1 or FIG. 16; light chain amino acid sequence shown in SEQ ID NO: 2 or FIG. 17) described in WO2015 / 098099 was prepared.

[0153] 1)-2 Evaluation of binding activity of anti-TROP2 antibody HT1-11 to anti-TROP2 antigen by ELISA The same as in Example 1)-2 of WO2023 / 153442. The antigen binding activity of HT1-11 was evaluated. As shown in Figure 2, HT1-11 bound to the human TROP2 antigen in a concentration-dependent manner.

[0154] Reference Example 2: Enrichment of mimotope peptides that bind to anti-TROP2 antibody HT1-11 The same as Example 2 of WO2023 / 153442. Mimotope peptides that bind to HT1-11 were enriched. As shown in Figure 3, when HT1-11 was immobilized, 2624-fold more mRNA was recovered than when human serum IgG was immobilized. This result suggested the enrichment of peptides that specifically bind to HT1-11.

[0155] (Reference Example 3) Screening for mimotope peptides that bind to anti-TROP2 antibodies 3)-1 Preparation of HT1-11 scFv fused with panning-derived peptides Same as WO2023 / 153442 Example 3)-1. HT1-11 scFv fused with panning-derived peptides was prepared. 3)-2 Acquisition of mimotope peptides Same as WO2023 / 153442 Example 3)-2. Binding activity to human TROP2 antigen was evaluated by ELISA in the same manner as in Reference Example 1)-2, and unique clones MHT1001 (SEQ ID NO: 3, Figure 18) and MHT1002 (SEQ ID NO: 4, Figure 19) were obtained. 3)-3 Evaluation of binding activity of positive clones Same as WO2023 / 153442 Example 3)-3. HT1-11-scFv-HL (SEQ ID NO: 5, FIG. 20), HT1-11-scFv-LH (SEQ ID NO: 6, FIG. 21), and the positive clones MHT1001 and MHT1002 identified in Reference Example 3)-2 were prepared, and their binding activity to the human TROP2 antigen was assessed by ELISA.

[0156] As shown in Figure 4-1(A) and Figure 4-1(B), HT1-11-scFv-HL and HT1-11-scFv-LH, which were not fused with a mimotope peptide, showed similar binding activity regardless of the addition of MMP1. Furthermore, MHT1001 and MHT1002, which were fused with a mimotope peptide, showed stronger binding activity in the presence of MMP1 than in the absence of MMP1, indicating that the EC ５０ The ratios (MMP1 non-addition condition / MMP1 addition condition) were 7.9 and 4.3, respectively (FIG. 4-2(C) and FIG. 4-2(D)).

[0157] (Reference Example 4) Preparation of anti-TROP2 masked antibody and evaluation of binding activity The same as Example 4 of WO2023 / 153442. The expression vectors for the anti-TROP2 masked antibody MHT1007 heavy chain (Table 2, SEQ ID NO: 7, FIG. 22) fused with the mimotope peptide of MHT1001 and an MMP cleavable linker (see Reference Example 3)-1), the MHT1008 heavy chain (Table 2, SEQ ID NO: 8, FIG. 23) in which the MMP cleavable linker was modified to a non-cleavable linker that cannot be cleaved by proteases, and the MHT1009 heavy chain (Table 2, SEQ ID NO: 9, FIG. 24) in which the uPA cleavable linker (amino acids 36 to 55 of the amino acid sequence shown in FIG. 24 and SEQ ID NO: 9) was modified) were combined with the light chain expression vector of HT1-11 to produce masked antibodies in the same manner as in Reference Example 1)-1, and named MHT1007, MHT1008, and MHT1009 (Table 2). The "pH-responsive linker" in the heavy chain sequence of each masked antibody in Table 2 corresponds to the second peptide of the target antigen-binding molecule of the present invention.

[0158]

[0159] The binding activity to human TROP2 antigen was evaluated by ELISA under protease-containing and protease-free conditions in the same manner as in Reference Examples 1)-2 and 3)-2, except for the following points. Under protease-containing conditions, MMP1 or active human uPA (Accession No.: P00749) was added to a final concentration of 300 nM to 3 μM antibody, and the mixture was incubated at 37°C. The antibody concentration was adjusted with ELISA buffer and then added to the wells where the human TROP2 antigen had been immobilized.

[0160] As shown in Figure 5-1 (A), HT1-11, which is not fused with a mimotope peptide, showed the same binding activity under the condition of MMP1 addition as under the condition of no addition. Furthermore, under the condition of no protease addition, the binding activity of MHT1007, which is fused with a mimotope peptide, was lower than that of HT1-11, indicating a masking effect similar to that of scFv. Furthermore, under the condition of MMP1 addition, it showed a stronger binding activity than under the condition of no MMP1 addition, indicating a higher EC ５０The ratio (MMP1 non-addition condition / MMP1 addition condition) was 104 (Fig. 5-1(B)). In MHT1008 equipped with a non-cleavable linker, the binding activity was reduced under MMP1 addition conditions as well as under non-addition conditions (Fig. 5-2(C)). In MHT1009 equipped with a uPA cleavable linker, the binding activity was stronger under uPA addition conditions than under uPA non-addition conditions, and the EC ５０ The ratio (uPA-free condition / uPA-added condition) was 83 (FIG. 5-2(D)). The above results demonstrated that the mimotope obtained in Reference Example 3 functions as a masking domain not only in the scFv state but also in the IgG state, and that the cleavable linker sequence can be modified while maintaining the masking effect.

[0161] To confirm that the obtained peptide was a mimotope, the Fab region of MHT1007 containing the masked peptide and an MMP-cleavable linker (see Reference Example 3-1) was prepared by a method known to those skilled in the art, and the Fab region was subjected to crystallization and X-ray crystal structure analysis. The results showed that the peptide bound to the CDR region of HT1-11 (data not shown). Furthermore, SPR confirmed that the chemically synthesized masked peptide bound to HT1-11 competitively with the TROP2 antigen (data not shown).

[0162] Reference Example 5: Design of a His Cluster-Equipped Linker and Evaluation of pH Responsiveness of Binding

[0063] The same as Example 5 of WO2023 / 153442. An anti-TROP2 masked antibody was designed with multiple His residues loaded within the linker, and its interaction with human TROP2 antigen was evaluated by ELISA in the same manner as in Reference Example 1)-2, except for the following: The antibody was diluted to 2000 nM with a neutral buffer (50 mM TRIS or 50 mM HEPES, 150 mM NaCL, 0.05% Tween-20, pH 7.5) or an acidic buffer (50 mM Bis-TRIS, 150 mM NaCL, 0.05% Tween-20, pH 6.5-5.5), incubated at 37°C, and then the concentration was adjusted with each buffer and added to wells containing immobilized human TROP2 antigen. Washing and dilution of horseradish peroxidase (HRP)-labeled anti-human IgG antibody (Jackson ImmunoResearch Laboratories) were also carried out under the same pH conditions.

[0163] As shown in Figure 6-1(A), MHT1008, which does not have a His residue, showed similar binding activity under neutral pH conditions (pH 7.5) and acidic pH conditions (pH 5.5). On the other hand, MHT1803 (Table 2, SEQ ID NO: 10, Figure 25), MHT1804 (Table 2, SEQ ID NO: 11, Figure 26), and MHT1805 (Table 2, SEQ ID NO: 12, Figure 27), which have one or two consecutive His residues (His clusters) in the linker, showed stronger binding under acidic pH conditions than under neutral pH conditions (Figures 6-1(B), 6-2(C), and 6-2(D)). The EC values ​​of binding for MHT1803, MHT1804, and MHT1805 were 0.01 and 0.02, respectively. ５０ The ratios (neutral pH condition / acidic pH condition) were 3.9, 3.6, and 2.3, respectively. MHT1806 (Table 2, SEQ ID NO: 13, FIG. 28), which has three His clusters, also showed strong binding activity under acidic pH conditions (FIG. 6-3(E)). ５０ The ratio (neutral pH condition / acidic pH condition) was 38.8.

[0164] To investigate the number of consecutive His residues forming a His cluster, the binding activity of MHT1808 (Table 2, SEQ ID NO: 14, Figure 29), MHT1809 (Table 2, SEQ ID NO: 15, Figure 30), and MHT1810 (Table 2, SEQ ID NO: 16, Figure 31) was evaluated. All clones showed stronger binding under acidic pH conditions than under neutral pH conditions (Figures 6-3(F), 6-4(G), and 6-4(H)), indicating that EC ５０ The ratios (neutral pH condition / acidic pH condition) were 24.4, 11.4, and 7.8, respectively.

[0165] The binding activity of MHT1811 (Table 2, SEQ ID NO: 17, FIG. 32) loaded with a known MMP substrate (Biopolymers.; 40(4):399-416(1996)) consisting of a His cluster and PLGLWA (SEQ ID NO: 40, FIG. 51) was evaluated. Even when a protease substrate was included, MHT1811 showed stronger binding under acidic pH conditions than under neutral pH conditions (FIG. 6-5(I)), and EC ５０ The ratio (neutral pH condition / acidic pH condition) was 28.1. In addition, strong binding activity was also observed under the condition of adding MMP1, and EC ５０ The ratio (MMP1 non-added condition / MMP1 added condition) was 48.4.

[0166] The acidic pH conditions under which binding activity is improved were analyzed. As shown in Figure 6-5(J), MHT1806 showed improved binding activity under acidic pH conditions of 6.5 or less compared to neutral pH conditions (pH 7.5). Furthermore, MHT1808 showed improved binding activity under acidic pH conditions of 6.0 or less compared to neutral pH conditions (pH 7.5) (Figure 6-6(K)).

[0167] To verify whether protonation of His residues contributes to the pH-dependent improvement in binding activity, we designed and prepared MHT1817 (Table 2, SEQ ID NO: 18, Figure 33), MHT1818 (Table 2, SEQ ID NO: 19, Figure 34), and MHT1819 (Table 2, SEQ ID NO: 20, Figure 35), each of which has a negatively charged Asp residue located within the His cluster. Each antibody was diluted to 20 nM in buffers of pH 7.5, pH 6.5, or pH 6.0, and incubated at 37°C for 30 minutes. The binding activity to the human TROP2 antigen was assessed by ELISA. While the binding activity of MHT1808 improved in a pH-dependent manner, the binding activity of MHT1817, MHT1818, and MHT1819 was not as improved as that of MHT1808 at either pH 6.5 or pH 6.0 (Figure 7). Furthermore, the change in binding activity with pH change became smaller as the number of Asp residues in the His cluster increased. This result indicates that it is preferable that the pH-responsive linker does not contain Asp residues.

[0168] Reference Example 6 Evaluation of Binding Activity of Anti-CD98 Antibody, Anti-EGFR Antibody, and Anti-GPRC5D Antibody 6)-1 Evaluation of Binding Activity of Anti-CD98 Antibody hM23H1L1 Same as Example 6)-1 of WO2023 / 153442. The binding activity of the known anti-CD98 antibody hM23H1L1 (heavy chain sequence (Table 3, SEQ ID NO: 21, Figure 36) and light chain sequence (Table 3, SEQ ID NO: 22, Figure 37)) described in WO2015 / 146132 to human CD98 antigen was evaluated by ELISA. As shown in Figure 8-1(A), hM23H1L1 bound to human CD98 antigen in a concentration-dependent manner.

[0169] 6)-2 Preparation and binding activity evaluation of anti-EGFR antibody Cetuximab The same as Example 6)-2 of WO2023 / 153442. The binding activity of the anti-EGFR antibody Cetuximab (Table 3, heavy chain sequence (SEQ ID NO: 23, Figure 38), light chain sequence (SEQ ID NO: 24, Figure 39)) to human EGFR was evaluated by ELISA. As shown in Figure 8-1(B), Cetuximab bound to the human EGFR antigen in a concentration-dependent manner.

[0170] 6)-3 Preparation of anti-GPRC5D antibodies and evaluation of binding activity The same as Example 6)-3 of WO2023 / 153442. The binding activity of the known anti-GPRC5D antibody C3022 (Table 3, heavy chain sequence (SEQ ID NO: 25, Figure 40), light chain sequence (SEQ ID NO: 26, Figure 41)) described in WO2018 / 147245 to human GPRC5D antigen was evaluated by ELISA. As shown in Figure 8-2(C), C3022 bound to human GPRC5D antigen in a concentration-dependent manner. The "pH-responsive linker" in the heavy chain sequence or light chain sequence of each masked antibody in Table 3 corresponds to the second peptide of the target antigen-binding molecule of the present invention.

[0171]

[0172] Reference Example 7: Enrichment of mimotope peptides against hM23H1L1, cetuximab, and C3022 7)-1 Enrichment of mimotope peptides binding to hM23H1L1 The same as Example 7)-1 of WO2023 / 153442. Mimotope peptides binding to hM23H1L1 were enriched. As shown in Figure 9-1(B), for both the Linear 15mer lib and ZPZP lib peptide libraries, when hM23H1L1 was immobilized, approximately 200-fold more mRNA was recovered than when human serum IgG was immobilized. This result suggests the enrichment of peptides that specifically bind to hM23H1L1.

[0173] 7)-2 Concentration of mimotope peptides for cetuximab The same as in Example 7)-2 of WO2023 / 153442. Mimotope peptides for cetuximab were concentrated. As shown in Figure 9-2(C), under the condition where cetuximab was immobilized, 276-fold more mRNA was recovered than under the condition where human serum IgG was immobilized. This result demonstrated the enrichment of peptides that specifically bind to cetuximab.

[0174] 7)-3 Enrichment of mimotope peptides for C3022 The same as in Example 7)-3 of WO2023 / 153442. Mimotope peptides for C3022 were enriched. As shown in Figure 9-2(D), when C3022 was immobilized, 503-fold more mRNA was recovered than when human serum IgG was immobilized. This result demonstrated the enrichment of peptides that specifically bind to C3022.

[0175] Reference Example 8: Versatility of application of pH-responsive linkers. The same as in Example 8 of WO 2023 / 153442. Mimotope peptides capable of inhibiting the binding of the anti-CD98 antibody, the anti-GPRC5D antibody, or Cetuximab to their target antigens were selected, and a pH-responsive linker equipped with three His clusters was used to verify whether the masked antibodies were universally activated under acidic pH conditions. The selected mimotopes were used to design the anti-CD98 masked antibody MhM8001 (Table 3, SEQ ID NO: 27, Figure 42), the anti-EGFR masked antibody MCE-2101 (Table 3, SEQ ID NO: 28, Figure 43), and the anti-GPRC5D masked antibody MC3-9001 (Table 3, SEQ ID NO: 29, Figure 44). Masked antibodies were prepared in the same manner as in Reference Example 1)-1, and their binding activity under neutral and acidic pH conditions was evaluated by ELISA in the same manner as in Reference Examples 5 and 6.

[0176] MhM8001, MCE-2101, and MC3-9001 all showed stronger binding under acidic pH conditions (pH 5.5) than under neutral pH conditions (pH 7.5), and EC ５０ The ratios (neutral pH condition / acidic pH condition) were 13.1, 39.0, and 40.4, respectively (Figures 10-1(A), 10-1(B), and 10-2(C)). These results suggest that masked antibodies carrying His clusters are generally activated under acidic pH conditions.

[0177] Reference Example 9 Preparation and Binding Activity Evaluation of Anti-TROP2 Masked Antibodies to be Converted into ADCs The same as Example 9 of WO2023 / 153442. Aiming to verify whether the incorporation of a His cluster into the linker moiety improves the cytocidal activity of masked antibodies in a pH-decreasing dependent manner, the following anti-TROP2 masked antibodies were designed: MHT1808, which is equipped with a His cluster; MHT1221 (Table 2, SEQ ID NO: 30, FIG. 45), which is not equipped with a His cluster; and MHT1008. Each antibody was prepared in the same manner as in Reference Example 1)-1, and its binding activity to human TROP2 antigen under neutral pH conditions (pH 7.5) and acidic pH conditions (pH 5.5) was evaluated by ELISA in the same manner as in Reference Example 5.

[0178] As shown in Figure 11, MHT1808 exhibited stronger binding under acidic pH conditions than under neutral pH conditions, and EC ５０ The ratio (neutral pH condition / acidic pH condition) was 25.4. On the other hand, MHT1221 and MHT1008 showed equivalent binding activity under both pH conditions, and the EC ５０ The ratios (neutral pH condition / acidic pH condition) were 1.7 and 1.2, respectively.

[0179] Reference Example 10: Preparation of antibody-drug conjugate The same as Example 10 of WO2023 / 153442. The drug linker for preparing the antibody-drug conjugate was N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide (Example 58, Step 8). WO 2014 / 057687, hereinafter referred to as "Linker 1") and N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo {so-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide (Example 2-1, WO 2020 / 196474, hereinafter referred to as "Linker 2") was used. See Example 10 of WO 2023 / 153442 for common procedures and Table 4 used in the preparation and identification of antibody-drug conjugates.

[0180] 10)-1 Synthesis of MhM1018-M1-DXd-ADC

[0181]

[0182] By the method described in WO2023 / 153442 Example 10 Step 1: Antibody-drug conjugate (1), an antibody-drug conjugate "MhM1018-M1-DXd-ADC" represented by Chemical Formula 1 was obtained. Chemical Formulas 1 to 8 in WO2023 / 153442 correspond to Chemical Formulas 1 to 8 of the present invention.

[0183] 10)-2 Synthesis of MhM1024-M1-DXd-ADC

[0184]

[0185] The antibody-drug conjugate "MhM1024-M1-DXd-ADC" represented by Chemical Formula 2 was obtained by the method described in WO2023 / 153442 Example 10 Step 1: Antibody-drug conjugate (2).

[0186] 10)-3 Synthesis of MHT1221-PBD-ADC

[0187]

[0188]

[0189] WO2023 / 153442 Example 10 "MHT1221-PBD-ADC" was obtained by the method described in Step 1: Preparation of (Fucα1,6)GlcNAc-MHT1221 antibody, Step 2: Preparation of glycochain-modified MHT1221 antibody, and Step 3: Conjugation of antibody and drug linker.

[0190] 10)-4 Synthesis of MHT1008-PBD-ADC

[0191]

[0192]

[0193] WO2023 / 153442 Example 10 "MHT1008-PBD-ADC" was obtained by the method described in Step 1: Preparation of (Fucα1,6)GlcNAc-MHT1008 antibody, Step 2: Preparation of glycochain-modified MHT1008 antibody, and Step 3: Conjugation of antibody and drug linker.

[0194] 10)-5 Synthesis of MHT1808-PBD-ADC

[0195]

[0196]

[0197] WO2023 / 153442 Example 10 "MHT1808-PBD-ADC" was obtained by the method described in Step 1: Preparation of (Fuca1,6)GlcNAc-MHT1808 antibody, Step 2: Preparation of glycochain-modified MHT1808 antibody, and Step 3: Conjugation of antibody and drug linker.

[0198] (Reference Example 11) Masking effect of anti-CD98 antibodies with different affinities 11)-1 Evaluation of binding affinity of anti-CD98 antibodies Same as Example 11)-1 of WO2023 / 153442. The binding affinity (K) of anti-CD98 antibodies (hM23H1L1 or hM23-M1 with point mutations introduced into the H chain CDR1 (Table 3, heavy chain sequence (SEQ ID NO: 31, Figure 46), light chain sequence (SEQ ID NO: 32, Figure 47)) to the CD98 antigen was evaluated. Ｄ As shown in Figure 13(A), the binding affinity (K Ｄ ) were 0.58 nM and 10.3 nM, respectively. 11)-2 Evaluation of the masking effect of anti-CD98 masked antibodies Same as Example 11)-2 of WO2023 / 153442. Anti-CD98 masked antibodies MhM1013 (Table 3) and MhM1013-M1 (Table 3), which have the heavy chain of hM23-M1 and the light chain of MhM1013, were prepared, and their binding activity to human CD98 antigen was evaluated by ELISA. As shown in Figure 13(B), MhM1013 and MhM1013-M1 fused with mimotope peptides showed stronger binding activity in the presence of MMP1 than in the absence of MMP1. EC ５０ The ratios (MMP1 non-added condition / MMP1 added condition) were 239 and 552, respectively.

[0199] (Reference Example 12) Preparation of anti-CD98 masked antibodies to be converted into ADCs and evaluation of binding activity The same as Example 12 of WO2023 / 153442. Anti-CD98 masked antibodies MhM1018-M1 (Table 3, SEQ ID NO: 34, Figure 49) not carrying a His cluster and MhM1024-M1 (Table 3, SEQ ID NO: 35, Figure 50) carrying a His cluster were designed and prepared, and their binding activity to human CD98 antigen was evaluated by ELISA.

[0200] As shown in Figure 14, MhM1018-M1 exhibited equivalent binding activity under both pH conditions, and EC ５０ The ratio (neutral pH condition / acidic pH condition) was 1.7. On the other hand, MhM1024-M1 showed stronger binding under acidic pH conditions than under neutral pH conditions, and EC ５０ The ratio (neutral pH condition / acidic pH condition) was 19.2.

[0201] (Reference Example 13) Evaluation of proliferation inhibitory activity of ADC The same as Example 13 of WO2023 / 153442. 13)-1 Cytotoxicity of anti-TROP2 masked PBD-ADC As shown in Figure 15-1 (A), MHT1808-PBD-ADC carrying a His cluster exhibited strong proliferation inhibitory activity in a pH-decreasing manner, and inhibited EC ５０ The ratio (pH 7.4 / pH 5.5) was 10.7. Furthermore, MHT1221-PBD-ADC without a His cluster exhibited comparable growth inhibitory activity under both conditions, and ５０ The ratio (pH 7.4 / pH 5.5) was 1.9 (Figure 15-1(B)). 13)-2 Cytotoxicity of anti-CD98 masked DXd-ADC As shown in Figure 15-2(C), MhM1024-M1-DXd-ADC carrying a His cluster exhibited strong growth inhibitory activity in a pH-dependent manner, and inhibited EC ５０ The ratio (pH 7.4 / pH 5.5) was 10.9. MhM1018-M1-DXd-ADC, which does not have a His cluster, showed comparable growth inhibitory activity under both conditions, and ５０ The ratio (pH 7.4 / pH 5.5) was 2.8 (FIG. 15-2(D)).

[0202] Reference Example 14: Improvement of pH responsiveness The same as Example 14 of WO2023 / 153442. For the purpose of improving pH responsiveness, the following anti-CD98 masked antibodies were designed in which a positively charged amino acid (Arg or Lys) was positioned near the His cluster: MhM1026-M1 (Table 4, SEQ ID NO: 68, Figure 57), MhM1028-M1 (Table 4, SEQ ID NO: 69, Figure 58), MhM1042-M1 (Table 4, SEQ ID NO: 70, Figure 59), MhM1043-M1 (Table 4, SEQ ID NO: 71, Figure 60), MhM1044-M1 (Table 4, SEQ ID NO: 72, Figure 61), MhM1045-M1 (Table 4, SEQ ID NO: 73, Figure 62), and MhM1046-M1 (Table 4, SEQ ID NO: 74, Figure 63). Masked antibodies were prepared in the same manner as in 1)-1 of Reference Example 1, and their binding activity under neutral and acidic pH conditions was evaluated by ELISA in the same manner as in Reference Examples 5 and 6.

[0203] As shown in Figure 56-1(A), at pH 7.5, MhM1026-M1, MhM1028-M1, MhM1042-M1, and MhM1043-M1 showed similar binding activity. On the other hand, at pH 5.5, MhM1028-M1, MhM1042-M1, and MhM1043-M1 showed stronger binding activity than MhM1026-M1 (Figure 56-1(B)). The EC ５０ A comparison of the ratio (neutral pH condition / acidic pH condition) showed that placing Arg near the His cluster improved the pH responsiveness by 4.0-5.2 times (Table 4).

[0204] Similarly, the binding activity of MhM1044-M1, MhM1045-M1, and MhM1046-M1, which have Lys residues located near the His cluster, was compared with that of MhM1026-M1. As shown in Figures 56-2 (C) and (D), all clones showed similar binding activity regardless of pH conditions. The EC ５０ Comparison of the ratios revealed no change between clones (Table 4).

[0205]

[0206] Example 1 Antibody Purification Using a pH-Responsive Linker Using the anti-TROP2 masked antibodies MHT1808 and MHT1007 prepared by the method described in Reference Example 1 and Dynabeads His-Tag Isolation (ThermoFisher Scientific), it was examined whether antibody purification using a pH-responsive linker was possible.

[0207] First, 20 μL (800 μg) of Dynabeads His-Tag Isolation was washed with binding buffer (20 mM Tris, 300 mM NaCl, pH 8.0) using a magnetic stand, and the supernatant was removed. Next, using a Zeba spin desalting column (ThermoFisher Scientific), the antibody (MHT1808 or MHT1007) was replaced with binding buffer and adjusted to a concentration of 1 mg / mL. After collecting 2 μL of antibody-containing sample as a pre-reaction fraction, 20 μL of the antibody-containing sample was added to the beads and allowed to react at room temperature for 15 minutes. The beads were collected using a magnetic stand, and 2 μL of the supernatant was collected as the unbound fraction. After washing twice with 100 μL of binding buffer, 20 μL of elution buffer (20 mM Tris, 300 mM NaCl, 500 mM imidazole, pH 8.0) was added and reacted at room temperature for 10 minutes. After that, the beads were recovered using a magnetic stand, and 2 μL of the supernatant was collected as the bound fraction.

[0208] For SDS-PAGE under reducing conditions, 2 μL of the collected sample was mixed with an equal volume of a reducing agent-containing sample buffer (0.125 M Tris-HCl (pH 6.8), 4 (w / v)% SDS, 20 (w / v)% glycerol, 0.01 (w / v)% Bromophenol Blue, 10 (v / v)% 3-mercapto-1,2-propandiol: FUJIFILM). For SDS-PAGE under non-reducing conditions, 2 μL of the collected sample was mixed with one-fifth the volume of a reducing agent-free sample buffer (Sample Buffer Solution without Reducing Reagent (6x) for SDS-PAGE pH 6.8). The gel was mixed with 0.45 μm filtered 0.375 M Tris-HCl (pH 6.8), 0.03 (w / v) % BPB, containing glycerin, and anionic surfactant (Nacalai tesque), then heat denatured and analyzed by SDS-PAGE using a method known to those skilled in the art. After electrophoresis, the gel was stained with GelCode Blue Safe Protein Stain (ThermoFisher Scientific). After destaining, the band intensity of the antibody heavy chain was quantified using ImageJ (version 1.53) using a method known to those skilled in the art, and the interaction between the antibody and Dynabeads His-Tag Isolation was analyzed.

[0209] As shown in Figures 65(A) and (B), the heavy chain band of MHT1808 equipped with a pH-responsive linker was observed in the pre-reaction fraction and the bound fraction, and the band intensity of the bound fraction was approximately 80% of the band intensity of the pre-reaction fraction. In contrast, the heavy chain band of MHT1007 equipped with no pH-responsive linker was observed in the pre-reaction fraction, unbound fraction, and bound fraction, and the band intensity of the bound fraction was approximately 20% of the band intensity of the pre-reaction fraction. These results demonstrate that the pH-responsive linker strongly interacts with Dynabeads His-Tag Isolation.

[0210] Similarly, the interaction of Dynabeads His-Tag Isolation with anti-TROP2 masked antibodies MHT1809-MHT1811 and MHT1803-MHT1806, which have different cluster sequences of histidine residues (His clusters), was analyzed. As a result, as shown in Figures 66(A) and (B), antibody heavy chain bands were confirmed in the pre-reaction fraction and the bound fraction for all clones. These results demonstrate that a wide variety of pH-responsive linkers interact with Dynabeads His-Tag Isolation, and that antibodies equipped with a wide variety of pH-responsive linkers can be purified by immobilized metal affinity chromatography (hereinafter referred to as "IMAC") purification.

[0211] (Example 2) Purification of various antibodies using a pH-responsive linker To verify the possibility of purifying various antibodies using a pH-responsive linker, an experiment similar to that in Example 1 was carried out using the anti-CD98 masked antibodies MhM1018-M1 and MhM1024-M1 and the anti-GPRC5D masked antibodies MC3-1901 (heavy chain amino acid sequence: SEQ ID NO: 85 (Figure 77), light chain amino acid sequence: SEQ ID NO: 26 (Figure 41)) and MC3-9001.

[0212] As shown in Figure 67, similar to the case of the anti-TROP2 masked antibody, antibody heavy chain bands were confirmed in the pre-reaction fraction and the bound fraction for MhM1024-M1 and MC3-9001 equipped with a pH-responsive linker, while antibody heavy chain bands were confirmed in the pre-reaction fraction and the unbound fraction for MhM1018-M1 and MC3-1901, which do not equipped with a pH-responsive linker. These results demonstrate that antibodies equipped with a pH-responsive linker generally interact with Dynabeads His-Tag Isolation, i.e., can be purified by IMAC.

[0213] Example 3 Expression and Purification of Fc Fusion Antibodies Having Different Antigen-Binding Sites The three types of plasmids (heavy chain 1, heavy chain 2, light chain) shown in Table 5 were mixed at a weight ratio of 1:1:2, and four types of antibodies, MHT1808_Fc, MHT1008_Fc, MhM1024-M1_HT1-11 scFvFc, and MhM1025-M1_HT1-11_scFvFc, were prepared according to the method described in Reference Example 1, and subjected to Examples 4 and 5. As shown in Figures 68 and 71, the expressed and purified Molecules 1 to 3 include heavy chain 1-heavy chain 1 homodimers, heavy chain 2-heavy chain 2 homodimers, and heavy chain 1-heavy chain 2 heterodimers. In these dimers containing heavy chain 2, heavy chain 2 interacts with the light chain to form a Fab region.

[0214]

[0215] Example 4 Purification of Antibodies with Monovalent Antigen-Binding Sites Under the conditions in which MHT1808_Fc was expressed and purified, three types of Fc-containing antibodies (Fc-containing molecules) shown in Figure 68 were present, with the number of antigen-binding sites and pH-responsive linkers differing among the three molecules. We examined whether antibodies with monovalent antigen-binding sites could be purified by utilizing the interaction between the pH-responsive linker and the IMAC purification resin. At the same time, an MHT1008_Fc-expressing sample without a pH-responsive linker was also evaluated in the same manner.

[0216] First, an antibody-containing sample was prepared at 1 mg / mL with Buffer A1 (20 mM Tris, 300 mM NaCl, 5 mM imidazole, pH 8.0), and 1 mg of the sample was loaded onto a 1 mL HisTrap HP column (Cytiva) using an AKTA pure 25 system (Cytiva). To purify MHT1808_Fc, the antibody was eluted by mixing with Buffer A1 under a linear gradient condition, with Buffer B1 (20 mM Tris, 300 mM NaCl, 500 mM imidazole, pH 8.0) concentration varying from 10% to 70% over 30 column volumes. When purifying MHT1008_Fc, the antibody was eluted by mixing with buffer A1 under linear gradient conditions so that the buffer B1 concentration was changed from 0% to 100% over 20 column volumes.

[0217] As a result, under the conditions for purifying MHT1808_Fc, three peaks, E1, E2, and E3, were observed as the concentration of buffer B1 in the mobile phase increased ( Figure 69(A) ). In contrast, under the conditions for purifying MHT1008_Fc, only one elution peak, E1, was observed ( Figure 69(B) ).

[0218] To identify the molecules in each elution peak, SDS-PAGE analysis was performed under reducing and non-reducing conditions using 2 μg of each elution sample, using techniques known to those skilled in the art. Under the conditions used to purify MHT1808_Fc, the results under non-reducing conditions showed that small single molecules, E1, E2, and E3, were eluted in this order (Figure 70(A)). Furthermore, the results under reducing conditions showed that the number of chains constituting E1, E2, and E3 was one, three, and two, respectively (Figure 70(B)). These results indicated that E1 was an Fc homodimer, E2 was a heterodimer of MHT1808 and Fc, and E3 was an MHT1808 homodimer.

[0219] The IMAC elution fraction of MHT1008_Fc was also analyzed by SDS-PAGE. The results confirmed the presence of three molecules with different molecular weights (Figures 70(A) and (B)). Comparison with MHT1808_Fc identified the three molecules, in order of molecular weight, as an Fc homodimer, an MHT1008-Fc heterodimer, and an MHT1008 homodimer.

[0220] This study demonstrated that IMAC purification of antibodies using pH-responsive linkers is possible, that the IMAC retention time varies depending on the number of pH-responsive linkers contained in the antibody, and that the difference in retention time can be used to purify antibodies according to the valency of the antigen-binding site.

[0221] Example 5 Purification of Bispecific Antibodies Carrying a pH-Responsive Linker Under the conditions under which MhM1024-M1_HT1-11 scFvFc was expressed and purified, three types of Fc fusion antibodies (Fc fusion molecules: Molecules 1 to 3) shown in Figure 71 were present, with the number of antigen-binding site 1, antigen-binding site 2, and pH-responsive linker differing among the three molecules. To verify whether a pH-responsive linker could be used to purify bispecific antibodies having different antigen-binding sites, MhM1024-M1_HT1-11 scFvFc and MhM1025-M1_HT1-11 scFvFc expressed and purified in Example 3 were subjected to IMAC purification by the method described in Example 4.

[0222] As a result, in IMAC purification of MhM1024-M1_HT1-11 scFvFc and MhM1025-M1_HT1-11 scFvFc, three peaks, E1, E2, and E3, were confirmed as the concentration of buffer B1 increased (FIG. 72 (A, B)).

[0223] To identify the molecules in each elution peak, SDS-PAGE analysis was performed under reducing and non-reducing conditions using 1 μg of each elution sample by a method known to those skilled in the art. When MhM1024-M1_HT1-11 scFvFc and MhM1025-M1_HT1-11 scFvFc were purified under non-reducing conditions, the results showed that small molecular weight molecules, E1, E2, and E3, were the main elutes (Figure 73(A)). The results under reducing conditions showed that the number of chains constituting E1, E2, and E3 was one, three, and two, respectively (Figure 73(B)). From the above results, E1 was identified as an HT1-11 scFvFc homodimer, E2 as a heterodimer of MhM1024-M1 or MhM1025-M1 and HT1-11 scFvFc, and E3 as a homodimer of MhM1024-M1 or MhM1025-M1.

[0224] This study demonstrated that by utilizing the fact that the IMAC retention time changes depending on the number of pH-responsive linkers, it is possible to separate bivalent antibodies with different target antigen-binding moieties, i.e., bispecific antibodies such as Molecule 2 in Figure 71, from monospecific bivalent antibodies such as Molecules 1 and 3.

[0225] (Example 6) Binding activity of Fc variants to Protein A A method for purifying a hetero-Fc dimer in which amino acids derived from human IgG3 (R435 or R435, F436) were grafted onto human IgG1 in order to improve the purification purity has been reported (Non-Patent Document 5).

[0226] Four types of anti-CD98 antibodies (hM23-M1_His, hM23-M1_I253D_His, hM23-M1_I253N_His, hM23-M1_H435R_His) each having a His-Tag attached to the C-terminus of the heavy chain and a different Fc sequence were expressed in the same manner as in Reference Example 1 and purified using Ni Sepharose Excel (ThermoFisher Scientific) by methods known to those skilled in the art. The amino acid sequences of each antibody are shown in Table 6.

[0227] The binding activity of purified antibodies to Protein A was evaluated by SPR. Antibody samples diluted to 10 μg / mL with HBS-EP+ buffer (Cytiva) were reacted on a Series S Sensor Chip Protein A (Cytiva) chip at a flow rate of 20 μL / min for 90 seconds, and the binding level was calculated. Between measurements, regeneration buffer (10 mM Gly-HCl, pH 1.5) was reacted with the chip to dissociate the antibody bound to Protein A from the chip.

[0228] As shown in Figure 78, hM23-M1_I253D_His, hM23-M1_I253N_His, and hM23-M1_H435R_His, in which mutations were introduced into the Fc, all showed significantly reduced binding compared to hM23-M1_His, which has a wild-type Fc sequence. This indicates that the I253D, I253N, and H435R mutations reduce the binding activity of the antibody to Protein A.

[0229]

[0230] (Example 7) Purification of antibodies with monovalent antigen-binding sites using Fc mutations Three types of plasmids (heavy chain 1: Fc (SEQ ID NO: 82, Figure 74), heavy chain 2: hM23-M1_I253N (SEQ ID NO: 90, Figure 85), light chain: MhM1024 (SEQ ID NO: 35, Figure 50)) were mixed at a weight ratio of 1:2:4, and MhM1024-M1_I253N_Fc was expressed according to the method described in Reference Example 1. Antibodies with monovalent antigen-binding sites were purified using two types of resins (Protein A resin or IMAC resin). The I253N mutation evaluated in Example 6 was introduced into the Fc of heavy chain 2.

[0231] 45 mL of the culture supernatant was loaded onto a 5 mL Hitrap MabSelect SuRe (Cytiva) column using an AKTA pure 25 system, washed with 10 column volumes of PBS, and then eluted with 6 column volumes of 0.1 M acetate buffer (pH 3.5). The collected peak was designated PI_1, as shown in Figure 79(A).

[0232] Next, PI_1 was diluted 5-fold with Buffer A (20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0) and loaded onto a 5 mL Histrap Excel (Cytiva) column using an AKTA pure 25 system. After washing with 10 column volumes of Buffer A, the antibody was eluted with 10 column volumes of Buffer B (20 mM Tris, 300 mM NaCl, 300 mM imidazole, pH 8.0). As shown in Figure 79(B), the collected peaks were designated PI_2 and PI_3.

[0233] Antibodies were purified from culture supernatants by changing the resin order. 42 mL of culture supernatant was loaded onto a 5 mL Histrap Excel column using an AKTA pure 25 system. After washing with 10 column volumes of buffer A, the antibody was eluted by mixing with buffer A under linear gradient conditions, with the buffer B concentration ranging from 0% to 100% over 10 column volumes. The collected peak was designated IP_1, as shown in Figure 79(C).

[0234] Next, IP_1 was loaded onto a 5 mL Hitrap MabSelect SuRe column using an AKTA pure 25 system. After washing with 10 column volumes of PBS, the antibody was eluted with 6 column volumes of 0.1 M acetate buffer (pH 3.5). As shown in Figure 79(D), the collected peaks were designated IP_2 and IP_3. The culture supernatant and the collected peaks during purification were analyzed by SDS-PAGE under non-reducing conditions (Figure 80). Each sample was loaded at 2 μg. Comparison of the culture supernatant with PI_1 or IP_1 with IP_2 demonstrated that the I253N-containing heavy chain 2 dimer did not bind to Protein A resin. Furthermore, the results of PI_3 and IP_3 showed that by combining a ProA affinity-reducing mutation with a pH-responsive linker, only molecules containing a pH-responsive linker bind to the IMAC resin, regardless of whether they are contacted with the Protein A resin or the IMAC resin first, and that only molecules containing an Fc region that does not contain a ProA affinity-reducing mutation bind to the Protein A resin. This demonstrates that antibodies with monovalent antigen-binding sites can be purified to a high degree of purity.

[0235] The present invention makes it possible to produce proteins using a new method.

[0236] SEQ ID NO: 1: heavy chain amino acid sequence of anti-TROP2 antibody (Figure 16) SEQ ID NO: 2: light chain amino acid sequence of anti-TROP2 antibody (Figure 17) SEQ ID NO: 3: amino acid sequence of MHT1001 (Figure 18) SEQ ID NO: 4: amino acid sequence of MHT1002 (Figure 19) SEQ ID NO: 5: amino acid sequence of HT1-11-scFv-HL (Figure 20) SEQ ID NO: 6: amino acid sequence of HT1-11-scFv-LH (Figure 21) SEQ ID NO: 7: heavy chain amino acid sequence of MHT1007 (Figure 22) SEQ ID NO: 8: heavy chain amino acid sequence of MHT1008 (Figure 23) SEQ ID NO: 9: heavy chain amino acid sequence of MHT1009 (Figure 24) SEQ ID NO: 10: heavy chain amino acid sequence of MHT1803 (Figure 25) SEQ ID NO: 11: heavy chain amino acid sequence of MHT1804 (Figure 26) SEQ ID NO: 12: Heavy chain amino acid sequence of MHT1805 (Figure 27) SEQ ID NO: 13: Heavy chain amino acid sequence of MHT1806 (Figure 28) SEQ ID NO: 14: Heavy chain amino acid sequence of MHT1808 (Figure 29) SEQ ID NO: 15: Heavy chain amino acid sequence of MHT1809 (Figure 30) SEQ ID NO: 16: Heavy chain amino acid sequence of MHT1810 (Figure 31) SEQ ID NO: 17: Heavy chain amino acid sequence of MHT1811 (Figure 32) SEQ ID NO: 18: Heavy chain amino acid sequence of MHT1817 (Figure 33) SEQ ID NO: 19: Heavy chain amino acid sequence of MHT1818 (Figure 34) SEQ ID NO: 20: Heavy chain amino acid sequence of MHT1819 (Figure 35) SEQ ID NO: 21: Heavy chain amino acid sequence of anti-CD98 antibody hM23H1L1 (Figure 36) SEQ ID NO: 22: Light chain amino acid sequence of anti-CD98 antibody hM23H1L1 (Figure 37) SEQ ID NO: 23: Heavy chain amino acid sequence of anti-EGFR antibody cetuximab (Figure 38) SEQ ID NO: 24: Light chain amino acid sequence of anti-EGFR antibody cetuximab (Figure 39) SEQ ID NO: 25: Heavy chain amino acid sequence of anti-GPRC5D antibody C3022 (Figure 40) SEQ ID NO: 26: Light chain amino acid sequence of anti-GPRC5D antibody C3022 (Figure 41) SEQ ID NO: 27: Light chain amino acid sequence of MhM8001 (Figure 42) SEQ ID NO: 28: Light chain amino acid sequence of MCE-2101 (Figure 43) SEQ ID NO: 29: Heavy chain amino acid sequence of MC3-9001 (Figure 44) SEQ ID NO: 30: Heavy chain amino acid sequence of MHT1221 (Figure 45) SEQ ID NO: 31: heavy chain amino acid sequence of hM23-M1 (Figure 46) SEQ ID NO: 32: light chain amino acid sequence of hM23-M1 (Figure 47)SEQ ID NO: 33: Light chain amino acid sequence of MhM1013 (Figure 48) SEQ ID NO: 34: Light chain amino acid sequence of MhM1018-M1 (Figure 49) SEQ ID NO: 35: Light chain amino acid sequence of MhM1024-M1 (Figure 50) SEQ ID NO: 36: Amino acid sequence 1 recognized by human uPA and serving as a substrate therefor (Figure 51) SEQ ID NO: 37: Amino acid sequence 2 recognized by human uPA and serving as a substrate therefor (Figure 51) SEQ ID NO: 38: Amino acid sequence 3 recognized by human uPA and serving as a substrate therefor (Figure 51) SEQ ID NO: 39: Amino acid sequence 1 recognized by human MMP1 and serving as a substrate therefor (Figure 51) SEQ ID NO: 40: Amino acid sequence 2 recognized by human MMP1 and serving as a substrate therefor (Figure 51) SEQ ID NO: 41: Amino acid sequence recognized by human MMP9 and serving as a substrate therefor (Figure 51) SEQ ID NO: 42: Amino acid sequence 1 of the second peptide (Figure 52) SEQ ID NO: 43: Amino acid sequence 2 of the second peptide (Figure 52) SEQ ID NO: 44: Amino acid sequence 3 of the second peptide (Figure 52) SEQ ID NO: 45: Amino acid sequence of the amino terminal peptide of human GPRC5D (Figure 53) SEQ ID NO: 46: Amino acid sequence 4 of the second peptide (Figure 54) SEQ ID NO: 47: Amino acid sequence 5 of the second peptide (Figure 54) SEQ ID NO: 48: Amino acid sequence 6 of the second peptide (Figure 54) SEQ ID NO: 49: Amino acid sequence 7 of the second peptide (Figure 54) SEQ ID NO: 50: Amino acid sequence 8 of the second peptide (Figure 54) SEQ ID NO: 51: Amino acid sequence 9 of the second peptide (Figure 54) SEQ ID NO: 52: Amino acid sequence 10 of the second peptide (Figure 54) SEQ ID NO: 53: Amino acid sequence 11 of the second peptide (Figure 54) SEQ ID NO: 54: Amino acid sequence 12 of the second peptide (Figure 54) SEQ ID NO: 55: Amino acid sequence 13 of the second peptide (Figure 54) SEQ ID NO: 56: Amino acid sequence 14 of the second peptide (Figure 54) SEQ ID NO: 57: Amino acid sequence 15 of the second peptide (Figure 54) SEQ ID NO: 58: Amino acid sequence 16 of the second peptide (Figure 54) SEQ ID NO: 59: Amino acid sequence 17 of the second peptide (Figure 54) SEQ ID NO: 60: Amino acid sequence 18 of the second peptide (Figure 54) SEQ ID NO: 61: Amino acid sequence 19 of the second peptide (Figure 54) SEQ ID NO: 62: Amino acid sequence 20 of the second peptide (Figure 54) SEQ ID NO: 63: Amino acid sequence 21 of the second peptide (Figure 54) SEQ ID NO: 64: Amino acid sequence 22 of the second peptide (Figure 54) SEQ ID NO: 65: Amino acid sequence 23 of the second peptide (Figure 54)SEQ ID NO: 66: Amino acid sequence 24 of the second peptide (Figure 54) SEQ ID NO: 67: Amino acid sequence recognized by human CAPN1 and serving as a substrate thereof (Figure 55) SEQ ID NO: 68: Light chain amino acid sequence of MhM1026-M1 (Figure 57) SEQ ID NO: 69: Light chain amino acid sequence of MhM1028-M1 (Figure 58) SEQ ID NO: 70: Light chain amino acid sequence of MhM1042-M1 (Figure 59) SEQ ID NO: 71: Light chain amino acid sequence of MhM1043-M1 (Figure 60) SEQ ID NO: 72: Light chain amino acid sequence of MhM1044-M1 (Figure 61) SEQ ID NO: 73: Light chain amino acid sequence of MhM1045-M1 (Figure 62) SEQ ID NO: 74: Light chain amino acid sequence of MhM1046-M1 (Figure 63) SEQ ID NO: 75: Amino acid sequence 25 of the second peptide (Figure 64) SEQ ID NO: 76: Amino acid sequence 26 of the second peptide (Figure 64) SEQ ID NO: 77: Amino acid sequence 27 of the second peptide (Figure 64) SEQ ID NO: 78: Amino acid sequence 28 of the second peptide (Figure 64) SEQ ID NO: 79: Amino acid sequence 29 of the second peptide (Figure 64) SEQ ID NO: 80: Amino acid sequence 30 of the second peptide (Figure 64) SEQ ID NO: 81: Amino acid sequence 31 of the second peptide (Figure 64) SEQ ID NO: 82: Amino acid sequence of human IgG1 Fc (wild type) (Figure 74) SEQ ID NO: 83: Amino acid sequence of HT1-11 scFvFc (Figure 75) SEQ ID NO: 84: Amino acid sequence of the light chain of MhM1025-M1 (Figure 76) SEQ ID NO: 85: Amino acid sequence of the heavy chain of MC3-1901 (Figure 77) SEQ ID NO: 86: Amino acid sequence of the heavy chain of hM23-M1_His (Figure 81) SEQ ID NO: 87: heavy chain amino acid sequence of hM23-M1_I253D_His (Figure 82) SEQ ID NO: 88: heavy chain amino acid sequence of hM23-M1_I253N_His (Figure 83) SEQ ID NO: 89: heavy chain amino acid sequence of hM23-M1_H435R_His (Figure 84) SEQ ID NO: 90: heavy chain amino acid sequence of hM23-M1_I253N (Figure 85) All publications, patents and patent applications cited herein are incorporated by reference in their entirety into the disclosure of this specification.

Claims

1. A method for producing a molecule (molecule 1) that binds to a target antigen 1 and comprises the following [a] part, [b] part, [c] part and optionally [e] part, the method comprising contacting a starting sample comprising molecule 1 and one or more molecules that are not molecule 1 (other molecules) with a carrier of immobilized metal affinity chromatography (IMAC) to separate molecule 1 from the other molecules, wherein the number of [c] parts contained in the other molecules is one or more greater or less than the number of [c] parts contained in molecule 1, the method: [a] part: part 1 that binds to target antigen 1; [b] part: a first peptide that recognizes target antigen binding site 1 contained in the [a] part; [c] part: a second peptide consisting of an amino acid sequence containing an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral conditions to acidic conditions in the range of pH 7.5 or less; [e] part: the Fc region of an antibody.

2. The molecule 1 is characterized by having a higher binding affinity for the target antigen under acidic conditions compared to neutral conditions, preferably with an EC ５０ ratio of 3 or more, more preferably 10 or more, and even more preferably 30 or more. The method according to claim 1.

3. The method according to claim 1 or 2, wherein molecule 1 comprises one or more of the [a] part, [b] part and [c] part, preferably the number of each of the [a] part, [b] part and [c] part is the same as each other, and more preferably each of the [a] part, [b] part and [c] part comprises one or two of each.

4. The method according to any one of claims 1 to 3, wherein the second peptide consists of an amino acid sequence containing four or more amino acid side chains whose charge changes from uncharged to positively charged in response to a change in pH from neutral conditions to acidic conditions in the range of pH 7.5 or less, and the pI value of the second peptide exceeds 6.4, preferably 6.8 or more, more preferably 7.2 or more, and even more preferably 7.6 or more.

5. The method according to any one of claims 1 to 4, wherein the second peptide consists of an amino acid sequence that does not contain an amino acid having a negative charge under acidic conditions, preferably an amino acid sequence that does not contain aspartic acid and / or glutamic acid.

6. The method according to any one of claims 1 to 5, wherein the second peptide consists of an amino acid sequence having the structures of the following (1) and (2): (1) (a) an amino acid sequence consisting of amino acids having two or more consecutive basic functional groups with a pKa of 7 or less in the side chain, or (b) an amino acid sequence in which one arginine or lysine is inserted or added at any position (including the amino terminus or carboxyl terminus) of the amino acid sequence of (a) (the amino acid sequence of (a) or (b) is referred to as a basic amino acid cluster). Preferably, it contains one or more amino acid sequences in which one arginine is inserted or added at any position (including the amino terminus or carboxyl terminus) of the amino acid sequence of (a) (b'). More preferably, it contains one amino acid sequence in which one arginine is inserted or added at any position (including the amino terminus or carboxyl terminus) of the amino acid sequence of (a) (b''). And (2) it contains four or more amino acids having a basic functional group with a pKa of 7 or less in the side chain in total.

7. The method according to any one of claims 1 to 6, wherein the second peptide consists of an amino acid sequence having the structures of the following (1) and (2): (1) it contains one amino acid sequence represented by RHHH, and (2) it contains four or more amino acids having a basic functional group with a pKa of 7 or less in the side chain in total.

8. The method according to any one of claims 1 to 6, wherein the second peptide consists of an amino acid sequence containing one or more amino acids having a basic functional group with a pKa of 7 or less in the side chain, and containing four or more amino acids having a basic functional group with a pKa of 7 or less in the side chain in total.

9. The method according to any one of claims 1 to 5, wherein the second peptide consists of a structure represented by an amino acid sequence containing an amino acid having a basic functional group with a pKa of 7 or less (preferably 5.0 to 7.0, more preferably 5.5 to 6.5) in the side chain at every other position.

10. The method according to any one of claims 6 to 9, wherein the amino acid is a natural amino acid, preferably histidine.

11. The method according to any one of claims 6 to 9, wherein the amino acid is a non-natural amino acid.

12. The method according to any one of claims 1 to 11, wherein the second peptide consists of an amino acid sequence containing 1 to 4 basic amino acid clusters.

13. The method according to any one of claims 1 to 12, wherein the second peptide consists of 10 to 60 amino acids, preferably 12 to 50 amino acids, more preferably 15 to 40 amino acids, and even more preferably 20 to 35 amino acids.

14. The method according to any one of claims 1 to 10, 12, and 13, wherein the second peptide contains an amino acid sequence represented by any one of SEQ ID NOs: 49 to 53, 55, 59 to 61, 65, 66, 75 to 81 (FIGS. 54 and 64), and preferably contains the amino acid sequence represented by SEQ ID NO: 53 (FIG. 54).

15. The method according to any one of claims 1 to 14, wherein the second peptide consists of an amino acid sequence containing an amino acid having a basic functional group with a pKa of 7 or less in the side chain and having 10 or less such amino acids.

16. The method according to any one of claims 1 to 15, wherein the amino acid sequence of the second peptide contains two or more basic amino acid clusters, and one basic amino acid cluster is located at the amino terminus or carboxyl terminus of the amino acid sequence, preferably at the amino terminus, and (i) none of the other basic amino acid clusters is located at the amino terminus or carboxyl terminus of the amino acid sequence, (ii) another basic amino acid cluster is located at the carboxyl terminus, or (iii) none of the other basic amino acid clusters is located at the amino terminus and carboxyl terminus of the amino acid sequence.

17. The method according to any one of claims 1 to 14, wherein the amino acid sequence of the second peptide contains an amino acid sequence cleaved by an intracellular and / or extracellular protease and an amino acid sequence containing a basic amino acid cluster, and the amino acid sequence is (1) linked in the order of an amino acid sequence containing a basic amino acid cluster and an amino acid sequence cleaved by an intracellular and / or extracellular protease from the amino terminus to the carboxyl terminus, or (2) linked in the order of an amino acid sequence cleaved by an intracellular and / or extracellular protease and an amino acid sequence containing a basic amino acid cluster from the amino terminus to the carboxyl terminus.

18. The method according to any one of claims 12 to 17, wherein the number of amino acids having a basic functional group with a pKa of 7 or less in the side chain contained in the amino acid sequence of the second peptide is 5% or more and 30% or less of the total number of amino acids contained in the amino acid sequence.

19. The method according to any one of claims 1 to 18, wherein molecule 1 is formed by linking a first peptide, a second peptide, and a moiety that binds to target antigen 1 in this order.

20. The method according to any one of claims 1 to 19, wherein the moiety that binds to target antigen 1 is a polypeptide consisting of an amino acid sequence not included in the first peptide and the second peptide.

21. The method according to any one of claims 1 to 20, wherein molecule 1 consists of a polypeptide.

22. The method according to claim 21, wherein molecule 1 is formed by binding a first peptide, a second peptide, and a moiety that binds to target antigen 1 in the direction from the amino terminus to the carboxyl terminus.

23. The method according to any one of claims 1 to 22, wherein any one selected from the group consisting of the first peptide, the second peptide, and the moiety that binds to the target antigen is bound to one or both of the other two via a linker or a spacer.

24. The method according to any one of claims 1 to 23, wherein the acidic condition is pH 6.5 or lower, preferably pH 6.0 or lower, more preferably pH 5.5 or lower.

25. The method according to any one of claims 1 to 24, wherein target antigen 1 is an antigen present in tumor tissue, endosome, or lysosome.

26. The method according to any one of claims 1 to 25, wherein a [d] moiety, which is another moiety, is bound to the moiety that binds to target antigen 1, and the [d] moiety does not include the first peptide and the second peptide.

27. The [d] moiety is one or more selected from the group consisting of an antibody or an antigen-binding fragment thereof that is not the [a] moiety, a peptide containing an amino acid sequence not contained in the first peptide and the second peptide, a cytokine, a toxin, a radioisotope, a labeling molecule, a photosensitive substance, an immune activating substance, an antitumor compound, a drug, a payload, and a polymer. Preferably, the antitumor compound is a camptothecin derivative or a pyrrolobenzodiazepine derivative, and the immune activating substance is a cyclic dinucleotide derivative. More preferably, the camptothecin derivative is N-[(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide. The method according to claim 26.

28. The method according to claim 26 or 27, wherein molecule 1 consists of a polypeptide or consists of the [d] moiety and a polypeptide.

29. The method according to any one of claims 1 to 28, wherein the first peptide is obtained by a method comprising the following steps (i) to (iii): (i) contacting a peptide library containing a repeating sequence of an aromatic amino acid and Pro with the portion that binds to the target antigen 1 described in claim 1; (ii) recovering the peptide that binds to the portion that binds to the target antigen 1; and (iii) preparing the obtained peptide by recombination, chemical synthesis, or peptide synthesis.

30. The method according to any one of claims 1 to 29, comprising the step of preparing a peptide comprising the amino acid sequence contained in the first peptide and / or the amino acid sequence contained in the second peptide by recombination, in vitro translation, chemical synthesis, or peptide synthesis.

31. Culturing a cell into which a polynucleotide comprising the amino acid sequence contained in the portion that binds to the target antigen 1 and optionally the amino acid sequence contained in the first peptide and / or the amino acid sequence contained in the second peptide is introduced, and recovering the polypeptide that binds to the target antigen from the culture. The method according to any one of claims 1 to 30.

32. The method according to any one of claims 1 to 31, wherein the molecule 1 contains n [c] moieties, and n is a positive integer.

33. The method according to claim 32, wherein the other molecule contains n + 1 or more [c] moieties or contains n - 1 or fewer [c] moieties.

34. The method according to claim 33, wherein the other molecule contains 1 or more [c] moieties.

35. The method according to any one of claims 32 to 34, wherein the other molecule comprises a molecule (other molecule 1) containing n + 1 or more [c] moieties and a molecule (other molecule 2) containing n - 1 or fewer [c] moieties.

36. The method according to any one of claims 32 to 35, wherein n is 1.

37. The method according to any one of claims 1 to 36, wherein the molecule 1 is a multispecific antibody, preferably a bispecific antibody, and optionally the multispecific antibody or bispecific antibody contains a [d] moiety.

38. The method according to claim 37, wherein the molecule 1 comprises a [a] moiety (anti-target antigen 1 antibody) which is a monovalent or higher antibody or an antigen-binding fragment thereof that binds to the target antigen 1, and a monovalent or higher antibody or an antigen-binding fragment thereof (anti-target antigen 2 antibody) that binds to the target antigen 2, and preferably the anti-target antigen 1 antibody and the anti-target antigen 2 antibody are monovalent antibodies or antigen-binding fragments thereof.

39. The method according to claim 38, wherein the molecule 1 is a heteromultimer, preferably a heterodimer.

40. The method according to any one of claims 37 to 39, wherein the other molecule 1 contains an anti-target antigen 1 antibody which is a [x×2]-valent monospecific antibody or an antigen-binding fragment thereof, x is an integer, and preferably x is 1.

41. The method according to any one of claims 37 to 40, wherein the other molecule 2 contains an anti-target antigen 2 antibody which is a [y×2]-valent monospecific antibody or an antigen-binding fragment thereof, y is 0 or a positive integer, and preferably y is 0 or 1.

42. The method according to any one of claims 37 to 41, wherein the other molecule 1 does not contain an anti-target antigen 2 antibody and the other molecule 2 does not contain an anti-target antigen 1 antibody.

43. The method according to any one of claims 37 to 42, wherein the other molecule 1 and the other molecule 2 are homomultimers, preferably homodimers.

44. The method according to claim 38, wherein one or both of the anti-target antigen 1 antibody and the anti-target antigen 2 antibody are scFv or VHH, and preferably the anti-target antigen 1 antibody or the anti-target antigen 2 antibody that is not scFv or VHH is a monovalent antibody or an antigen-binding fragment thereof.

45. The method according to claim 44, wherein molecule 1 is a hetero-oligomer, preferably a hetero-dimer.

46. The method according to claim 44 or 45, wherein another molecule 1 contains an anti-target antigen 1 antibody and an anti-target antigen 2 antibody, and another molecule 2 does not contain an anti-target antigen 1 antibody and an anti-target antigen 2 antibody.

47. The method according to any one of claims 44 to 46, wherein another molecule 1 and another molecule 2 are homo-oligomers, preferably homo-dimers.

48. The method according to any one of claims 1 to 36, wherein the [a] moiety is a monospecific antibody or an antigen-binding fragment thereof, and molecule 1 does not contain an antibody or an antigen-binding fragment thereof other than the [a] moiety.

49. The method according to claim 48, wherein the [a] moiety is a monovalent antibody or an antigen-binding fragment thereof.

50. The method according to claim 48 or 49, wherein another molecule 1 contains a bivalent monospecific antibody or an antigen-binding fragment thereof (anti-target antigen 1 monospecific antibody) that binds to target antigen 1, and another molecule 2 does not contain an anti-target antigen 1 monospecific antibody.

51. The method according to any one of claims 1 to 50, wherein IMAC is column chromatography.

52. The method according to claim 51, wherein the mobile phase in IMAC may contain imidazole or histidine.

53. The method according to claim 52, wherein the imidazole concentration or histidine concentration, preferably the imidazole concentration, in the mobile phase is linearly or stepwise increased in the step of separating molecule 1 from other molecules.

54. The method according to claim 53, wherein a molecule containing more [c] moieties is eluted later than a molecule containing fewer [c] moieties.

55. A method for producing a molecule (molecule 1) that binds to target antigen 1 and comprises the following [a] moiety, [b] moiety, [c] moiety, and optionally [e] moiety, the method comprising a step of contacting a starting sample comprising molecule 1 with a carrier of immobilized metal affinity chromatography (IMAC): [a] moiety: moiety 1 that binds to target antigen 1; [b] moiety: a first peptide that recognizes target antigen binding site 1 contained in the [a] moiety; [c] moiety: a second peptide consisting of an amino acid sequence containing an amino acid side chain whose charge changes from uncharged to positively charged in response to a change in pH from neutral to acidic conditions in the range of pH 7.5 or lower; [e] moiety: the Fc region of an antibody.

56. The method according to claim 55, wherein the starting sample comprises one or more molecules other than molecule 1, and the number of [c] moieties contained in the other molecule is one or more greater than or less than the number of [c] moieties contained in molecule 1.

57. The method according to claim 56, wherein the other molecule is the other molecule defined in any one of claims 33 - 35, 40 - 42, 46, 47 or 50.

58. The method according to any one of claims 1 - 57, wherein molecule 1 contains an [e] moiety, and the [e] moiety contains one or more amino acid mutations that reduce the affinity of the wild - type Fc region for the Protein A carrier.

59. The method according to claim 58, wherein the [e] moiety has only one amino acid mutation that reduces the affinity of the wild - type Fc region for the Protein A carrier.

60. The method according to claim 58 or 59, wherein the one amino acid mutation is a substitution of Ile at position 253 (EU numbering) with Asn.

61. The method according to any one of claims 58 - 60, further comprising a chromatography step or a fractionation step other than IMAC.

62. The method according to claim 61, wherein the chromatography step or the fractionation step other than IMAC is a Protein A chromatography step.

63. The method according to claim 62, wherein the Protein A chromatography step comprises contacting the starting sample with a Protein A carrier, and contacting a fraction that has passed through the carrier or a fraction obtained by eluting the components adsorbed on the carrier with the starting sample contacted with the IMAC carrier, with the Protein A carrier.

64. The method according to any one of claims 51, 52, and 56 - 63, wherein in IMAC, the elution of the components adsorbed on the IMAC carrier is an elution using a single mobile phase.

65. The method according to claim 62, wherein the starting sample is a fraction obtained by contacting a sample containing molecule 1 with a Protein A carrier and eluting the components adsorbed on the carrier.

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