Compound or salt thereof, and antibody obtained by the same

Abstract

To provide a compound that makes it easy to regioselectively modify an antibody by a functional substance, and to control a bond ratio between the antibody and the functional substance within a desired range, or a salt thereof, and an antibody that can be produced by using the same.SOLUTION: Provided are a compound expressed by the following formula (I): [in the formula, X denotes a leaving group, Y denotes affinity peptide which has a binding region in a CH2 domain in an antibody, O denotes an oxygen atom, S denotes a sulfur atom, W denotes an oxygen atom or a sulfur atom, La denotes a first linker, Lb denotes a second linker, and the total of an atomic number constituting a main chain in the first linker and an atomic number constituting a main chain in the second linker is 5 to 7] or a salt thereof, and an antibody that can be produced by using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a compound or a salt thereof, and an antibody obtained therefrom, etc. [Background technology]

[0002] In recent years, research and development of antibody drug conjugates (ADCs) has been actively pursued. As the name suggests, ADCs are drugs in which a drug (e.g., an anticancer drug) is conjugated to an antibody, and have direct cytocidal activity against cancer cells and the like. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)), jointly developed by Immunogene and Roche.

[0003] Since their inception, ADCs, including T-DM1, have faced the challenge of heterogeneity. Because small molecule drugs are randomly conjugated to the approximately 70–80 lysine residues in an antibody, the drug-antibody ratio (DAR) and conjugation site are inconsistent. This random conjugation method typically results in a DAR ranging from 0 to 8, resulting in multiple antibody drugs with different numbers of drugs attached. In recent years, it has been reported that altering the number and site of drug attachments in an ADC can affect pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs require control over the number and site of conjugated drugs. Maintaining a consistent number and site is believed to achieve the desired efficacy and address regulatory issues such as variations in conjugated drugs and lot-to-lot differences.

[0004] Research into site-selective modification of antibodies is being conducted worldwide, but most of the research involves genetic engineering or enzyme-based modification. While genetic engineering modification methods can control site and number selectivity, problems have been pointed out, such as reduced expression efficiency of the antibody itself (reducing the overall yield when preparing ADCs). Another problem is that it takes a long time to establish an antibody expression system.

[0005] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed, which enables site-selective modification of antibodies using chemical synthesis techniques (Patent Document 1). This method successfully achieves site-selective modification of antibodies by reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide. However, the ADCs produced by this method conjugate the antibody and drug via a linker containing a peptide moiety. The peptide moiety has potential immunogenicity and is easily hydrolyzed in blood. Therefore, the ADCs produced by this method have room for improvement in terms of including a peptide moiety in the linker.

[0006] As an improvement over the C-CAP method, techniques have been reported that use chemical synthesis techniques using specific compounds containing affinity peptides to prepare antibodies that do not contain a peptide moiety as a linker and that regioselectively carry functional substances (e.g., drugs) (Patent Documents 2 to 6). Avoiding the use of linkers containing peptide moieties is desirable for clinical applications. These techniques propose several positions in the antibody that can be regioselectively modified with drugs, corresponding to various amino acid residues in the CH2 and CH3 domains (e.g., lysine, tyrosine, serine, and threonine residues). However, it is not always easy to regioselectively modify an antibody with a functional substance and control the binding ratio between the antibody and the functional substance within a desired range. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 186206 [Patent Document 2] International Publication No. 2018 / 199337 [Patent Document 3] International Publication No. 2019 / 240287 [Patent Document 4] International Publication No. 2019 / 240288 [Patent Document 5] International Publication No. 2020 / 009165 [Patent Document 6] International Publication No. 2020 / 090979 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to site-selectively modify an antibody with a functional substance and to control the binding ratio between the antibody and the functional substance within a desired range. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have found that by selecting the lysine residue at positions 288 / 290 of the heavy chain in an immunoglobulin unit as the modification site of an antibody and using a specific compound that enables site-specific modification of the lysine residue, it is possible to regioselectively modify an antibody with a functional substance and to easily control the average ratio of bonds between immunoglobulin units and functional substances (number of functional substances / immunoglobulin unit) to a desired range (1.5 to 2.5). Such a specific compound corresponds to a compound in which the total number of atoms constituting the main chain linking the reactive moiety [X(leaving group)-C=O] with the lysine residue at positions 288 / 290 of the heavy chain in an immunoglobulin unit to the binding moiety [O=CY(affinity peptide)] is 7 to 9 (i.e., a compound represented by formula (I) in which the total number of atoms constituting the main chain of the first linker and the main chain of the second linker is 5 to 7). Based on these findings, the present inventors have succeeded in developing a compound represented by formula (I) or a salt thereof, and an antibody derivatization reagent containing the compound. The present inventors have also discovered that the use of a compound represented by formula (I) or a salt thereof makes it possible to prepare specific antibodies (antibody intermediates, thiol group-introduced antibody derivatives, and conjugates of antibodies and functional substances) in which the lysine residues at positions 288 / 290 in the antibody are regioselectively modified with modifying groups and the average ratio of bonds between immunoglobulin units and modifying groups (number of modifying groups / immunoglobulin units) is highly controlled within a desired range (1.5 to 2.5), thereby completing the present invention.

[0010] That is, the present invention is as follows. [1] A compound represented by formula (I) or a salt thereof: [2] The compound of [1] or a salt thereof, wherein the leaving group is selected from the following: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom); or (c)R A -(R B -)N(where R A and R B each independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom; or (d) Halogen atoms. [3] The compound or salt thereof according to [1] or [2], wherein the immunoglobulin unit is a human immunoglobulin unit. [4] The compound or salt thereof according to any one of [1] to [3], wherein the immunoglobulin unit is human IgG. [5] The affinity peptide contains a lysine residue and binds to the lysine residue via the amino group in the side chain. , which forms an amide bond with the carbonyl group (C=O) adjacent to Y, or a salt thereof. [6] Any of the compounds of [1] to [5] or a salt thereof, wherein the affinity peptide is any one of the following: (A) an affinity peptide comprising the amino acid sequence of CQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 1); or (B) An affinity peptide comprising an amino acid sequence of CQRRFYEALHDPNLNEEQRNARIRSIKDDC (sequence number 1) in which 1 to 5 amino acid residue mutations selected from the group consisting of amino acid residue substitution, insertion, deletion, and addition (wherein the lysine residue at position 27 and the two cysteine ​​residues at positions 1 and 30 are maintained). [7] The compound of [6] or a salt thereof, wherein the affinity peptide (B) is selected from the group consisting of: (a) an affinity peptide comprising the amino acid sequence FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 2); (b) an affinity peptide comprising the amino acid sequence FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC (SEQ ID NO: 3); (c) an affinity peptide comprising the amino acid sequence FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 4); (d) an affinity peptide comprising the amino acid sequence NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); (e) an affinity peptide comprising the amino acid sequence of MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 6); and (f) An affinity peptide comprising the amino acid sequence QCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 7). [8] The N-terminal and C-terminal amino acid residues in the affinity peptide may be protected, and The compound of [6] or [7] or a salt thereof, wherein the two thiol groups in the side chains of the two cysteine ​​residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker. [9] A compound or a salt thereof according to any one of [1] to [8], wherein the compound represented by formula (I) is represented by formula (I'):

[10] The compound of [9] or a salt thereof, wherein the compound represented by formula (I') is represented by formula (I'').

[11] A reagent for antibody derivatization, comprising a compound represented by formula (I) or a salt thereof.

[12] The reagent according to

[11] , wherein the compound represented by formula (I) is represented by formula (I').

[13] The reagent according to

[12] , wherein the compound represented by formula (I') is represented by formula (I'').

[14] An antibody intermediate or a salt thereof comprising a structural unit represented by formula (II):

[15] The antibody intermediate or a salt thereof according to

[14] , wherein the antibody intermediate is a human antibody intermediate.

[16] The antibody intermediate or a salt thereof according to

[14] or

[15] , wherein the antibody intermediate is a human IgG intermediate.

[17] The antibody intermediate or a salt thereof according to any one of

[14] to

[16] , wherein the structural unit represented by formula (II) is represented by formula (II').

[18] The antibody intermediate of

[17] or a salt thereof, wherein the structural unit represented by formula (II') is represented by formula (II'').

[19] A thiol group-introduced antibody derivative or a salt thereof, which comprises a structural unit represented by formula (III):

[20] The thiol group-introduced antibody derivative or a salt thereof according to

[19] , wherein specific amino acid residues other than the lysine residues at positions 288 / 290 in the two heavy chains are further modified.

[21] The thiol group-introduced antibody derivative or a salt thereof according to

[20] , wherein the specific amino acid residues are lysine residues located at positions 246 / 248 in the two heavy chains.

[22] The thiol group-introduced antibody derivative or a salt thereof according to any one of

[19] to

[21] , wherein the structural unit represented by formula (III) is represented by formula (III').

[23] The thiol group-introduced antibody derivative or a salt thereof according to

[22] , wherein the structural unit represented by formula (III') is represented by formula (III'').

[24] A conjugate of an antibody and a functional substance, or a salt thereof, comprising a structural unit represented by formula (IV):

[25] The conjugate or salt thereof according to

[24] , wherein specific amino acid residues other than the lysine residues at positions 288 / 290 in the two heavy chains are further modified.

[26] The conjugate or salt thereof according to

[25] , wherein the specific amino acid residues are lysine residues located at positions 246 / 248 in the two heavy chains.

[27] A conjugate or a salt thereof according to any one of

[24] to

[26] , wherein the structural unit represented by formula (IV) is represented by formula (IV').

[28] The conjugate or salt thereof according to

[27] , wherein the structural unit represented by formula (IV') is represented by formula (IV'').

[29] A compound represented by formula (V) or a salt thereof:

[30] The compound of

[29] or a salt thereof, wherein the compound represented by formula (V) is represented by formula (V'):

[31] The compound or a salt thereof according to

[30] , wherein the compound represented by formula (V') is represented by formula (V'').

[32] The compound or a salt thereof according to

[31] , wherein the compound represented by formula (V'') is represented by formula (V''-1) or (V''-2):

[33] A compound represented by formula (VI) or a salt thereof:

[34] The compound or salt thereof according to

[33] , wherein the leaving group having a higher ability to leave than the leaving group X is a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyloxy group.

[35] The compound of

[33] or

[34] , or a salt thereof, wherein the compound of formula (VI) is represented by formula (VI'):

[36] The compound of

[35] or a salt thereof, wherein the compound of formula (VI') is represented by formula (VI'').

[37] The compound of

[36] or a salt thereof, wherein the compound of formula (VI'') is represented by formula (VI''-1) or (VI''-2):

[38] A method for producing an antibody intermediate or a salt thereof, comprising reacting a compound represented by formula (I) or a salt thereof with an antibody containing an immunoglobulin unit to produce an antibody intermediate or a salt thereof containing a structural unit represented by formula (II).

[39] (1) reacting a compound represented by formula (I) or a salt thereof with an antibody containing an immunoglobulin unit to produce an antibody intermediate containing a structural unit represented by formula (II) or a salt thereof; and (2) A method for producing a thiol group-introduced antibody derivative or a salt thereof, comprising subjecting an antibody intermediate or a salt thereof to a thioester cleavage reaction to produce a thiol group-introduced antibody derivative or a salt thereof, which comprises a structural unit represented by formula (III).

[40] (1) reacting a compound represented by formula (I) or a salt thereof with an antibody containing an immunoglobulin unit to produce an antibody intermediate or a salt thereof containing a structural unit represented by formula (II); (2) subjecting the antibody intermediate or its salt to a thioester cleavage reaction to produce a thiol group-introduced antibody derivative or its salt, which contains a structural unit represented by formula (III); and (3) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting a thiol group-introduced antibody derivative or a salt thereof with a functional substance to produce a conjugate of an antibody and a functional substance or a salt thereof, which contains a structural unit represented by formula (IV).

[41] A compound represented by formula (VI) or a salt thereof, comprising two heavy chains and two light chains. The method according to any one of

[38] to

[40] , further comprising reacting the compound represented by formula (I) or a salt thereof with an affinity peptide having a binding region in the CH2 domain of an immunoglobulin unit containing the compound represented by formula (I).

[42] (1') Reacting a compound represented by formula (V) or a salt thereof with a carboxyl group-modifying reagent to produce a compound represented by formula (VI) or a salt thereof; and (2') The method according to any one of

[38] to

[40] , further comprising reacting the compound represented by formula (VI) or a salt thereof with an affinity peptide having a binding region in a CH2 domain in an immunoglobulin unit comprising two heavy chains and two light chains to produce the compound represented by formula (I) or a salt thereof.

[43] A method for producing a compound represented by formula (VI) or a salt thereof, comprising reacting a compound represented by formula (V) or a salt thereof with a carboxyl group-modifying reagent to produce a compound represented by formula (VI) or a salt thereof.

[44] A method for producing a thiol group-introduced antibody derivative or a salt thereof, comprising: subjecting an antibody intermediate containing a structural unit represented by formula (II) or a salt thereof to a thioester cleavage reaction to produce a thiol group-introduced antibody derivative or a salt thereof containing a structural unit represented by formula (III).

[45] (1) subjecting an antibody intermediate or a salt thereof containing a structural unit represented by formula (II) to a thioester cleavage reaction to produce a thiol group-introduced antibody derivative or a salt thereof containing a structural unit represented by formula (III); and (2) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting a thiol group-introduced antibody derivative or a salt thereof with a functional substance to produce a conjugate of an antibody and a functional substance or a salt thereof, which contains a structural unit represented by formula (IV).

[46] A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting a thiol group-introduced antibody derivative or a salt thereof, which contains a structural unit represented by formula (III), with a functional substance to produce a conjugate of an antibody and a functional substance or a salt thereof, which contains a structural unit represented by formula (IV). [Effects of the Invention]

[0011] The compound represented by formula (I) or its salt can specifically and highly modify the lysine residues at positions 288 / 290 of the heavy chain in the immunoglobulin unit so that the average ratio of bonds between immunoglobulin units and affinity peptide-containing groups (number of affinity peptide-containing groups / immunoglobulin unit) falls within the desired range (1.5 to 2.5). Therefore, the compound represented by formula (I) or its salt is useful as a reagent for antibody derivatization. Furthermore, the compound represented by formula (I) or a salt thereof can provide an antibody intermediate represented by formula (II) or a salt thereof, in which the lysine residues at positions 288 / 290 of the heavy chain in the immunoglobulin unit are specifically modified with affinity peptide-containing groups, and the average ratio of bonds between immunoglobulin units and affinity peptide-containing groups (number of affinity peptide-containing groups / immunoglobulin unit) is highly controlled within a desired range. Furthermore, antibodies produced using the antibody intermediate represented by formula (II) or its salt as a starting material can inherit the regioselectivity and average binding ratio of the antibody intermediate or its salt. Thus, it is possible to provide a thiol group-introduced antibody derivative represented by formula (III) or its salt, and a conjugate of an antibody represented by formula (IV) and a functional substance or its salt, which have the regioselectivity and average binding ratio described above. Also provided are compounds represented by formulas (V) and (VI) or salts thereof, which are synthetic intermediates that enable efficient production of the compound represented by formula (I) or salts thereof. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram (part 1) illustrating the concept of modifying an immunoglobulin unit with a compound of the present invention represented by formula (I) or a salt thereof. First, the compound of the present invention represented by formula (I) or a salt thereof associates with the CH2 domain of an immunoglobulin unit via the affinity peptide (Y). Next, the compound of the present invention represented by formula (I) or a salt thereof reacts with the side chain of a specific amino acid residue in the CH2 domain (the amino group in the side chain of a lysine residue in the drawing) via the activated carbonyl group having a leaving group (X) to produce an antibody intermediate or a salt thereof. [Figure 2] 2 is a schematic diagram (part 2) illustrating the concept of immunoglobulin unit modification with the compound of the present invention represented by formula (I) or a salt thereof. Cleavage of the thioester group generates a thiol group-introduced antibody derivative or a salt thereof. [Figure 3] 3 is a schematic diagram (part 3) illustrating the concept of modifying an immunoglobulin unit with the compound of the present invention represented by formula (I) or a salt thereof. A conjugate of an antibody and a functional substance (Z) is produced by reaction of the thiol group in the thiol group-introduced antibody derivative or its salt with a functional substance (Z). [Figure 4] FIG. 4 is a diagram showing an overview of one embodiment of the present invention. [Figure 5] FIG. 5 is a diagram showing an overview of a preferred embodiment of the present invention. [Figure 6] FIG. 6 is a diagram showing an outline of a more preferred embodiment of the present invention. [Figure 7] FIG. 7 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (anti-HER2 IgG antibody) (Example 1). [Figure 8]FIG. 8 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 1). [Figure 9] FIG. 9 shows (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 8), and (2) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 9). [Figure 10] Figure 10 shows the MS spectrum (measured value: m / z 577.03606, theoretical value: 577.03557, tetravalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 10), a peptide consisting of 18 amino acid residues including a modification site on a lysine residue by trypsin digestion of trastuzumab (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) (Example 1-9-4). [Figure 11] FIG. 11 shows the CID spectrum of the product ion at m / z 682.13 (theoretical value: 682.01), which corresponds to trivalent y16 and indicates modification of the lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering (Example 1-9-4). [Figure 12] FIG. 12 shows the results of searching for peptide fragments containing a thiol-introduced product (+145.019 Da) that had been modified at a lysine residue (carbamidomethylated with iodoacetamide) in a tryptic digest of trastuzumab using BioPharma Finder (Example 1-9-4). The horizontal axis shows the identified lysine residues, and the vertical axis shows intensity. [Figure 13] FIG. 13 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 2). [Figure 14] FIG. 14 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 2). [Figure 15]Figure 15 shows the MS spectrum (measured value: m / z 577.03571, theoretical value: 577.03557, tetravalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 10), a peptide consisting of 18 amino acid residues including a modification site on a lysine residue by trypsin digestion of trastuzumab (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) (Example 2-7-4). [Figure 16] FIG. 16 shows the CID spectrum of the product ion at m / z 682.41 (theoretical value: 682.01), which corresponds to trivalent y16 and indicates modification of the lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering (Example 2-7-4). [Figure 17] FIG. 17 shows the results of searching for peptide fragments containing a thiol-introduced product (+145.019 Da) that had been modified at a lysine residue (carbamidomethylated with iodoacetamide) in a tryptic digest of trastuzumab using BioPharma Finder (Example 2-7-4). The horizontal axis shows the identified lysine residues, and the vertical axis shows intensity. [Figure 18] FIG. 18 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 3). [Figure 19] FIG. 19 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 3). [Figure 20] FIG. 20 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 4). [Figure 21] FIG. 21 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 4). [Figure 22] FIG. 22 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 5). [Figure 23]FIG. 23 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 5). [Figure 24] FIG. 24 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 6). [Figure 25] FIG. 25 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 6). [Figure 26] FIG. 26 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 7). [Figure 27] FIG. 27 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 7). [Figure 28] Figure 28 shows the MS spectrum (measured value: m / z 769.04506, theoretical value: 769.04482, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 10), a peptide consisting of 18 amino acid residues including a modification site on a lysine residue by trypsin digestion of trastuzumab (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) (Example 7-6-4). [Figure 29] Figure 29 shows the CID spectrum of the product ion at m / z 1022.71 (theoretical value: 1022.51), which corresponds to divalent y16, indicating modification of the lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering (Example 7-6-4). [Figure 30] FIG. 30 shows the results of searching for peptide fragments containing a thiol-introduced product (+145.019 Da) that had been modified at a lysine residue (carbamidomethylated with iodoacetamide) in a tryptic digest of trastuzumab using BioPharma Finder (Example 7-6-4). The horizontal axis shows the identified lysine residues, and the vertical axis shows intensity. [Figure 31]FIG. 31 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 8). [Figure 32] FIG. 32 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 8). [Figure 33] FIG. 33 shows ESI-TOFMS analysis of specific modifications (number of binding peptides introduced) of trastuzumab (Example 9). [Figure 34] FIG. 34 shows ESI-TOFMS analysis of specific modifications (heavy chain selectivity) of trastuzumab (Example 9). DETAILED DESCRIPTION OF THE INVENTION

[0013] 1. General definitions In the present invention, the term "antibody" is as follows. Furthermore, the term "immunoglobulin unit" corresponds to a divalent monomer unit that is the basic component of such antibodies, and is a unit containing two heavy chains and two light chains. Therefore, the definitions, examples, and preferred examples of the origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of lysine residue, and regioselectivity of the immunoglobulin unit are the same as those of the antibody described below.

[0014] The origin of the antibody is not particularly limited, and may be derived from animals such as mammals or birds (e.g., chickens). Preferably, the immunoglobulin unit is derived from a mammal. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pets (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, and more preferably humans.

[0015] The antibody may be a polyclonal or monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent or higher antibody (e.g., IgA, IgM). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies with a specific glycosylation chain added (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, full-length antibodies or antibody fragments containing the variable region and CH1 and CH2 domains can be used as monoclonal antibodies, with full-length antibodies being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0016] Any antigen can be used as the antigen for the antibody. Examples of such antigens include proteins (including oligopeptides and polypeptides, and may also be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low-molecular-weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0017] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0018] (1) Oncology PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1(CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfelin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Ruis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 inhibitory factor, TAG-72 (CA72-4), CD70,

[0019] (2) Autoimmune diseases and inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP(Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT

[0020] (3) Neurological disorders CGRP, CD20, β-amyloid, β-amyloid protofibrin, calcitonin gene-related peptide receptor, LINGO (Ig domain containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0021] (4) Infectious disease Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0022] (5) Genetic and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin Chin

[0023] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0024] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0025] (8) Hematological diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0026] (9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

[0027] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, and ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, and daratumumab). , ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (if no IgG subtype is mentioned, it refers to IgG1).

[0028] The positions of lysine residues in antibodies and in the heavy chain constant region (e.g., CH2 domain) follow EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). Therefore, when targeting human IgG, the lysine residue at position 288 corresponds to the 58th residue in the CH2 region of human IgG, and the lysine residue at position 290 corresponds to the 60th residue in the CH2 region of human IgG. The designation 288 / 290 indicates that the lysine residue at position 288 or 290 is the target.

[0029] According to the present invention, the lysine residues at positions 288 / 290 of an antibody can be regioselectively modified. As used herein, "regioselective" or "regioselectivity" refers to the fact that a specific structural unit capable of binding to a specific amino acid residue in an antibody is preferentially located in a specific region of the antibody, even though that specific amino acid residue is not preferentially located in a specific region of the antibody. Therefore, expressions related to regioselectivity, such as "regioselectively possessed," "regioselective binding," and "regioselective binding," mean that the retention or binding rate of a specific structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues homologous to the specific amino acid residues in the target region. Such regioselectivity is 50% or higher, preferably 60% or higher, more preferably 70% or higher, even more preferably 80% or higher, and particularly preferably 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, or 100%. According to the present invention, the lysine residues at positions 288 / 290 can be regioselectively modified without using a peptide-containing linker. Peptide moieties have potential immunogenicity and are easily hydrolyzed in blood. Therefore, avoiding the use of linkers containing peptide moieties is desirable for clinical applications.

[0030] In the present invention, as long as the lysine residues at positions 288 / 290 in the antibody are regioselectively modified, specific amino acid residues at other positions may also be regioselectively modified.For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in WO / 2018 / 199337, WO / 2019 / 240288, WO / 2019 / 240287, and WO / 2020 / 090979. Such specific amino acid residues include amino acid residues (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, and cysteine ​​residues) having a side chain that is easily modified (e.g., an amino group, a carboxy group, an amide group, a hydroxy group, or a thiol group). Lysine residues having a side chain containing an amino group, tyrosine residues, serine residues, and threonine residues having a side chain containing a hydroxy group, or cysteine ​​residues having a side chain containing a thiol group are preferred. Lysine residues are more preferred. Lysine residues at positions 246 / 248 or 317 are even more preferred. Lysine residues at positions 246 / 248 are particularly preferred. The notation "positions 246 / 248" indicates that the lysine residues at positions 246 or 248 are the target.

[0031] In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0032] 2. Compound or salt thereof The present invention provides a compound represented by the following formula (I) or a salt thereof: [ka] [During the ceremony, X represents a leaving group, Y represents an affinity peptide having a binding region in the CH2 domain of an immunoglobulin unit comprising two heavy chains and two light chains; O represents an oxygen atom, S represents a sulfur atom, W represents an oxygen atom or a sulfur atom; La represents the first linker, Lb represents a second linker, The total number of atoms constituting the main chain of the first linker and the total number of atoms constituting the main chain of the second linker is 5 to 7.

[0033] In formula (I) and other formulas presented in connection with the present invention, a hyphen (-) indicates that the two units on either side of it are covalently bonded. Thus, in formula (I), X is covalently bonded to the carbon atom constituting the carbonyl group, La is covalently bonded to the carbon atom constituting the carbonyl group and S, S is covalently bonded to La and the carbon atom constituting the carbonyl group, Lb is covalently bonded to the two carbon atoms constituting the two adjacent carbonyl groups, and Y is covalently bonded to the carbon atom constituting the carbonyl group.

[0034] The leaving group represented by X is a group that can be eliminated by a reaction between the carbon atom of the carbonyl group adjacent to X and an amino group. Those skilled in the art can appropriately select such a leaving group. Examples of such leaving groups include the following: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom); (c)R A -(R B -)N(where R A and R B each independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom; or (d) Halogen atoms.

[0035] Preferably, the leaving group represented by X may be: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom); or (c)R A -(R B -)N(where R A and R B each independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group;

[0036] More preferably, the leaving group represented by X may be: (a) RS (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom); or (b) RO (wherein R represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom).

[0037] Even more preferably, the leaving group represented by X may be: (a) RS (wherein R represents a monovalent aromatic hydrocarbon group which may have a substituent, or a monovalent aromatic heterocyclic group which may have a substituent, and S represents a sulfur atom).

[0038] Particularly preferably, the leaving group represented by X may be: (a') RS (wherein R represents an optionally substituted monovalent aromatic hydrocarbon group (eg, phenyl), and S represents a sulfur atom).

[0039] Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms.

[0040] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0041] A monovalent chain hydrocarbon group refers to a hydrocarbon group that is composed only of a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0042] The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0043] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0044] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0045] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0046] A monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not have to be composed only of alicyclic hydrocarbons, and it may also contain a chain structure as part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0047] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 or 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0048] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 or 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0049] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 or 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0050] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0051] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed of only aromatic rings, and it may contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, an aryl group having 6 to 12 carbon atoms is preferred, an aryl group having 6 to 10 carbon atoms is more preferred, and an aryl group having 6 carbon atoms is even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of the substituent. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0052] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0053] Among these, alkyl, cycloalkyl and aryl are preferred as the monovalent hydrocarbon group.

[0054] A monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic group preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0055] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0056] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0057] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0058] Above R, R A , and R B The number of "substituents" in the "monovalent hydrocarbon group which may have a substituent" and the "monovalent heterocyclic group which may have a substituent" represented by the formula (I) may be, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2. Examples of such substituents include the following: (i) halogen atoms; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) aralkyl; (v)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or a monovalent hydrocarbon group; or (vi)NR b R c -, NR b R c-C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; (vii) Nitro, sulfate, sulfonate, cyano, and carboxyl groups.

[0059] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, and monovalent heterocyclic group in the above substituents are the same as those of the above R and R, respectively. A , and R B The monovalent hydrocarbon group and the monovalent heterocyclic group are the same as those described above.

[0060] Aralkyl refers to arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0061] Preferably, the substituents may be: (i) halogen atoms; (ii) alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or alkyl having 1 to 12 carbon atoms; (vi) NR b R c -, NR b R c -C(=O)-, NR b Rc -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (vii) The same groups as those listed in (vii) above.

[0062] More preferably, the substituents may be: (i) halogen atoms; (ii) alkyl having 1 to 12 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0063] Even more preferably, the substituents may be: (i) halogen atoms; (ii) alkyl having 1 to 6 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0064] Particularly preferably, the substituents may be: (i) halogen atoms; (ii) alkyl having 1 to 4 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0065] The affinity peptide represented by Y has a binding region in the CH2 domain of an immunoglobulin unit comprising two heavy chains and two light chains. Any peptide having a binding region in the CH2 domain of an immunoglobulin unit can be used as the affinity peptide. The affinity peptide contains an amino acid residue (e.g., lysine residue, proline residue, tryptophan residue, tyrosine residue, serine residue, threonine residue) having a side chain containing a moiety (e.g., amino group, hydroxy group) capable of bonding with the carbonyl group (C=O) adjacent to Y (e.g., methylamino acid ... Examples of such affinity peptides that can be used include those disclosed in International Publication Nos. 2016 / 186206, 2018 / 199337, 2019240288, 2019 / 240287, and 2020 / 090979, as well as the literature cited in these international publications.

[0066] Preferably, the affinity peptide may be: (A) CQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 1) an affinity peptide comprising the amino acid sequence of (B) An affinity peptide comprising an amino acid sequence of CQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 1) containing mutations of 1 to 5 (i.e., 1, 2, 3, 4, or 5) amino acid residues selected from the group consisting of amino acid residue substitution, insertion, deletion, and addition (wherein the lysine residue at position 27 and the two cysteine ​​residues at positions 1 and 30 are maintained). An affinity peptide comprising the amino acid sequence of SEQ ID NO: 1 is a suitable peptide having a binding region in the CH2 domain of an immunoglobulin unit, and therefore, the use of such an affinity peptide is preferred in the present invention. The N-terminal and C-terminal amino acid residues of the affinity peptide may be protected, and the thiol groups of the side chains of the two cysteine ​​residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.

[0067] Preferably, the affinity peptide (B) may be selected from the group consisting of: (a) an affinity peptide comprising the amino acid sequence FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 2); (b) an affinity peptide comprising the amino acid sequence FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC (SEQ ID NO: 3); (c) an affinity peptide comprising the amino acid sequence FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 4); (d) an affinity peptide comprising the amino acid sequence NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); (e) an affinity peptide comprising the amino acid sequence of MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 6); and (f) An affinity peptide comprising the amino acid sequence QCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 7). The N-terminal and C-terminal amino acid residues of the affinity peptide may be protected, and the thiol groups of the side chains of the two cysteine ​​residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.

[0068] At least two spaced cysteine ​​residues in each amino acid sequence of the affinity peptide can form a cyclic peptide via a disulfide bond. Alternatively, the thiol groups of the two cysteine ​​residues in the peptide can be linked by a carbonyl group-containing linker shown below.

[0069] [ka]

[0070] The dashed line portion of the carbonyl group-containing linker shown above represents the bond with the thiol group. This linker is more stable against reduction reactions and the like than a typical disulfide bond. Such peptides can be prepared, for example, by the method described in WO 2016 / 186206.

[0071] The amino acids constituting the affinity peptide may be either L- or D-isomers, but L-isomers are preferred (in the examples, all amino acid residues constituting the peptide are L-isomers). The affinity peptide may be linked to the compound of formula (I) or a salt thereof by modifying a specific amino acid residue with a crosslinker. Examples of such specific amino acid residues include lysine, aspartic acid, and glutamic acid residues, with lysine being preferred. Examples of crosslinkers include crosslinkers containing preferably two or more succinimidyl groups, such as DSG (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate); crosslinkers containing preferably two or more imidate moieties, such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2HCl), and DMS (dimethyl suberimidate·2HCl); and crosslinkers having an S-S bond, such as DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., WO 2016 / 186206).

[0072] The amino and carboxyl groups at the termini of the affinity peptide may be protected. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl groups, and arylalkyl (aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). The acetyl group is preferred as the protecting group for the N-terminal amino group. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid may have a pyroglutamic acid-type cyclic structure. When the N-terminal amino acid is glutamine, the protected N-terminal glutamine may have a pyroglutamic acid-type cyclic structure. Examples of protecting groups for the C-terminal carboxy group include groups capable of forming esters or amides. Examples of the group capable of forming an ester or amide include alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups. As a protecting group for the C-terminal carboxy group, an amino group is preferred.

[0073] The first linker and second linker, represented by La and Lb, respectively, are divalent groups as can be seen from the chemical structure of formula (I). The total number of atoms constituting the main chain of the first linker and the main chain of the second linker is 5 to 7. Use of first linkers and second linkers having such numbers of atoms allows regioselective modification of the lysine residues at positions 288 / 290 of the heavy chain in the immunoglobulin unit with affinity peptide-containing groups, and also facilitates precise control of the average bond ratio between the antibody and the affinity peptide-containing group within the desired range (1.5 to 2.5). In the present invention, the average bond ratio between the antibody and a predetermined group (e.g., affinity peptide-containing group) can be confirmed by analyzing MS analysis data using a DAR calculator (Agilent software).

[0074] Considering that the total number of atoms constituting the main chain of the first linker and the main chain of the second linker is 5 to 7, the number of atoms constituting the main chain of the first linker is 1 to 6, and the number of atoms constituting the main chain of the second linker is 1 to 6. More specifically, the relationship between the numbers of atoms constituting the main chain of the first linker and the second linker is as follows:

[0075] [Table 1]

[0076] The main chains of the first linker and the second linker are composed of a chain structure, a cyclic structure, or a structure containing a combination thereof. When the main chain is a chain structure that does not contain a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure. On the other hand, when the main chain is a structure that contains a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms constituting the cyclic structure as the number of atoms in the main chain. Specifically, the number of atoms in the main chain in a cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (see, for example, the bolded paths in (a) to (d) below). When the main chain is a structure that contains a combination of a chain structure and a cyclic structure, the number of atoms in the main chain can be determined by adding the number of atoms in the chain structure that does not contain a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure. [ka] · is a bond. In the case of (a), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 2. In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 3. In the case of (c), both paths are the shortest paths (equidistant), so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4. In the case of (d), the path of the condensation site is the shortest path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.

[0077] The main chains of the first linker and the second linker are each formed by the number of atoms constituting the main chain as described above. Therefore, the main chain in the first linker and the second linker is set to have 1 to 6 of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR d -(R d represents a hydrogen atom or a substituent.) may be composed of —O—, —S—, or a group consisting of a combination of two or more (for example, two or three) of these.

[0078] The divalent straight chain hydrocarbon group is a straight chain alkylene, a straight chain alkenylene, or a straight chain alkynylene. The straight-chain alkylene is a straight-chain alkylene having 1 to 6 carbon atoms, and preferably a straight-chain alkylene having 1 to 4 carbon atoms. Examples of straight-chain alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, and preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethylenylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, and preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of linear alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. The divalent linear hydrocarbon group is preferably a linear alkylene group.

[0079] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. By appropriately setting the two bonds in such a divalent cyclic hydrocarbon group, the number of atoms constituting the main chain as described above can be set to 1 to 6 (the same applies hereinafter to groups having a cyclic structure). The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene group.

[0080] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocycle preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferable. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. The divalent heterocyclic group is preferably a divalent aromatic heterocyclic group.

[0081] W represents an oxygen atom or a sulfur atom, and preferably represents an oxygen atom.

[0082] La and Lb represent a first linker and a second linker, respectively. The main chains of the first linker and the second linker may be composed of only carbon atoms, or may be composed of a combination of carbon atoms and heteroatoms (e.g., oxygen atoms, nitrogen atoms, sulfur atoms). However, from the viewpoint of ease of organic synthesis and improved stability, they may be composed of only carbon atoms. In certain cases, the main chains of the first linker and the second linker may be composed of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, or a group consisting of a combination of two or more of these (e.g., 2 to 4, preferably 2 or 3).

[0083] In a specific embodiment, the main chain of the first linker represented by La is preferably designed so that the number of atoms constituting the main chain as described above is 2 to 4. In this case, the main chain of the second linker represented by Lb is preferably designed so that the number of atoms constituting the main chain as described above is 1 to 5.

[0084] In another specific embodiment, the main chain of the first linker represented by La is preferably designed so that the number of atoms constituting the main chain as described above is 2. In this case, the main chain of the second linker represented by Lb is preferably designed so that the number of atoms constituting the main chain as described above is 3 to 5.

[0085] The main chain of the first linker represented by La is preferably composed of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. Because La is a moiety contained in the thiol group-introduced antibody derivative and the conjugate of the antibody and the functional substance, the thiol group-introduced antibody derivative and the conjugate preferably do not contain -C(=O)- or -C(=S)-, which are relatively less stable (i.e., more reactive) than divalent linear hydrocarbon groups, divalent cyclic hydrocarbon groups, and divalent heterocyclic groups. Because La is a moiety contained in the thiol group-introduced antibody derivative, and the group is preferably low in steric hindrance so as not to interfere with the reaction between the thiol group of the thiol group-introduced antibody derivative and the functional substance, and in light of ease of organic synthesis, the main chain of the first linker represented by La is more preferably composed of a divalent linear hydrocarbon group having 2 to 4 carbon atoms. The main chain of the first linker represented by La is even more preferably an ethylene group, a propylene group, or a butylene group, with an ethylene group being particularly preferred.

[0086] On the other hand, the main chain of the second linker represented by Lb is contained in the antibody intermediate, but is not contained in the thiol group-introduced antibody derivative produced from the antibody intermediate, or in the conjugate of the antibody and the functional substance, so the stability of the main chain is unlikely to be a problem. Furthermore, when producing a thiol group-introduced antibody derivative from an antibody intermediate, the thiocarbonyl group (S- Although the thiol group generated by cleavage between the thio group and the carbonyl group in C(=O) remains in the antibody, the second linker-containing structural unit represented by C(=W)-Lb-C(=O)-Y does not remain in the antibody, and therefore, there is no problem even if this second linker is decomposed during the production. In other words, unlike the main chain in the first linker, the stability of the main chain in the second linker is less likely to be a problem. Therefore, the main chain in the second linker can be a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR d -(R drepresents a hydrogen atom or a substituent.) It can be suitably composed of -O-, -S-, or a group consisting of a combination of two or more (e.g., 2 or 3) of these. The number of atoms constituting the main chain of the second linker is preferably 1 to 5, and more preferably 3 to 5.

[0087] Preferably, the main chain of the second linker represented by Lb is a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, -C(=O)-, -C(=S)-, -NR d -(R d represents a hydrogen atom or a substituent.) It may be composed of -O-, -S-, or a group consisting of a combination of two or more (e.g., two or three) of these. The number of atoms constituting the main chain of the second linker is preferably 1 to 5, and more preferably 3 to 5.

[0088] More preferably, the main chain of the second linker represented by Lb may be composed of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, -C(=O)-, -C(=S)-, -O-, -S-, or a combination of two or more (e.g., two or three) of these. The number of atoms constituting the main chain of the second linker is preferably 1 to 5, and more preferably 3 to 5.

[0089] Even more preferably, the main chain of the second linker represented by Lb may be composed of a straight-chain alkylene, arylene, -C(=O)-, -C(=S)-, -O-, -S-, or a combination of two or more (e.g., two or three) of these. The number of atoms constituting the main chain of the second linker is preferably 1 to 5, and more preferably 3 to 5.

[0090] Particularly preferably, the main chain in the second linker represented by Lb may be propylene or m-phenylene.

[0091] The groups constituting the main chain in the first linker and the second linker may have, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2 substituents. Furthermore, the above-mentioned Rd, which is one of the groups constituting the main chain in the first linker and the second linker, may represent a substituent, as described above. Examples of such substituents include the following: (i') a halogen atom; (ii') a monovalent hydrocarbon group; (iii') aralkyl; (iv') a monovalent heterocyclic group; (v')R e -O-, R e -C(=O)-, R e -OC(=O)- or R e -C(=O)-O-(R e represents a hydrogen atom or a monovalent hydrocarbon group; or (vi')NR f R g -, NR f R g -C(=O)-, NR f R g -C(=O)-O- or R f -C(=O)-NR g -(R f and R g are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; (vii') Nitro groups, sulfate groups, sulfonate groups, cyano groups, and carboxyl groups.

[0092] The halogen atoms, monovalent hydrocarbon groups, aralkyls, and monovalent heteroatoms in the above substituents The definitions, examples, and preferred examples of the ring group are the same as those of R and R A , and R B The same applies to the halogen atoms, monovalent hydrocarbon groups, aralkyls, and monovalent heterocyclic groups explained in (i) to (iv) above.

[0093] Preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii') aralkyl having 3 to 15 carbon atoms; (iv') a 5- or 6-membered heterocycle; (v')R e -O-, R e -C(=O)-, R e -OC(=O)- or R e -C(=O)-O-(R e represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (vi')NR f R g -, NR f R g -C(=O)-, NR f R g -C(=O)-O- or R f -C(=O)-NR g -(R f and R g are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (vii') The same groups as those listed in (vii') above.

[0094] More preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 12 carbon atoms; (iii')R e -O-, R e -C(=O)-, R e -OC(=O)- or R e -C(=O)-O-(R e represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (iv')NR f R g -, NR f R g -C(=O)-, NR f R g -C(=O)-O- or R f -C(=O)-NR g -(R fand R g are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (v') The same groups as those listed in (vii') above.

[0095] Even more preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 6 carbon atoms; (iii')R e -O-, R e -C(=O)-, R e -OC(=O)- or R e -C(=O)-O-(R e represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; (iv')NR f R g -, NR f R g -C(=O)-, NR f R g -C(=O)-O- or R f -C(=O)-NR g -(R f and R g are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms; or (v') The same groups as those listed in (vii') above.

[0096] Particularly preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 4 carbon atoms; (iii')R e -O-, R e -C(=O)-, R e -OC(=O)- or R e -C(=O)-O-(R e represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; (iv')NR f R g -, NR f Rg -C(=O)-, NR f R g -C(=O)-O- or R f -C(=O)-NR g -(R f and R g are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms; or (v') The same groups as those listed in (vii') above.

[0097] Preferably, the compound represented by formula (I) may be a compound represented by the following formula (I'): [ka] [During the ceremony, X, Y, O, S and W are the same as those in formula (I), Lb represents a second linker having 3 to 5 atoms constituting the main chain; R1, R2, R3, and R4 each independently represent a hydrogen atom or a substituent.

[0098] In formula (I'), the definitions, examples and preferred examples of the substituents represented by R1, R2, R3 and R4 are the same as those of the above-mentioned substituents that may be possessed by the group constituting the main chain in the first linker.

[0099] More preferably, the compound represented by formula (I') may be a compound represented by the following formula (I''). [ka] [During the ceremony, X, Y, O, S and W are the same as those in formula (I), Lb is the same as in formula (I').

[0100] The compounds of the present invention represented by formulas (I) to (I") or salts thereof can be obtained, for example, by synthesizing a compound or salt thereof represented by formulas (I) to (I") in which Y is substituted with a leaving group (a leaving group having a higher leaving ability than X), and then reacting the synthesized compound or salt with an affinity peptide. For example, such a reaction can be carried out in an appropriate reaction system (e.g., an organic solvent, an aqueous solution, or a mixed solvent thereof) at an appropriate temperature (e.g., about 15 to 200°C). The reaction system may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0101] Preferably, in the compound represented by formula (I) to (I″) or a salt thereof, the moiety Y is The compound substituted with a leaving group (a leaving group having a higher leaving ability than X) or a salt thereof may be a compound represented by the following formula (VI) or a salt thereof. [ka] [During the ceremony, X represents a leaving group, X' represents a leaving group having a higher ability to leave than the leaving group X; O represents an oxygen atom, S represents a sulfur atom, W represents an oxygen atom or a sulfur atom; La represents the first linker, Lb represents a second linker, The total number of atoms constituting the main chain of the first linker and the total number of atoms constituting the main chain of the second linker is 5 to 7.

[0102] The leaving group represented by X′, which has a higher leaving ability than the leaving group X, is not particularly limited as long as it is a leaving group which has a higher leaving ability than the leaving group X, and examples thereof include a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, and an N-succinimidyloxy group.

[0103] The definitions, examples, and preferred examples of symbols, terms, and expressions such as X (leaving group), W (oxygen atom or sulfur atom), La (first linker), and Lb (second linker) in formula (VI) are the same as those in the above formula.

[0104] The compound represented by formula (VI) or a salt thereof is useful, for example, as a synthetic intermediate for efficiently producing the compound represented by formula (I) or a salt thereof.

[0105] More preferably, the compound represented by formula (VI) may be a compound represented by the following formula (VI'). [ka] [During the ceremony, X, X', O, S and W are the same as those in formula (VI), Lb represents a second linker having 3 to 5 atoms constituting the main chain; R1, R2, R3, and R4 each independently represent a hydrogen atom or a substituent.

[0106] In formula (VI'), the definitions, examples and preferred examples of the substituents represented by R1, R2, R3 and R4 are the same as those of the above-mentioned substituents that may be possessed by the group constituting the main chain in the first linker.

[0107] Even more preferably, the compound represented by formula (VI') may be a compound represented by the following formula (VI''). [ka] [During the ceremony, X, X', O, S and W are the same as those in formula (VI), Lb is the same as that in formula (VI').

[0108] Particularly preferably, the compound represented by formula (VI'') may be a compound represented by the following formula (VI''-1) or (VI''-2). [ka] [During the ceremony, O, S and W are the same as in formula (VI), F is a fluorine atom.

[0109] The compound represented by formula (VI) or a salt thereof may be a compound represented by the following formula (V) or a salt thereof: It can be prepared from the salt of [ka] [During the ceremony, X represents a leaving group, O represents an oxygen atom, OH represents a hydroxy group; S represents a sulfur atom, W represents an oxygen atom or a sulfur atom; La represents the first linker, Lb represents a second linker, The total number of atoms constituting the main chain of the first linker and the total number of atoms constituting the main chain of the second linker is 5 to 7.

[0110] The definitions, examples, and preferred examples of symbols, terms, and expressions such as X (leaving group), W (oxygen atom or sulfur atom), La (first linker), and Lb (second linker) in formula (V) are the same as those in the above formula.

[0111] The compound represented by formula (VI) or a salt thereof can be obtained by reacting the compound represented by formula (V) or a salt thereof with a carboxyl group-modifying reagent. Examples of the carboxyl group-modifying reagent include pentafluorophenylating reagents (e.g., pentafluorophenyl trifluoroacetate), tetrafluorophenylating reagents (e.g., tetrafluorophenyl trifluoroacetate), paranitrophenylating reagents (e.g., paranitrophenyl trifluoroacetate), and N-succinimidylating reagents (e.g., N-succinimidyl trifluoroacetate). For example, such a reaction can be carried out in an appropriate organic solvent (e.g., an organic solvent containing an alkyl halide (e.g., methyl halide) such as CHCl and an amine such as triethylamine) at an appropriate temperature (e.g., about −10 to 30°C). The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0112] The compound represented by formula (V) or a salt thereof is useful, for example, as a synthetic intermediate for efficiently producing the compound represented by formula (VI) or a salt thereof.

[0113] More preferably, the compound represented by formula (V) may be a compound represented by the following formula (V'). [ka] [During the ceremony, X, O, OH, S and W are the same as those in formula (V), Lb represents a second linker having 3 to 5 atoms constituting the main chain; R1, R2, R3, and R4 each independently represent a hydrogen atom or a substituent.

[0114] In formula (V'), the definitions, examples, and preferred examples of the substituents represented by R1, R2, R3, and R4 are the same as those of the above-mentioned substituents that may be possessed by the group constituting the main chain in the first linker.

[0115] More preferably, the compound represented by formula (V') may be a compound represented by the following formula (V''). [ka] [During the ceremony, X, O, OH, S and W are the same as those in formula (V), Lb is the same as that in formula (V').

[0116] Particularly preferably, the compound represented by formula (V'') may be a compound represented by the following formula (V''-1) or (V''-2). [ka] [During the ceremony, O, OH, S and W are the same as those in formula (V).

[0117] The compound represented by formula (V) can be obtained by reacting a compound represented by XC(=O)-La-SH with a dicarboxylic acid compound represented by HO-C(=W)-Lb-C(=O)-OH, or a cyclic compound produced by an intramolecular condensation reaction of the dicarboxylic acid compound (see, for example, Examples (1-3) and (2-1)). For example, such a reaction can be carried out in an appropriate organic solvent at an appropriate temperature (e.g., about 4 to 60°C). The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0118] The production of the above-mentioned series of compounds or salts thereof can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Such compounds or salts thereof can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0119] 3. Antibody intermediate or salt thereof The present invention provides an antibody intermediate or a salt thereof comprising a structural unit represented by the following formula (II): [ka] [During the ceremony, Ig denotes an immunoglobulin unit containing two heavy chains and two light chains, and Eu The amino groups in the side chains of the lysine residues at positions 288 / 290 in the two heavy chains (according to the Ig numbering system) form amide bonds with the carbonyl groups adjacent to the Ig. Y represents an affinity peptide having a binding region in the CH2 domain of an immunoglobulin unit; O represents an oxygen atom, S represents a sulfur atom, W represents an oxygen atom or a sulfur atom; La represents the first linker, Lb represents a second linker, the total number of atoms constituting the main chain of the first linker and the total number of atoms constituting the main chain of the second linker is 5 to 7; The average ratio r of the above amide bonds per two heavy chains is 1.5 to 2.5.

[0120] The immunoglobulin unit represented by Ig is as described above. The definitions, examples, and preferred examples of symbols, terms, and expressions such as Y (affinity peptide), W (oxygen atom or sulfur atom), La (first linker), and Lb (second linker) in formula (II) are the same as those in the formula above.

[0121] In formula (II), the average ratio (r) of the amide bonds per two heavy chains represents the average ratio of bonds between immunoglobulin units and affinity peptide-containing groups (number of affinity peptide-containing groups / immunoglobulin units). This average ratio is 1.5 to 2.5. This average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. This average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, this average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1.

[0122] Preferably, the structural unit represented by formula (II) may be a structural unit represented by the following formula (II'). [ka] [During the ceremony, Ig, Y, O, S, W and r are the same as those in formula (II), Lb represents a second linker having 3 to 5 atoms constituting the main chain; R1, R2, R3, and R4 each independently represent a hydrogen atom or a substituent.

[0123] In formula (II'), the definitions, examples, and preferred examples of the substituents represented by R1, R2, R3, and R4 are the same as those of the above-mentioned substituents that may be possessed by the group constituting the main chain of the first linker.

[0124] More preferably, the structural unit represented by formula (II') may be a structural unit represented by the following formula (II''). [ka] [During the ceremony, Ig, Y, O, S, W and r are the same as those in formula (II), Lb is the same as that in formula (II').

[0125] The antibody intermediate or salt thereof of the present invention can be obtained by reacting the compound or salt thereof of the present invention with an antibody containing the above-described immunoglobulin unit. In the reaction, the compound or salt thereof of the present invention is first mixed with the antibody. This allows the compound or salt thereof to associate with the antibody via an affinity peptide having affinity for the antibody. After the antibody association, the carbonyl group (XC=O) having a leaving group (X) can regioselectively react with the amino group in the side chain of the lysine residue at positions 288 / 290 of the antibody. This reaction bonds the amino group with the carbonyl group carbon atom, and the leaving group (X) is eliminated from the carbonyl group, thereby obtaining the antibody intermediate or salt thereof of the present invention. The molar ratio of the compound or salt thereof of the present invention to the antibody in the reaction (compound or salt thereof of the present invention / antibody) is not particularly limited, as it varies depending on factors such as the type of compound or salt thereof and antibody. It is, for example, 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 50, and particularly preferably 6 to 30.

[0126] Such a reaction can be carried out appropriately under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in an appropriate reaction system, such as a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0127] The production of an antibody intermediate or a salt thereof can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Regioselectivity can be confirmed by, for example, peptide mapping. Peptide mapping can be performed, for example, by protease treatment and mass spectrometry. The protease is preferably an endoprotease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of affinity peptides introduced can be confirmed by, for example, electrophoresis, chromatography, or mass spectrometry, preferably mass spectrometry. The antibody intermediate or a salt thereof can be appropriately purified by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0128] 4. Thiol group-introduced antibody derivative or salt thereof The present invention provides a thiol group-introduced antibody derivative or a salt thereof, which comprises a structural unit represented by the following formula (III): [ka] [During the ceremony, Ig denotes an immunoglobulin unit containing two heavy chains and two light chains, and Eu The amino groups in the side chains of the lysine residues at positions 288 / 290 in the two heavy chains (according to the Ig numbering system) form amide bonds with the carbonyl groups adjacent to the Ig. O represents an oxygen atom, SH represents a thiol group, La represents the first linker, the number of atoms constituting the main chain of the first linker is 2 to 4, The average ratio r of the above amide bonds per two heavy chains is 1.5 to 2.5.

[0129] The immunoglobulin unit represented by Ig is as described above. The definitions, examples, and preferred examples of symbols, terms, and expressions such as La (first linker) in formula (III) are the same as those in the above formula.

[0130] In formula (III), the average ratio (r) of the amide bonds per two heavy chains indicates the average ratio of bonds between immunoglobulin units and thiol-containing groups (number of thiol-containing groups / immunoglobulin unit). Such an average ratio is 1.5 to 2.5. Such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio is preferably It may be 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1.

[0131] Preferably, the structural unit represented by formula (III) may be a structural unit represented by the following formula (III'). [ka] [During the ceremony, Ig, O, SH and r are the same as those in formula (III), R1, R2, R3, and R4 each independently represent a hydrogen atom or a substituent.

[0132] In formula (III'), the definitions, examples, and preferred examples of the substituents represented by R1, R2, R3, and R4 are the same as those of the above-mentioned substituents that may be possessed by the group constituting the main chain in the first linker.

[0133] More preferably, the structural unit represented by formula (III') may be a structural unit represented by the following formula (III''). [ka] [During the ceremony, Ig, O, SH, and r are the same as those in formula (III).

[0134] The thiol group-introduced antibody derivative or a salt thereof of the present invention can be obtained by subjecting the antibody intermediate or a salt thereof of the present invention to a thioester cleavage reaction. The thioester cleavage reaction can be carried out under conditions (mild conditions as described above) that do not cause denaturation or degradation (e.g., cleavage of amide bonds) of proteins (immunoglobulins / antibodies). More specifically, the thiol group-introduced antibody derivative or a salt thereof can be obtained by subjecting the antibody intermediate or a salt thereof of the present invention to a thioester cleavage reaction. The thioester cleavage reaction can be carried out under conditions that do not cause denaturation or degradation (e.g., cleavage of amide bonds) of proteins (immunoglobulins / antibodies) (e.g., mild conditions as described above). More specifically, the thiol group-introduced antibody derivative or a salt thereof can be obtained by subjecting the antibody intermediate or a salt thereof to a thioester cleavage reaction. The thioester cleavage reaction can be carried out under conditions (mild conditions as described above) that do not cause denaturation or degradation (e.g., cleavage of amide bonds) of proteins (immunoglobulins / antibodies). More specifically, the thiol group-introduced antibody derivative or a salt thereof can be obtained by subjecting the antibody intermediate or a salt thereof to a thioester cleavage reaction. It can be cleaved by stirring in a hydrochloride solution for 1 hour (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160.).

[0135] The production of a thiol group-introduced antibody derivative or a salt thereof can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw material and the molecular weight of the product. Regioselectivity can be confirmed, for example, by peptide mapping. Peptide mapping can be performed, for example, by treatment with a protease (e.g., trypsin, chymotrypsin) and mass spectrometry. Preferred proteases are endoproteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of thiol groups introduced can be confirmed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The thiol group-introduced antibody derivative or a salt thereof can be appropriately purified by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0136] 5. Conjugate of antibody and functional substance or salt thereof The present invention provides a conjugate of an antibody and a functional substance, or a salt thereof, which comprises a structural unit represented by the following formula (IV): [ka] [During the ceremony, Ig denotes an immunoglobulin unit containing two heavy chains and two light chains, and Eu The amino groups in the side chains of the lysine residues at positions 288 / 290 in the two heavy chains (according to the Ig numbering system) form amide bonds with the carbonyl groups adjacent to the Ig. O represents an oxygen atom, S represents a sulfur atom, Z represents a functional substance, La represents the first linker, the number of atoms constituting the main chain of the first linker is 2 to 4, The average ratio r of the above amide bonds per two heavy chains is 1.5 to 2.5.

[0137] The immunoglobulin unit represented by Ig is as described above. The definitions, examples, and preferred examples of symbols, terms, and expressions such as La (first linker) in formula (IV) are the same as those in the above formula.

[0138] The functional substance is not particularly limited as long as it is a substance that imparts a desired function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred. The functional substance may also be a single functional substance, or two or more functional substances linked together. It may also be a substance.

[0139] The drug may be a drug for any disease. Examples of such diseases include cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infection, viral infection), genetic and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver disease, kidney disease, lung disease, circulatory system disease, digestive system disease). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0140] More specifically, the drug is an anticancer drug. Examples of anticancer drugs include chemotherapeutic agents, toxins, radioisotopes, and substances containing the same. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioactive isotopes of hydrogen atoms (e.g., 3 H), radioactive isotopes of carbon atoms (e.g., 14 C), radioactive isotopes of phosphorus atoms (e.g., 32 P), radioactive isotopes of sulfur atoms (e.g., 35 S), radioactive isotopes of yttrium (e.g., 90 Y), radioactive isotopes of technetium (e.g., 99m Tc), radioactive isotopes of indium (e.g., 111 In), radioactive isotopes of iodine atoms (e.g., 123 I, 125 I, 129 I, 131 I), radioactive isotopes of samarium (e.g., 153 Sm), rhenium radiation Sex isotopes (e.g., 186 Re), radioactive isotopes of astatine (e.g., 211 At), radioactive isotopes of bismuth (e.g., 212 More specifically, examples of the drug include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBDs (pyrrolobenzodiazepines), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0141] A labeling substance is a substance that enables detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), and substances containing the same.

[0142] Stabilizers are substances that enable antibody stabilization, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0143] The functional substance may also be a peptide, a protein, a nucleic acid, a low-molecular-weight organic compound, a sugar chain, a lipid, a high-molecular-weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Examples of nucleic acids also include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of low-molecular-weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.

[0144] If the functional substance does not have a functional group that easily reacts with a thiol group, the functional substance may be derivatized to have such a functional group. Derivatization is common knowledge in the art (e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be performed using any crosslinking agent. Alternatively, derivatization may be performed using a specific linker having a desired functional group. For example, such a linker may be one that allows the functional substance and the antibody to be separated by cleavage in an appropriate environment (e.g., intracellular or extracellular). Examples of such linkers include peptidyl linkers that are degraded by specific proteases (e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes) or extracellular proteases (e.g., secreted proteases) (e.g., U.S. Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), and linkers that can be cleaved at local acidic sites present in vivo (e.g., U.S. Pat. Nos. 5,622,929, 5,122,368, and 5,824,805). The linker may also be self-immolative (e.g., WO 02 / 083180, WO 04 / 043493, and WO 05 / 112919). In the present invention, a derivatized functional substance is also simply referred to as a "functional substance."

[0145] In formula (IV), the average ratio (r) of the amide bonds per two heavy chains represents the average ratio of bonds between immunoglobulin units and functional substance-containing groups (number of functional substance-containing groups / immunoglobulin units). This average ratio is 1.5 to 2.5. This average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. This average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, this average ratio may be preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1.9 to 2.1.

[0146] Preferably, the structural unit represented by formula (IV) may be a structural unit represented by the following formula (IV'). [ka] [During the ceremony, Ig, O, S, Z and r are the same as those in formula (IV), R1, R2, R3, and R4 each independently represent a hydrogen atom or a substituent.

[0147] In formula (IV'), the definitions, examples and preferred examples of the substituents represented by R1, R2, R3 and R4 are the same as those of the above-mentioned substituents that may be possessed by the group constituting the main chain in the first linker.

[0148] Preferably, the structural unit represented by formula (IV') may be a structural unit represented by the following formula (IV''). [ka] [During the ceremony, Ig, O, S, Z and r are the same as those in formula (IV).

[0149] The conjugate or salt thereof of the present invention can be obtained by reacting the thiol group-introduced antibody derivative or salt thereof of the present invention with a functional substance. This reaction can be carried out under conditions (such as mild conditions as described above) that do not cause denaturation or degradation (e.g., cleavage of amide bonds) of the protein (immunoglobulin / antibody). The functional substance can be any functional group capable of reacting with a thiol group under mild conditions, but a functional group that readily reacts with a thiol group is preferred. Examples of such functional groups include maleimide groups, disulfide groups, α-haloketones, α-haloamides, benzyl bromides, and iodoalkyls. Alternatively, if the functional substance does not have a functional group readily reactive with a bioorthogonal functional group, a functional substance derivatized as described above can be used. In the reaction, the molar ratio of the functional substance to the thiol group-introduced antibody derivative or its salt (functional substance / thiol group-introduced antibody derivative or its salt) is not particularly limited, as it varies depending on factors such as the type of thiol group-introduced antibody derivative or its salt and functional substance, and the reaction time, but is, for example, 2 or more, preferably 3 or more, and more preferably 5 or more. To ensure that the functional substance reacts sufficiently with the thiol group of the thiol group-introduced antibody derivative in a short reaction time, a sufficient amount (e.g., an excess amount) of the functional substance can be used relative to the thiol group-introduced antibody derivative or its salt.

[0150] The production of a conjugate or a salt thereof can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Regioselectivity can be confirmed, for example, by peptide mapping. Peptide mapping can be performed, for example, by treatment with a protease (e.g., trypsin, chymotrypsin) and mass spectrometry. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of functional substances introduced can be confirmed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The conjugate or a salt thereof can be appropriately purified by any method, such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0151] 6.Applications The compound of the present invention or a salt thereof can regioselectively modify, for example, the lysine residues at positions 288 / 29 of an antibody. Thus, the present invention provides a reagent for derivatizing an antibody, comprising the compound of the present invention or a salt thereof.

[0152] The reagent of the present invention may be provided in the form of a composition further comprising other components. Such other components include, for example, a solution and a stabilizer (e.g., an antioxidant, a preservative). The solution is preferably an aqueous solution. Examples of aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), and buffer solutions (e.g., aqueous phosphate solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), with buffer solutions being preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[0153] The antibody intermediate or salt thereof of the present invention, and the thiol group-introduced antibody derivative or salt thereof of the present invention are useful, for example, as intermediates for preparing conjugates of antibodies and functional substances or salts thereof.

[0154] The conjugate or salt thereof of the present invention is useful, for example, as a pharmaceutical or reagent (e.g., diagnostic agent, research reagent). In particular, the conjugate or salt thereof of the present invention, which is regioselectively modified with a functional substance and in which the average binding ratio between the antibody and the functional substance is highly controlled within a desired range (1.5 to 2.5), is useful as a pharmaceutical. It has been reported that changing the number and binding position of drugs in antibody-drug conjugates (ADCs) can affect the pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs require control of the number and position of drugs to be conjugated. It is believed that maintaining a constant number and position can achieve the expected efficacy and resolve problems such as variations in conjugated drugs and lot-to-lot differences, known as "regulation." Therefore, the conjugate or salt thereof of the present invention can resolve such regulation problems.

[0155] The conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate of the present invention or a salt thereof, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; flavorings such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene. The conjugate of the present invention or a salt thereof may also have any modification that confers stability (eg, PEGylation).

[0156] Suitable formulations for oral administration include solutions in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granule; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution of an effective amount of the active ingredient is dispersed and emulsified in a suitable dispersion medium.

[0157] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0158] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc. of the subject, but can be set appropriately. [Example]

[0159] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0160] Example 1: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity substance for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab with the compound and its analysis. (1-1) Synthesis of IgG1 Fc-binding peptide The peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2), an affinity substance for soluble proteins, was synthesized by Fmoc solid-phase synthesis. A CEM Liberty Blue peptide synthesizer was used. All reagents were from Watanabe Chemical Industry. The resin used was Fmoc-NH-SAL-PEG Resin, HL. Arginine (R), cysteine ​​(C), and histidine (H) were double-coupled. Cleavage from the resin was carried out in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol (94:2.5:1.0:2.5) with stirring for 3 hours. After cleavage, the resin was removed by filtration, and trifluoroacetic acid was removed. Diethyl ether was added to the resulting crystals to perform ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in 0.1% trifluoroacetic acid and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group-bonded silica gel as the packing, eluted with a mixture of water and acetonitrile containing 0.1% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove only the acetonitrile, followed by lyophilization.

[0161] (1-2) Formation of an intramolecular disulfide bond between Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2) The peptide synthesized in (1-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Oxidized glutathione was added to this solution and stirred at room temperature for 20 hours. 2M aqueous trifluoroacetic acid was added to the reaction solution to terminate the reaction, and this was dissolved in 0.05% aqueous trifluoroacetic acid and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material. Elution was performed with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove the acetonitrile, and then frozen. The mixture was lyophilized and dried to obtain the target product (20.0 mg, 4.70 μmol).

[0162] MS(ESI)m / z:z=4 1063.65[M+4H] 4+ ,z=5 851.15[M+5H] 5+

[0163] (1-3) Synthesis of thioester linker (1-3-1) [ka] 3,3'-Dithiodipropionic acid (1 g, 5.0 mmol) was dissolved in THF (10 mL). DMF (100 μL) and oxalyl chloride (1.5 mL, 15.0 mmol) were added at 0 °C. The mixture was stirred at 0 °C for 15 minutes and at room temperature for 1 hour. Benzenethiol (1.53 mL, 15.0 mmol), pyridine (4 mL, 50 mmol), and CHCl (10 mL) were then added at 0 °C and stirred at room temperature for 3 hours. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the crystals were removed, extracted with ethyl acetate and 1 M aqueous hydrochloric acid, and the organic layer was concentrated. The mixture was eluted with a mixture of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fractions containing the product were collected, concentrated under reduced pressure to remove the organic solvent, and then dried in vacuo to obtain the above compound (1.2 g, 3.04 mmol).

[0164] (1-3-2) [ka]

[0165] The compound synthesized in (1-3-1) (1.2 g, 3.04 mmol) was dissolved in a mixed solvent of DMF / HO = 5 / 1, and TCEP·HCl (1.74 g, 6.08 mmol) was added and stirred at room temperature for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (1.18 g, 6.5 mmol).

[0166] (1-3-3) [ka] The compound synthesized in (1-3-2) (200 mg, 1.10 mmol) was dissolved in acetonitrile. (2.0 mL), glutaric anhydride (125.5 mg, 1.10 mmol), DMAP (6.72 mg, 0.06 mmol), and pyridine (0.22 mL) were added and stirred at room temperature for 4 hours. After confirming the reaction by TLC (dichloromethane / methanol = 10 / 1), the mixture was extracted with ethyl acetate and 0.5 M HCl, and the organic layer was concentrated. Elution was performed with a mixed solution of dichloromethane and methanol, and each fraction was confirmed by TLC (dichloromethane / methanol = 10 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (135 mg, 0.45 mmol).

[0167] (1-3-4) [ka]

[0168] The compound synthesized in (1-3-3) (134 mg, 0.45 mmol) was dissolved in CHCl (2.25 mL) and triethylamine (157 μL, 1.13 mmol). Pentafluorophenyl trifluoroacetic acid (154 μL, 0.90 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (96 mg, 0.20 mmol). 1 H NMR (400 MHz, Chloroform-d) δ = 7.44 (s, 5H), 3.23 (t, J=6.9, 2H), 3.01 (t, J=6.9, 2H), 2.77 (dt, J=14.5, 7.3, 4H), 2.16 (t, J=7.3, 2H).

[0169] (1-4) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:2.

[0170] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized in (1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and the thioester linker synthesized in (1-3) (96.0 mg, 201 μmol) was added. The mixture was stirred at room temperature for 24 hours. This was then dissolved in 0.05% aqueous trifluoroacetic acid and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as the packing. Elution was performed with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid. Each fraction was analyzed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above peptide thioester linker conjugate-thiophenol activated compound (15.8 mg, 3.48 μmol).

[0171] MS(ESI)m / z:z=4 1136.80[M+4H] 4+

[0172] (1-5) Specific modification of anti-HER2 IgG antibody trastuzumab and its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (1-4) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer, and mass analysis was performed using ESI-TOFMS. A peak at 148223 was observed for the starting trastuzumab. The product peaks were confirmed to be 152660 (indicating one binding peptide), 157093 (indicating two binding peptides), and 161528 (indicating three binding peptides) (Figure 7).

[0173] (1-6) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (1-5), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The product had a heavy chain with one linker introduced at 55033, and a light chain peak at 23439, the same as the starting material (Figure 8).

[0174] (1-7) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (1-5) using the DAR calculator (software from Agilent), and the results are shown in Table 2. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 2 was 2.0. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0175] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 2, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0176] [Table 2]

[0177] (1-8) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (1-7) was added to a hydroxylamine solution as previously reported (WO2019 / 240287A1) and left to stand at room temperature for 1 hour. After 2 hours, the solution was replaced with 20 mM PBS buffer, 10 mM EDTA (pH 7.4), to obtain a thiol-functionalized antibody derivative. Mass measurement by ESI-TOFMS confirmed a peak at 148409, indicating the progress of the cleavage reaction.

[0178] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0179] (1-9) Peptide mapping by trypsin treatment Peptide mapping was performed on the trastuzumab thiol-introduced construct obtained in (1-8) using the following steps.

[0180] (1-9-1) Trypsinization of trastuzumab thiol-conjugate 10 μL of sample solution, 50 mM ammonium bicarbonate buffer, and 10 μL of 20 mM dithiothreitol in 40% trifluoroethanol were added to a 1.5 mL low-adsorption microtube and heated at 65°C for 1 hour. Then, 10 μL of 50 mM iodoacetamide was added and incubated at room temperature for 30 minutes in the dark. After the incubation, 40 μL of 50 mM ammonium bicarbonate buffer was added and stirred. 10 μL of 20 ng / μL trypsin solution was added and incubated at 37°C for 16 hours. After digestion, 2 μL of 20% trifluoroacetic acid solution was added to stop the reaction, and LC-MS / MS analysis was performed.

[0181] (1-9-2) LC-MS / MS measurement of trastuzumab (Analyzer) Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific) Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

[0182] (HPLC analysis conditions) Trap column: Acclaim PepMap® 100, 75 μm x 2 cm (Thermo Fisher Scientific) Analysis column: ESI-column (NTCC-360 / 75-3-125, 75μm×12.5cm, 3μm (Nikkyo Technos)) Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid in water Flow rate: 300nL / min Sample injection volume: 1 μL Gradient conditions (B%): 2% (0.0-0.5 min), 2% → 30% (0.5-23.5 min), 30% → 75% (23.5-25.5 min), 75% (25.5-35.0 min)

[0183] (Mass spectrometer analysis conditions) Ionization method: ESI, positive mode Scan Type: Data Dependent Acquisition Activation Type: Collision Induced Dissociation (CID) Data acquisition was performed using the accompanying software, Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune. This was performed using Application 2.0 (Thermo Fisher Scientific).

[0184] (1-9-3) Analysis of modification sites of trastuzumab Modification site analysis for the LC-MS / MS measurement results was performed using BioPharma Finder 3.0 (Thermo Fisher Scientific). Analysis using BioPharma Finder was performed with an S / N threshold of 1 and an MS noise level of 0.01% of the peak-top intensity. The digestive enzyme was trypsin, and specificity was set to high. Static modifications included carbamidomethyl (+57.021 Da) modification of cysteine ​​residues with iodoacetamide. Dynamic modifications included oxidation of methionine residues (+15.995 Da) and modification of lysine residues (thiol-introduced carbamidomethylated with iodoacetamide (+145.019 Da)). Filters were set to include only those with a confidence score of 80 or higher, a mass accuracy of 5 ppm or less at the time of peptide identification, and MS / MS observations. Regarding the residue numbers of lysine residues, those on the heavy chain VH domain and light chain are indicated by the numbers in the sequence (i.e., the N-terminal amino acid is number 1; the same applies below), while those on the heavy chain CH1, CH2, and CH3 domains are indicated using EU numbering. In addition, (1) and (2) shown in Figure 9 were used as data of the amino acid sequences to be searched for modification sites.

[0185] (1-9-4) Analysis of modification sites of trastuzumab by LC-MS / MS LC-MS / MS analysis revealed the MS spectrum (measured m / z 577.03606, theoretical m / z 577.03557, tetravalent) of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 10), which contains the lysine residue modification site (+145.019 Da) following trypsin digestion of trastuzumab (Fig. 10). The CID spectrum also revealed a product ion at m / z 682.13 (theoretical m / z 682.01), corresponding to the trivalent y16, indicating modification of the lysine residues at positions 288 and 290 in the EU numbering system of the heavy chain (Fig. 11). Analysis using BioPharma Finder also demonstrated highly selective modification of the lysine residues at positions 288 and 290 (Fig. 12). These results indicated that the trastuzumab thiol-introduced complex obtained in (1-8) above was conjugated site-selectively to Lys288 and Lys290 in the EU numbering system on the heavy chain of the antibody.

[0186] Example 2: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab with the compound and its analysis. (2-1) Synthesis of thioester linker (2-1-1) [ka]

[0187] The compound synthesized in (1-3-2) (295 mg, 1.62 mmol) was dissolved in CHCl (13.5 mL), and 3-(tert-Butoxycarbonyl)benzoic acid (300 mg, 1.35 mmol), DIPEA (700 μL, 2.03 mmol), and PyBOP (843 mg, 1.62 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated. Elution was performed with a mixture of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent. The mixture was then dried in vacuo to obtain the above compound (259 mg, 0.64 mmol).

[0188] (2-1-2) [ka]

[0189] The compound synthesized in (2-1-1) (259 mg, 0.64 mmol) was dissolved in a mixed solution of CHCl / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming that the solution had fallen to the origin by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated and then vacuum dried to obtain the above compound (227 mg, 0.66 mmol).

[0190] (2-1-3) [ka]

[0191] The compound synthesized in (2-1-2) (227 mg, 0.66 mmol) was dissolved in CHCl (3.3 mL) and triethylamine (230 μL, 1.65 mmol). Pentafluorophenyl trifluoroacetic acid (225 μL, 1.32 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (175.3 mg, 0.34 mmol). 1 H NMR (400 MHz, Chloroform-d) δ = 8.79 (t, J=1.8, 1H), 8.43 (dt, J=7.8, 1.5, 1H), 8.30 (dt, J=7.9, 1.5, 1H), 7.70 (t, J=7.8, 1H), 7.45 (s, 4H), 3.45 (t, J=6.9, 2H), 3.14 (t, J=6.9, 2H).

[0192] (2-2) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:2.

[0193] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) synthesized in (1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and the linker (72.0 mg, 141 μmol) was added. The mixture was stirred at room temperature for 24 hours. This was then dissolved in 0.05% aqueous trifluoroacetic acid and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing. Elution was performed with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid. Each fraction was analyzed by LC-MS. The product-containing fractions were collected, concentrated under reduced pressure to remove the acetonitrile, and lyophilized to obtain the above peptide thioester linker-thiophenol-activated product (10.0 mg, 2.19 μmol).

[0194] MS(ESI)m / z:z=4 1145.6[M+4H] 4+

[0195] (2-3) Specific modification of anti-HER2 antibody trastuzumab and its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (2-2) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer. Mass analysis by ESI-TOFMS revealed a peak at 148223 for the starting trastuzumab. Peaks were also confirmed for 152691, where one binding peptide was introduced, 157163, where two binding peptides were introduced, and 161634, where three binding peptides were introduced (Figure 13).

[0196] (2-4) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (2-3), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 55067, and the light chain had a peak at 23439, the same as the starting material (Figure 14).

[0197] (2-5) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (2-3) using the DAR calculator (software from Agilent), and the results are shown in Table 3. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 3 was 2.0. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0198] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 2, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0199] [Table 3]

[0200] (2-6) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody derivative having a thiol group introduced therein can be prepared by subjecting the antibody intermediate obtained in (2-5) to the cleavage reaction of the thioester group described in (1-8). The group-introduced antibody derivative was obtained.

[0201] (2-7) Peptide mapping by trypsin treatment Peptide mapping was performed on the trastuzumab thiol-introduced construct obtained in (2-6) using the following steps.

[0202] (2-7-1) Trypsinization of trastuzumab thiol-conjugated complex The trastuzumab thiol-introduced complex obtained in (2-6) was treated with trypsin in the same manner as in (1-9-1).

[0203] (2-7-2) LC-MS / MS measurement of trastuzumab LC-MS / MS measurement was carried out under the same conditions as in (1-9-2).

[0204] (2-7-3) Analysis of modification sites of trastuzumab The analysis was carried out in the same manner as in (1-9-3).

[0205] (2-7-4) Analysis of modification sites of trastuzumab by LC-MS / MS LC-MS / MS analysis revealed the MS spectrum (measured m / z 577.03571, theoretical m / z 577.03557, tetravalent) of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 10), which contains the lysine residue modification site (+145.019 Da) resulting from trypsin digestion of trastuzumab (Fig. 15). The CID spectrum also revealed a product ion at m / z 682.41 (theoretical m / z 682.01), corresponding to the trivalent y16 ion, indicating modification of the lysine residues at positions 288 and 290 in the EU numbering system of the heavy chain (Fig. 16). Analysis using BioPharma Finder also demonstrated highly selective modification of the lysine residues at positions 288 and 290 (Fig. 17). These results indicated that the trastuzumab thiol-introduced antibody obtained in (2-6) above was conjugated site-selectively to Lys288 and Lys290 in the EU numbering system on the heavy chain of the antibody.

[0206] Example 3: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab with the compound and its analysis. (3-1) Synthesis of thioester linker (3-1-1) [ka]

[0207] The compound synthesized in (1-3-2) (220 mg, 1.21 mmol) was dissolved in CH2Cl2( The mixture was dissolved in 12.0 mL of toluene, and Adipic Acid (530 mg, 3.63 mmol), DIPEA (314 μL, 1.82 mmol), and PyBOP (755 mg, 1.45 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction by TLC (dichloromethane / methanol = 10 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of dichloromethane and methanol, and each fraction was confirmed by TLC (dichloromethane / methanol = 10 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (205 mg, 0.63 mmol).

[0208] (3-1-2) [ka]

[0209] The compound synthesized in (3-1-1) (205 mg, 0.63 mmol) was dissolved in CHCl (3.15 mL) and triethylamine (220 μL, 1.58 mmol). Pentafluorophenyl trifluoroacetic acid (215 μL, 1.26 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (200 mg, 0.41 mmol). 1 H NMR (400 MHz, Chloroform-d) δ = 7.44 (s, 5H), 3.20 (t, J=7.0, 2H), 3.00 (t, J=6.9, 2H), 2.66 (dt, J=28.3, 7.4, 5H), 1.79 (ddt, J=20.4, 15.2, 7.5, 5H), 1.57 - 1.38 (m, 3H).

[0210] (3-2) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:2.

[0211] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form an intramolecular disulfide bond) synthesized in (1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and the linker (69.0 mg, 141 μmol) was added. The mixture was stirred at room temperature for 24 hours. This was then dissolved in 0.05% aqueous trifluoroacetic acid and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-bonded silica gel as a packing. Elution was performed with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid. Each fraction was analyzed by LC-MS. The product-containing fractions were collected, concentrated under reduced pressure to remove the acetonitrile, and lyophilized to obtain the above peptide thioester linker-thiophenol-activated product (7.5 mg, 1.65 μmol).

[0212] MS (ESI) m / z: z = 4 1140.50 [M + 4H] 4+

[0213] (3-3) Specific modification of anti-HER2 antibody trastuzumab and its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (3-2) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer. Mass analysis by ESI-TOFMS revealed a peak at 148223 for the starting trastuzumab. Peaks were confirmed for 152676, where one binding peptide was introduced, 157126, where two binding peptides were introduced, and 161572, where three binding peptides were introduced (Figure 18).

[0214] (3-4) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (3-3), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 55048, and the light chain had a peak at 23439, the same as the starting material (Figure 19).

[0215] (3-5) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (3-3) using the DAR calculator (software from Agilent), and the results are shown in Table 4. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 4 was 1.9. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 1.9) represented by the following structural formula was confirmed.

[0216] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 2, The average ratio r of these amide bonds per two heavy chains is 1.9.

[0217] [Table 4]

[0218] (3-6) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (3-5) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein.

[0219] Example 4: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab with the compound and its analysis. (4-1) Synthesis of thioester linker (4-1-1) [ka]

[0220] tBu-3-Sulfanylpropanoate (500 mg, 3.08 mmol) was dissolved in tetrahydrofuran (7 mL), triethylamine (0.64 mL, 4.62 mmol) was added, and then malonyl chloride (0.15 mg, 1.54 mmol) was added at 0°C and stirred for 3 hours. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (104 mg, 0.26 mmol).

[0221] (4-1-2) [ka]

[0222] The compound synthesized in (4-1-1) (104 mg, 0.26 mmol) was dissolved in a mixed solution of CHCl / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming that the solution had fallen to the origin by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated and then vacuum dried to obtain the above compound (104 mg, 0.37 mmol).

[0223] (4-1-3) [ka]

[0224] The compound synthesized in (4-1-2) (104 mg, 0.37 mmol) was dissolved in CHCl (1.85 mL) and triethylamine (130 μL, 0.93 mmol). N-Succinimidyl Trifluoroacetate (156 mg, 0.74 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 1 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 1 / 1). The fractions containing the product were collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (66.8 mg, 0.14 mmol). 1 H NMR (400 MHz, Chloroform-d) δ = 3.84 (s, 2H), 3.28 (t, J=6.9, 2H), 3.19 (t, J=6.8, 2H), 3.00 (t, J=6.8, 2H), 2.74 (t, J=6.8, 2H).

[0225] (4-2) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:2.

[0226] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2) (29.7 mg, 7.00 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized in (1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and a linker (66.8 mg, 0.14 mmol) was added and stirred at room temperature for 4 hours. This was then dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing. The product was subjected to system chromatography, eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above peptide thioester linker conjugate-NHS activated compound (13 mg, 2.82 μmol).

[0227] MS(ESI)m / z:z=4 1153.10[M+4H] 4+

[0228] (4-3) Specific modification of anti-HER2 antibody trastuzumab and its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (4-2) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer. Mass analysis by ESI-TOFMS revealed a peak at 148223 for the starting trastuzumab. Peaks were also confirmed for 152722, where one binding peptide was introduced, 157215, where two binding peptides were introduced, and 161708, where three binding peptides were introduced (Figure 20).

[0229] (4-4) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (4-3), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50594 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 55091, and the light chain had a peak at 23439, the same as the starting material (Figure 21).

[0230] (4-5) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (4-3) using the DAR calculator (software from Agilent), and the results are shown in Table 5. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 5 was 1.8. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 1.8) represented by the following structural formula was confirmed.

[0231] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 2, The average ratio r of these amide bonds per two heavy chains is 1.8.

[0232] [Table 5]

[0233] (4-6) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (4-5) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein.

[0234] Example 5: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab using the compound and its analysis. (5-1) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:3.

[0235] A linker was attached to Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC-NH2 (sequence number 3) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (1-1) in the same manner as in Example 2 (2-2), to obtain the above peptide thioester linker conjugate-thiophenol activated compound (10.0 mg, 2.19 μmol).

[0236] MS(ESI)m / z:z=4 1145.6[M+4H] 4+

[0237] (5-2) Specific Modification of Anti-HER2 Antibody Trastuzumab and Analysis by ESI-TOFMS The peptide linker conjugate synthesized in (5-1) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was The buffer was replaced with 0 mM ammonium acetate. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks were confirmed at 152707, where one binding peptide was introduced, at 157188, where two binding peptides were introduced, and at 161676, where three binding peptides were introduced (Figure 22).

[0238] (5-3) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (5-2), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 55077, and the light chain had a peak at 23439, the same as the starting material (Figure 23).

[0239] (5-4) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (5-2) using the DAR calculator (software from Agilent), and the results are shown in Table 6. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 6 was 2.0. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0240] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 3, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0241] [Table 6]

[0242] (5-5) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (5-4) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein.

[0243] Example 6: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity substance for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab with the compound and its analysis. (6-1) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:4.

[0244] A linker was attached to Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (1-1) in the same manner as in Example 2 (2-2), to obtain the above peptide thioester linker conjugate-thiophenol activated compound (22.2 mg, 4.82 μmol).

[0245] MS(ESI)m / z:z=4 1152.4[M+4H] 4+

[0246] (6-2) Specific Modification of Anti-HER2 Antibody Trastuzumab and Analysis by ESI-TOFMS The peptide linker conjugate synthesized in (6-1) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was The buffer was replaced with 0 mM ammonium acetate. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks were confirmed at 152720, where one binding peptide was introduced, 157216, where two binding peptides were introduced, and 161716, where three binding peptides were introduced (Figure 24).

[0247] (6-3) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (6-2), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 55091, and the light chain had a peak at 23439, the same as the starting material (Figure 25).

[0248] (6-4) Confirmation of the peptide / antibody binding ratio of a specific modified form of trastuzumab using a DAR calculator The peptide / antibody binding ratio of the MS data analyzed in (6-2) was confirmed using a DAR calculator (Agilent software), and the results are shown in Table 7. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 7 was 2.0. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0249] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 4, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0250] [Table 7]

[0251] (6-5) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (6-4) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein.

[0252] Example 7: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab using the compound and its analysis. (7-1) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:5.

[0253] A linker was attached to Ac-NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 5) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (1-1) in the same manner as in Example 2 (2-2), to obtain the above peptide thioester linker conjugate-thiophenol activated compound (12.0 mg, 2.69 μmol).

[0254] MS(ESI)m / z:z=4 1115.8[M+4H] 4+

[0255] (7-2) Specific Modification of Anti-HER2 Antibody Trastuzumab and Analysis by ESI-TOFMS The peptide linker conjugate synthesized in (7-1) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer. Mass analysis by ESI-TOFMS revealed a peak at 148223 for the starting trastuzumab. Peaks at 152573 for the compound with one binding peptide introduced and 156927 for the compound with two binding peptides introduced were confirmed (Figure 26).

[0256] (7-3) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex prepared in (7-2) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, a heavy chain peak was observed at 50596 and a light chain peak was observed at 23439 for the raw material trastuzumab. The reaction product had a heavy chain with one linker introduced at 54942, and the light chain had a peak at 23439, the same as the raw material (Figure 27).

[0257] (7-4) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (7-2) using the DAR calculator (software from Agilent), and the results are shown in Table 8. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 8 was 2.0. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0258] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 5, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0259] [Table 8]

[0260] (7-5) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (7-4) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein.

[0261] (7-6) Peptide mapping by trypsin treatment Peptide mapping was performed on the trastuzumab thiol-introduced construct obtained in (7-5) using the following steps.

[0262] (7-6-1) Trypsinization of trastuzumab thiol-conjugate The trastuzumab thiol-introduced complex obtained in (7-5) was treated with trypsin in the same manner as in (1-9-1).

[0263] (7-6-2) LC-MS / MS measurement of trastuzumab LC-MS / MS measurement was carried out under the same conditions as in (1-9-2).

[0264] (7-6-3) Analysis of modification sites of trastuzumab The analysis was carried out in the same manner as in (1-9-3).

[0265] (7-6-4) Analysis of modification sites of trastuzumab by LC-MS / MS LC-MS / MS analysis revealed the MS spectrum (measured m / z 769.04506, theoretical m / z 769.04482, trivalent) of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 10), an 18-residue peptide containing a lysine residue modification site (a thiol-introduced product (+145.019 Da)) following trypsin digestion of trastuzumab (Figure 28). The CID spectrum also revealed a product ion at m / z 1022.71 (theoretical m / z 1022.51), corresponding to the divalent y16, indicating modification of the lysine residues at positions 288 and 290 in the EU numbering system of the heavy chain (Figure 29). Analysis using BioPharma Finder also demonstrated highly selective modification of the lysine residues at positions 288 and 290 (Figure 30). These results indicated that the trastuzumab thiol-introduced antibody obtained in (7-5) above was conjugated site-selectively to Lys288 and Lys290 in the EU numbering system on the heavy chain of the antibody.

[0266] Example 8: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity substance for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab using the compound and its analysis. (8-1) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:6.

[0267] Ac-MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 6) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (1-1) was conjugated with a linker in the same manner as in Example 2 (2-2) to obtain the peptide thioester linker conjugate-thiophenol activated complex (16.8 mg, 3.87 μmol). obtained.

[0268] MS(ESI)m / z:z=4 1087.3[M+4H] 4+

[0269] (8-2) Specific Modification of Anti-HER2 Antibody Trastuzumab and Analysis by ESI-TOFMS The peptide linker conjugate synthesized in (8-1) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer. Mass analysis by ESI-TOFMS revealed a peak at 148223 for the starting trastuzumab. Peaks were confirmed at 152457 for one binding peptide, 156692 for two binding peptides, and 160929 for three binding peptides (Figure 31).

[0270] (8-3) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (8-2), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 54829, and the light chain had a peak at 23439, the same as the starting material (Figure 32).

[0271] (8-4) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was determined for the MS data analyzed in (8-2) using the DAR calculator (software from Agilent), and the results are shown in Table 9. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 9 was 2.0. Therefore, the formation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0272] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 6, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0273] [Table 9]

[0274] (8-5) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (8-4) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein described in (1-8).

[0275] Example 9: Synthesis of a compound (peptide thioester linker-thiophenol-activated compound) having an affinity for soluble proteins, a cleavable moiety, and a reactive group, and modification of the anti-HER2 antibody trastuzumab using the compound and its analysis. (9-1) Binding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:7.

[0276] A linker was attached to Ac-QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 7) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (9-1) in the same manner as in Example 2 (2-2), to obtain the above peptide thioester linker conjugate-thiophenol activated compound (14.1 mg, 3.35 μmol).

[0277] MS(ESI)m / z:z=4 1054.4[M+4H] 4+

[0278] (9-2) Specific Modification of Anti-HER2 Antibody Trastuzumab and Analysis by ESI-TOFMS The peptide linker conjugate synthesized in (9-1) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 10 mM of the peptide test compound was added. 3.38 μL of the drug (10 equivalents relative to the antibody) was added and stirred at room temperature for 1 hour. The reaction solution was then replaced with 20 mM ammonium acetate buffer. Mass analysis using ESI-TOFMS revealed a peak at 148223 for the raw trastuzumab. Peaks were also confirmed for 152236 (where one binding peptide was introduced), 156430 (where two binding peptides were introduced), and 160541 (where three binding peptides were introduced) (Figure 33).

[0279] (9-3) Confirmation of heavy chain selectivity of specific modified trastuzumab by ESI-TOFMS analysis under reducing conditions To the antibody-peptide complex produced in (9-2), 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added and stirred at room temperature for 15 minutes. Mass measurement by ESI-TOFMS revealed that the starting trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439. The reaction product had a heavy chain with one linker introduced at 54698, and the light chain had a peak at 23439, the same as the starting material (Figure 34).

[0280] (9-4) Confirmation of peptide / antibody binding ratio of specific modified trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (9-2). The results are shown in Table 10. The average peptide / antibody binding ratio calculated from the DAR peak and % Area in Table 10 was 2.0. Therefore, the antibody intermediate (average peptide / antibody binding ratio: 2.0) represented by the following structural formula was 2.0) was confirmed to be generated.

[0281] [ka] [wherein Ig represents an immunoglobulin unit (IgG) comprising two heavy chains and two light chains, and an amide bond is formed between the amino group in the side chain of the lysine residue at position 288 / 290 in the two heavy chains according to EU numbering and the carbonyl group adjacent to Ig; Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 7, The average ratio r of the above amide bonds per two heavy chains is 2.0.

[0282] [Table 10]

[0283] (9-5) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (9-4) was subjected to the cleavage reaction of the thioester group described in (1-8) to obtain the antibody derivative having a thiol group introduced therein.

[0284] (Summary of Examples 1 to 9) The relationship between linker length and peptide / antibody binding ratio (PAR) in the example compounds was investigated. As a result, compounds having a linker length of 7 to 9 atoms, which is the number of atoms constituting the main chain connecting the reactive moiety [X(leaving group)-C=O] with the lysine residue at position 288 / 290 of the heavy chain in the immunoglobulin unit to the binding moiety [O=CY(affinity peptide)] with the affinity peptide (compounds represented by formula (I) in which the total number of atoms constituting the main chain in the first linker and the main chain in the second linker is 5 to 7 atoms), showed an average PAR in the desired range (1.5 to 2.0).

[0285] [Table 11] 1) Linker length: The number of atoms constituting the main chain connecting the reactive moiety [X (leaving group)-C=O] with the lysine residue at position 288 / 290 of the heavy chain in the immunoglobulin unit and the binding moiety [O=CY (affinity peptide)] with the affinity peptide 2) Binding ratio: binding ratio between peptide and antibody (peptide / antibody) (Same below)

[0286] [Table 12]

[0287] [Reference examples 1~5] The following peptide thioester linker conjugate-NHS-activated product or peptide thioester linker conjugate-thiophenol-activated product was synthesized with reference to the above Examples, and specific modification of the anti-HER2 antibody trastuzumab and analysis by ESI-TOFMS were performed. Furthermore, as in the Examples, the peptide / antibody binding ratio (PAR) of the specifically modified trastuzumab was confirmed using a DAR calculator. Compounds with linker lengths of 6 or less or 11 or more atoms (compounds represented by formula (I) where the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker corresponds to less than 4 or 9 or more atoms) showed an average PAR of 0.5 or less.

[0288] [Table 13]

[0289] Example 10: Site-selective modification of multiple distinct target regions of IgG1 Fc with IgG1 Fc affinity peptide reagents and synthesis of antibody-drug conjugates (10-1) Site-selective modification of anti-HER2 antibody trastuzumab followed by cleavage of the thioester group to prepare a thiol-introduced antibody derivative The following peptide reagent, as previously reported (WO2019 / 240287A1), was dissolved in dimethylformamide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 20 mM sodium acetate buffer (pH 5.5), and 3.38 μL (10 equivalents relative to the antibody) of the 10 mM peptide reagent was added and stirred at room temperature for 1 hour. Hydroxylamine solution was then added as previously reported (WO2019 / 240287A1), and the mixture was allowed to stand at room temperature for 1 hour. The mass of the resulting thiol-introduced antibody was measured by ESI-TOFMS, revealing a peak at 148760, indicating the progression of the hydroxylamine cleavage reaction. Considering that the peptide reagent below is a reagent that enables modification of the lysine residues at positions 246 / 248 of the IgG heavy chain, it is considered that the lysine residues at positions 246 / 248 of the IgG heavy chain were modified with the peptide reagent below.

[0290] [ka] The above amino acid sequence is the amino acid sequence of SEQ ID NO:11.

[0291] (10-2) Site-selective modification of multiple distinct target regions of the anti-HER2 antibody trastuzumab The thiol-incorporated antibody solution synthesized in (10-1) was replaced with 50 mM HEPES buffer (pH 8.2). To this solution, 10 equivalents of the dimethylformamide solution of the peptide linker conjugate synthesized in (3-2) were added relative to the antibody, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. The mass of the resulting antibody was measured by ESI-TOFMS, and a peak at 157531 was confirmed, indicating that two binding peptides had been introduced into the thiol-incorporated antibody synthesized in (3-2).

[0292] (10-3) Preparation of thiol-group-introduced antibody derivatives by cleavage of thioester groups To the antibody intermediate obtained in (10-2), a hydroxylamine solution was added according to a previous report (WO2019 / 240287A1), and the mixture was allowed to stand at room temperature for 1 hour. After 2 hours, the mixture was replaced with 20 mM PBS buffer, 10 mM EDTA (pH 7.4), to obtain a thiol-group-introduced antibody derivative. Mass measurement by ESI-TOFMS confirmed a peak at 148760, indicating the progress of the cleavage reaction. Therefore, the obtained thiol-group-introduced antibody derivative was confirmed to have the following structure.

[0293] [ka] (Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the amino groups in the side chains of the lysine residues at positions 288 and 290 in the two heavy chains (according to Eu numbering) form amide bonds with the carbonyl groups adjacent to the Ig. Similarly, the amino groups in the side chains of the lysine residues at positions 246 and 248 in the two heavy chains (according to Eu numbering) form amide bonds with the carbonyl groups adjacent to the Ig. The average ratios of the above amide bonds per two heavy chains, r (positions 288 and 290) and n (positions 246 and 248), were evaluated to be 1.9 and 2.0, respectively.)

[0294] (10-4) Preparation of thiol-group-introduced antibody derivatives by cleavage of drug-mimicking thioester groups A 20 μM solution of the thiolated antibody (10-3) in pH 7.4 PBS buffer was added with 10 equivalents of a 1.25 mM DMF solution of MC-VC-PAB-MMAE (as previously reported (Org. Process Res. Dev. 2019, 23, 12, 2647-2654). The mixture was left at room temperature for 2 hours, and then purified using NAP-5 Columns (GE Healthcare) to obtain the ADC. Mass analysis by ESI-TOFMS confirmed a peak at 154029, indicating the presence of four MC-VC-PAB-MMAE units.

[0295] [ka]

Claims

1. The following formula (IV): 【Chemical 1】 [During the ceremony, Ig represents an antibody, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue present at positions 288 / 290 in the two heavy chains according to EU numbering; O represents an oxygen atom; S represents a sulfur atom; Z represents a functional substance, La represents a first linker; the number of atoms constituting the main chain of the first linker is 2 to 4, The average ratio r of the amide bonds per two heavy chains is 1.5 to 2.

5. A conjugate of an antibody and a functional substance, or a salt thereof, comprising a structural unit represented by the following formula:

2. The conjugate of an antibody and a functional substance according to claim 1 , or a salt thereof, wherein the antibody is an Fc region protein or an Fc fusion protein.

3. The conjugate or salt thereof according to claim 1, wherein specific amino acid residues other than the lysine residues at positions 288 / 290 in the two heavy chains are further modified.

4. The conjugate or a salt thereof according to claim 3, wherein the specific amino acid residues are lysine residues located at positions 246 / 248 in the two heavy chains.

5. The structural unit represented by the formula (IV) is represented by the following formula (IV′): 【Chemistry 2】 [During the ceremony, Ig, O, S, Z and r are the same as those in formula (IV), R １ , R ２ , R ３ , and R ４ and each independently represent a hydrogen atom or a substituent.

6. The structural unit represented by the formula (IV′) is represented by the following formula (IV″): 【Chemistry 3】 [During the ceremony, The conjugate or salt thereof according to claim 5 , wherein Ig, O, S, Z and r are the same as those in formula (IV).

7. (1) The following formula (I): 【Chemistry 4】 [During the ceremony, X represents a leaving group; Y represents an affinity peptide having a binding region to the CH2 domain of the antibody, O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom or a sulfur atom; La represents a first linker; Lb represents a second linker; the total number of atoms constituting the main chain of the first linker and the main chain of the second linker is 5 to 7, The following formula (II): 【Chemistry 5】 [During the ceremony, Ig represents the antibody, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue located at positions 288 / 290 in the two heavy chains according to EU numbering, Y, O, S, W, La, and Lb are the same as those in formula (I), the average ratio r of amide bonds per two heavy chains is 1.5 to 2.5; (2) subjecting the antibody intermediate or a salt thereof to a thioester cleavage reaction, The following formula (III): 【Chemistry 6】 [During the ceremony, SH represents a thiol group; wherein Ig, La, and r are the same as those defined in formula (II), and a thiol group-introduced antibody derivative or a salt thereof is produced, the thiol group-introduced antibody derivative or a salt thereof comprising a structural unit represented by the formula (II):

8. (1) The following formula (I): 【Chemistry 7】 [During the ceremony, X represents a leaving group; Y represents an affinity peptide having a binding region to the CH2 domain of the antibody, O represents an oxygen atom; S represents a sulfur atom; W represents an oxygen atom or a sulfur atom; La represents a first linker; Lb represents a second linker; the total number of atoms constituting the main chain of the first linker and the main chain of the second linker is 5 to 7, The following formula (II): 【Chemistry 8】 [During the ceremony, Ig represents the antibody, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue located at positions 288 / 290 in the two heavy chains according to EU numbering, Y, O, S, W, La, and Lb are the same as those in formula (I), the average ratio r of amide bonds per two heavy chains is 1.5 to 2.5; (2) subjecting the antibody intermediate or a salt thereof to a thioester cleavage reaction, The following formula (III): 【Chemistry 9】 [During the ceremony, SH represents a thiol group; wherein Ig, O, La, and r are the same as those in formula (II), or a salt thereof; (3) reacting the thiol group-introduced antibody derivative or a salt thereof with a functional substance, The following formula (IV): 【Chemistry 10】 [During the ceremony, S represents a sulfur atom; Z represents a functional substance, wherein Ig, O, La, and r are the same as those in formula (III).

9. The following formula (III): 【Chemistry 11】 [During the ceremony, Ig represents an antibody, and forms an amide bond with a carbonyl group adjacent to Ig via an amino group in the side chain of a lysine residue present at positions 288 / 290 in the two heavy chains according to EU numbering; O represents an oxygen atom; SH represents a thiol group; La represents a first linker; the number of atoms constituting the main chain of the first linker is 2 to 4, the average ratio r of the amide bonds per two heavy chains is 1.5 to 2.

5. The following formula (IV): 【Chemistry 12】 [During the ceremony, S represents a sulfur atom; Z represents a functional substance, wherein Ig, O, La, and r are the same as those in formula (III).

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