A compound or a salt thereof, and an antibody obtained therefrom

Abstract

The present invention provides: a compound which makes it possible to modify an antibody with a functional substance in a regioselective manner and also makes it possible to easily adjust the binding ratio between the antibody and the functional substance to a value falling within a desired range, or a salt of the compound; and an antibody produced using the compound or the salt thereof. More specifically, the present invention provides: a compound represented by formula (I): [wherein X represents a leaving group; Y represents an affinity peptide that has a binding region in a CH2 domain in an immunoglobulin unit containing 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; and La represents a first linker, and Lb represents a second linker, in which the total of the number of atoms constituting a main chain in the first linker and the number of atoms constituting a main chain in the second linker is 5 to 7] or a salt thereof; and an antibody produced using the compound or the salt thereof.

Description

Technical Field

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

Background Art

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

[0003] The heterogeneity of ADCs such as T-DM1 has been a problem since the beginning of their development. That is, since a small molecule drug is randomly reacted with about 70 to 80 lysine residues in the antibody, the drug-antibody ratio (DAR) and the conjugation position are not constant. Usually, in such a random conjugation method, the DAR is in the range of 0 to 8, and it has been found that a plurality of antibody drugs with different drug binding numbers are generated. In recent years, it has been reported that when the drug binding number and binding position of an ADC are changed, the pharmacokinetics, drug release rate, and effect change. From these facts, it is required to control the number and position of the conjugated drugs in the next-generation ADC. If the number and position are constant, it is considered that problems such as expected efficacy, variations in conjugated drugs, and lot-to-lot differences, so-called regulation problems, can be solved.

[0004] Methods for site-selective modification of antibodies have been studied worldwide, and most of them are genetic engineering methods or modification methods using enzymes. Regarding genetic engineering modification methods, although site selectivity and number selectivity can be controlled, problems such as a decrease in the expression efficiency of the antibody itself (a decrease in the overall yield when preparing an ADC) have been pointed out. In addition, it has been a problem that it takes a long time to construct an antibody expression system.

[0005] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method, which enables site-selective modification of antibodies by a chemical synthesis method, has been developed (Patent Document 1). In this method, a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide is reacted with an antibody, and site-selective modification of the antibody has been successful. However, in the ADC prepared by this method, the antibody and the drug are bound via a linker containing a peptide moiety. The peptide moiety has potential immunogenicity and is easily hydrolyzed in the blood. Therefore, the ADC prepared by this method has room for improvement in that it contains a peptide moiety in the linker.

[0006] As an improved method of the above C-CAP method, a technique has been reported that can prepare an antibody having a site-selective functional substance (e.g., drug) without containing a peptide moiety as a linker by a chemical synthesis method using a predetermined compound containing an affinity peptide (Patent Documents 2 to 6). Avoiding the use of a linker containing a peptide moiety is desirable in clinical applications. In these techniques, as positions of amino acid residues in the antibody that can be site-selectively modified with a drug, a plurality of positions corresponding to various amino acid residues (e.g., lysine residue, tyrosine residue, serine residue, and threonine residue) in the CH2 and CH3 domains have been proposed. However, it is not always easy to site-selectively 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 Problems to be Solved by the Invention

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

[0009] As a result of intensive studies, the present inventors have selected the lysine residues at positions 288 / 290 of the heavy chain in the immunoglobulin unit as the modification positions of the antibody, and by using a specific compound as a compound that enables site-specific modification of the lysine residues, the antibody can be site-selectively modified with a functional substance, and moreover, it has been found that it becomes easy to highly control the average ratio of the binding between the immunoglobulin unit and the functional substance (number of functional substances / immunoglobulin unit) within a desired range (1.5 to 2.5). Such a specific compound has a total of 7 to 9 atoms constituting the main chain that connects the reactive moiety [X (leaving group)-C=O] with the lysine residues at positions 288 / 290 of the heavy chain in the immunoglobulin unit and the binding moiety [O=C-Y (affinity peptide)] with the affinity peptide (that is, in the compound represented by formula (I), the total number of atoms constituting the main chain in the first linker and the total number of atoms constituting the main chain in the second linker is 5 to 7). Based on such findings, the present inventors have succeeded in developing a compound represented by formula (I) or a salt thereof, and a reagent for derivatizing an antibody containing them. The present inventors have also found that by using a compound represented by formula (I) or a salt thereof, a specific antibody, that is, an antibody in which the lysine residues at positions 288 / 290 in the antibody are site-selectively modified with a modifying group, and the average ratio of the binding between the immunoglobulin unit and the modifying group (number of modifying groups / immunoglobulin unit) is highly controlled within a desired range (1.5 to 2.5) (antibody intermediate, thiol group-introduced antibody derivative, and conjugate of antibody and functional substance) can be prepared, and thus the present invention has been completed.

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

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

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

[12] The compound represented by formula (I) is a reagent of

[11] represented by formula (I').

[13] The compound represented by formula (I') is a reagent of

[12] represented by formula (I'').

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

[15] The antibody intermediate of

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

[16] The antibody intermediate of

[14] or

[15] or a salt thereof, wherein the antibody intermediate is a human IgG intermediate. The structural unit represented by the formula (II) is an antibody intermediate represented by the formula (II') among

[14] to

[16] or a salt thereof. The structural unit represented by the formula (II') is an antibody intermediate represented by the formula (II'') among

[17] or a salt thereof. A thiol group-introduced antibody derivative containing a structural unit represented by the formula (III) or a salt thereof. The thiol group-introduced antibody derivative according to

[19] or a salt thereof, wherein specific amino acid residues other than the lysine residues present at positions 288 / 290 in the two heavy chains are further modified. The thiol group-introduced antibody derivative according to

[20] or a salt thereof, wherein the specific amino acid residue is a lysine residue present at positions 246 / 248 in the two heavy chains. The structural unit represented by the formula (III) is a thiol group-introduced antibody derivative according to any one of

[19] to

[21] represented by the formula (III') or a salt thereof. The structural unit represented by the formula (III') is a thiol group-introduced antibody derivative according to

[22] represented by the formula (III'') or a salt thereof. An antibody and a conjugate of a functional substance containing a structural unit represented by the formula (IV) or a salt thereof. The conjugate according to

[24] or a salt thereof, wherein specific amino acid residues other than the lysine residues present at positions 288 / 290 in the two heavy chains are further modified. The conjugate according to

[25] or a salt thereof, wherein the specific amino acid residue is a lysine residue present at positions 246 / 248 in the two heavy chains. The structural unit represented by the formula (IV) is a conjugate according to any one of

[24] to

[26] represented by the formula (IV') or a salt thereof. The structural unit represented by the formula (IV') is a conjugate according to

[27] represented by the formula (IV'') or a salt thereof. A compound represented by the formula (V) or a salt thereof. The compound represented by the formula (V) is a compound according to

[29] represented by the formula (V') or a salt thereof. The compound represented by formula (V’) is the compound of

[30] represented by formula (V’’) or a salt thereof. The compound represented by formula (V’’) is the compound of

[31] represented by formula (V’’-1) or (V’’-2) or a salt thereof. The compound represented by formula (VI) or a salt thereof. The leaving group having a higher leaving ability than the leaving group X is a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyloxy group, and the compound of

[33] or a salt thereof. The compound represented by formula (VI) is the compound of

[33] or

[34] represented by formula (VI’) or a salt thereof. The compound represented by formula (VI’) is the compound of

[35] represented by formula (VI’’) or a salt thereof. The compound represented by formula (VI’’) is the compound of

[36] represented by formula (VI’’-1) or (VI’’-2) or a salt thereof. A method for producing an antibody intermediate or a salt thereof, which comprises reacting the compound represented by formula (I) or a salt thereof with an antibody containing an immunoglobulin unit to produce an antibody intermediate containing the structural unit represented by formula (II) or a salt thereof. 〔39〕(1) Reacting the compound represented by formula (I) or a salt thereof with an antibody containing an immunoglobulin unit to produce an antibody intermediate containing the structural unit represented by formula (II) or a salt thereof; and (2) Subjecting the antibody intermediate or a salt thereof to a cleavage reaction of a thioester to produce a thiol group-introduced antibody derivative containing the structural unit represented by formula (III) or a salt thereof. A method for producing a thiol group-introduced antibody derivative or a salt thereof. 〔40〕(1) Reacting the compound represented by formula (I) or a salt thereof with an antibody containing an immunoglobulin unit to produce an antibody intermediate containing the structural unit represented by formula (II); (2) subjecting the antibody intermediate or a salt thereof to a thiolester cleavage reaction to produce a thiol group-introduced antibody derivative or a salt thereof containing a structural unit represented by formula (III); and (3) reacting the 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 containing a structural unit represented by formula (IV). A method for producing a conjugate of an antibody and a functional substance or a salt thereof.

[41] Further comprising reacting a compound represented by formula (VI) or a salt thereof with an affinity peptide having a binding region in the CH2 domain in an immunoglobulin unit containing two heavy chains and two light chains to produce a compound represented by formula (I) or a salt thereof. The method according to any one of

[38] to

[40] .

[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’) reacting a compound represented by formula (VI) or a salt thereof with an affinity peptide having a binding region in the CH2 domain in an immunoglobulin unit containing two heavy chains and two light chains to produce a compound represented by formula (I) or a salt thereof. The method according to any one of

[38] to

[40] .

[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 or a salt thereof containing a structural unit represented by formula (II) to a thiolester 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 thiolester 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 an antibody and a functional substance conjugate or a salt thereof, which comprises reacting a thiol group-introduced antibody derivative or a salt thereof with a functional substance to produce an antibody and a functional substance conjugate or a salt thereof containing a structural unit represented by the formula (IV).

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

[0011] The compound represented by the formula (I) or a salt thereof 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 the binding of the immunoglobulin unit to the affinity peptide-containing group (number of affinity peptide-containing groups / immunoglobulin unit) is within a desired range (1.5 to 2.5). Therefore, the compound represented by the formula (I) or a salt thereof is useful as a reagent for antibody derivatization. Further, according to the compound represented by the formula (I) or a salt thereof, an antibody intermediate represented by the 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 an affinity peptide-containing group and the average ratio of the binding of the immunoglobulin unit to the affinity peptide-containing group (number of affinity peptide-containing groups / immunoglobulin unit) is highly controlled within a desired range can be provided. Furthermore, an antibody prepared using the antibody intermediate represented by the formula (II) or a salt thereof as a raw material can inherit the site selectivity and the average ratio of the binding of the antibody intermediate or a salt thereof. Therefore, a thiol group-introduced antibody derivative represented by the formula (III) or a salt thereof having the above site selectivity and average ratio of binding, and an antibody and a functional substance conjugate represented by the formula (IV) or a salt thereof can be provided. Also provided are compounds represented by formula (V) and (VI) or salts thereof, which are synthetic intermediates enabling efficient production of the compound represented by formula (I) or a salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] 1. Definition of General Terms In the present invention, the term "antibody" is as follows. Also, the term "immunoglobulin unit" corresponds to the divalent monomer unit that is the basic component of such an antibody, and is a unit containing two heavy chains and two light chains. Therefore, for the immunoglobulin unit, the origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of lysine residue, and definitions, examples, and preferred examples of position selectivity 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, birds (e.g., chickens), etc. 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), draft animals (e.g., horses, sheep), preferably primates or rodents, and more preferably humans.

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

[0016] Any antigen can be used as the antigen of the antibody. For example, such antigens include proteins [including oligopeptides and polypeptides. It may be a protein modified with a biomolecule such as a sugar (e.g., glycoprotein)], sugar chains, nucleic acids, and low molecular weight compounds. Preferably, the antibody may be an antibody using a protein as an antigen. 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) Cancer region 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, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, 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-cadherin, 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, transferrin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lewis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 integrin, 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 diseases CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0021] (4) Infectious diseases Clostridium Difficile toxin B, Cytomegalovirus, RS 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) Hereditary and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

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

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

[0025] (8) Blood 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, sirukumab, dinutuximab, ortuxizumab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemrolizumab (IgG4), mepolizumab, elotuzumab, daratumumab, ikesekiizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, lirilumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesvacumab, brodalumab (IgG2), olaratumab). (When not referring to the IgG subtype, it indicates IgG1).

[0028] For the position of lysine residues in the antibody and the position of the constant region of the heavy chain (e.g., CH2 domain), follow EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). Thus, for human IgG, the lysine residue at position 288 corresponds to the 58th residue of the human IgG CH2 region, and the lysine residue at position 290 corresponds to the 60th residue of the human IgG CH2 region. The notation of positions 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 in an antibody can be site-selectively modified. As used herein, "site-selective" or "site-selectivity" means that a given structural unit capable of binding to a specific amino acid residue in an antibody is preferentially distributed in a specific region of the antibody, even though the specific amino acid residue is not preferentially distributed in a specific region in the antibody. Thus, expressions related to site-selectivity such as "having site-selectively", "site-selective binding", "binding with site-selectivity", etc. mean that the retention rate or binding rate of a given structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention rate or binding rate of the structural unit in a non-target region containing a plurality of amino acid residues of the same type as the specific amino acid residue in the target region. Such site-selectivity may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or even 100%. According to the present invention, the lysine residues at positions 288 / 290 can be site-selectively modified without using a linker containing a peptide. The peptide moiety has potential immunogenicity and is easily hydrolyzed in the blood. Therefore, avoiding the use of a linker containing a peptide moiety is desirable in clinical applications.

[0030] In the present invention, as long as the lysine residues at positions 288 / 290 in the antibody are site-specifically modified, specific amino acid residues at other positions may be further site-specifically modified. For example, methods for site-specifically modifying specific amino acid residues at a predetermined position in an antibody are described in International Publication No. WO 2018 / 199337, International Publication No. WO 2019 / 240288, International Publication No. WO 2019 / 240287, and International Publication No. WO 2020 / 090979. Such specific amino acid residues include amino acid residues having side chains that are easy to modify (e.g., amino group, carboxy group, amide group, hydroxy group, thiol group) (e.g., lysine residue, aspartic acid residue, glutamic acid residue, asparagine residue, glutamine residue, threonine residue, serine residue, tyrosine residue, cysteine residue), preferably lysine residues having side chains containing an amino group, tyrosine residues having side chains containing a hydroxy group, serine residues, and threonine residues, or cysteine residues having side chains containing a thiol group, more preferably lysine residues, even more preferably lysine residues at positions 246 / 248, or lysine residue at position 317, and particularly preferably lysine residues at positions 246 / 248. The notation of 246 / 248 indicates that the lysine residue at position 246 or 248 is the target.

[0031] In the present invention, examples of the term "salt" include 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 salts with ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkyl ethanolamine, dialkyl ethanolamine, 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 its salt The present invention provides a compound represented by the following formula (I) or a salt thereof.

Chemical formula

[0033] In formula (I) and other formulas presented in connection with the present invention, the -(hyphen) indicates that the two units present on both sides thereof 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 carbonyl groups present on both adjacent sides, 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 the reaction between the carbon atom of the carbonyl group adjacent to X and the amino group. Those skilled in the art can appropriately set such a leaving group. Examples of such a leaving group include the following: (a) R-S (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and S represents a sulfur atom); (b) R-O (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and O represents an oxygen atom); (c) R A -(R B -)N (wherein R A and R B each independently represent a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and N represents a nitrogen atom); or (d) A halogen atom.

[0035] Preferably, the leaving group represented by X may be the following: (a) R-S (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and S represents a sulfur atom); (b) R-O (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and O represents an oxygen atom); or (c) R A -(R B -)N (wherein R A and R B each independently represent a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent). It may be.

[0036] More preferably, the leaving group represented by X may be as follows: (a) R-S (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and S represents a sulfur atom); or (b) R-O (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and O represents an oxygen atom).

[0037] Even more preferably, the leaving group represented by X may be as follows: (a) R-S (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 as follows: (a’) R-S (wherein R represents a monovalent aromatic hydrocarbon group which may have a substituent (e.g., phenyl), and S represents a sulfur atom).

[0039] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[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] The monovalent chain hydrocarbon group means a hydrocarbon group composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain hydrocarbon group include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl, and alkynyl may be either linear or branched.

[0042] As the alkyl, an alkyl having 1 to 12 carbon atoms is preferable, an alkyl having 1 to 6 carbon atoms is more preferable, and an alkyl having 1 to 4 carbon atoms is even more preferable. The carbon atoms of the substituent are not included in the above carbon atom numbers. Examples of the 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] As the alkenyl, an alkenyl having 2 to 12 carbon atoms is preferable, an alkenyl having 2 to 6 carbon atoms is more preferable, and an alkenyl having 2 to 4 carbon atoms is even more preferable. The carbon atoms of the substituent are not included in the above carbon atom numbers. Examples of the alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0044] As the alkynyl, an alkynyl having 2 to 12 carbon atoms is preferable, an alkynyl having 2 to 6 carbon atoms is more preferable, and an alkynyl having 2 to 4 carbon atoms is even more preferable. The carbon atoms of the substituent are not included in the above carbon atom numbers. Examples of the alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0045] As the monovalent chain hydrocarbon group, alkyl is preferable.

[0046] The monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as the ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be either a monocyclic or polycyclic one. However, it is not necessary to be composed only of alicyclic hydrocarbons, and a chain structure may be included in a part thereof. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, and these may be either monocyclic or polycyclic.

[0047] As the cycloalkyl, cycloalkyl having 3 to 12 carbon atoms is preferable, cycloalkyl having 3 to 6 carbon atoms is more preferable, and cycloalkyl having 5 to 6 carbon atoms is even more preferable. The carbon atoms of the substituents are not included in the above carbon atom numbers. Examples of the cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0048] As the cycloalkenyl, cycloalkenyl having 3 to 12 carbon atoms is preferable, cycloalkenyl having 3 to 6 carbon atoms is more preferable, and cycloalkenyl having 5 to 6 carbon atoms is even more preferable. The carbon atoms of the substituents are not included in the above carbon atom numbers. Examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0049] As the cycloalkynyl, cycloalkynyl having 3 to 12 carbon atoms is preferable, cycloalkynyl having 3 to 6 carbon atoms is more preferable, and cycloalkynyl having 5 to 6 carbon atoms is even more preferable. The carbon atoms of the substituents are not included in the above carbon atom numbers. Examples of the cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutinyl, cyclopentynyl, and cyclohexynyl.

[0050] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferable.

[0051] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it does not necessarily consist only of aromatic rings, and may contain a chain structure or an alicyclic hydrocarbon in part, and the aromatic ring may be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, aryl having 6 to 12 carbon atoms is preferable, aryl having 6 to 10 carbon atoms is more preferable, and aryl having 6 carbon atoms is even more preferable. The carbon atoms of the substituent are not included in the above carbon atom numbers. Examples of aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

[0052] As the monovalent aromatic hydrocarbon group, phenyl is preferable.

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

[0054] The monovalent heterocyclic group means a group obtained by removing one hydrogen atom from the heterocyclic ring 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 selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom.

[0055] As the monovalent aromatic heterocyclic group, an aromatic heterocyclic group having 1 to 15 carbon atoms is preferable, an aromatic heterocyclic group having 1 to 9 carbon atoms is more preferable, and an aromatic heterocyclic group having 1 to 6 carbon atoms is even more preferable. The carbon atoms of the substituent are not included in the above carbon atom numbers. 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 carbon atoms of the substituents are not included in the number of carbon atoms. 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, as the monovalent heterocyclic group, a 5- or 6-membered heterocyclic group is preferable.

[0058] The above R, R A , and R B In the "optionally substituted monovalent hydrocarbon group" and "optionally substituted monovalent heterocyclic group" represented by, the number of "substituents" 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 atom; (ii) monovalent hydrocarbon group; (iii) monovalent heterocyclic group; (iv) aralkyl; (v) R a -O-, R a -C(=O)-, R a -O-C(=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 each represents a hydrogen atom or a monovalent hydrocarbon group.); (vii) Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

[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 monovalent hydrocarbon group and monovalent heterocyclic group described for R, R A , and R B above, respectively.

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

[0061] Preferably, the substituent may be as follows: (i) Halogen atom; (ii) Alkyl, phenyl, or naphthyl having 1 to 12 carbon atoms; (iii) Aralkyl having 3 to 15 carbon atoms; (iv) 5- or 6-membered heterocycle; (v) R a -O-, R a -C(=O)-, R a -O-C(=O)-, or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl having 1 to 12 carbon atoms.); (vi) NR b R c -, NR b R c -C(=O)-, NRb 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 an alkyl group having 1 to 12 carbon atoms.); or (vii) the same group as those listed in (vii) above.

[0062] More preferably, the substituent may be as follows: (i) a halogen atom; (ii) an alkyl group having 1 to 12 carbon atoms; (iii) R a -O-, R a -C(=O)-, R a -O-C(=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 an alkyl group having 1 to 12 carbon atoms.); or (v) the same group as those listed in (vii) above.

[0063] Even more preferably, the substituent may be as follows: (i) a halogen atom; (ii) an alkyl group having 1 to 6 carbon atoms; (iii) R a -O-, R a -C(=O)-, R a -O-C(=O)-, or R a -C(=O)-O-(R arepresents 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 an alkyl group having 1 to 6 carbon atoms.); or (v) the same group as those listed in (vii) above.

[0064] Particularly preferably, the substituent may be as follows: (i) a halogen atom; (ii) an alkyl group having 1 to 4 carbon atoms; (iii) R a -O-, R a -C(=O)-, R a -O-C(=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 an alkyl group having 1 to 4 carbon atoms.); or (v) the same group as those listed in (vii) above.

[0065] The affinity peptide represented by Y has a binding region to the CH2 domain in an immunoglobulin unit containing two heavy chains and two light chains. As the affinity peptide, any peptide having a binding region to the CH2 domain in the immunoglobulin unit can be used. The affinity peptide includes an amino acid residue (e.g., lysine residue, proline residue, tryptophan residue, tyrosine residue, serine residue, threonine residue) containing a side chain including a moiety (e.g., amino group, hydroxy group) capable of binding to a carbonyl group (C=O) adjacent to Y, and may form an amide bond with the carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue. Preferably, the affinity peptide contains a lysine residue and may form an amide bond with the carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue. As such an affinity peptide, for example, those disclosed in International Publication No. WO2016 / 186206, International Publication No. WO2018 / 199337, International Publication No. WO2019 / 240288, International Publication No. WO2019 / 240287, and International Publication No. WO2020 / 090979, as well as various affinity peptides disclosed in the documents cited in these international publications can be used.

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

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

[0068] At least two separated cysteine residues in each amino acid sequence of the above-mentioned affinity peptide can form a cyclic peptide by a disulfide bond. Alternatively, in the above-mentioned peptide, the thiol groups in the two cysteine residues may be linked by a carbonyl group-containing linker represented by the following.

[0069]

Chemical formula

[0070] The dashed portion of the carbonyl group-containing linker represented above means the bonding portion with a thiol group. Such a linker is more stable against a reduction reaction or the like than an ordinary disulfide bond. Such a peptide can be prepared, for example, by the method described in International Publication No. 2016 / 186206.

[0071] The amino acids constituting the above affinity peptide may be either L-form or D-form, but the L-form is preferred (in the examples, all amino acid residues constituting the peptide are in the L-form). The above affinity peptide may be modified at specific amino acid residues by a crosslinking agent and linked to the compound of formula (I) or a salt thereof. Examples of such specific amino acid residues include lysine residues, aspartic acid residues, and glutamic acid residues, but preferably lysine residues. Examples of the crosslinking agent include crosslinking agents preferably containing two or more succinimidyl groups such as DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), crosslinking agents preferably containing two or more imidic acid moieties such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2HCl), and DMS (dimethyl suberimidate·2HCl), and crosslinking agents having an SS bond such as DTBP (dimethyl 3,3’-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., International Publication No. 2016 / 186206).

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

[0073] The first linker and the second linker represented by La and Lb, respectively, are divalent groups as understood from the chemical structure of formula (I). The total number of atoms constituting the main chain in the first linker and the total number of atoms constituting the main chain in the second linker are 5 to 7. By using the first linker and the second linker having such a number of atoms, the lysine residues at positions 288 / 290 of the heavy chain in the immunoglobulin unit can be site-selectively modified with an affinity peptide-containing group, and moreover, it becomes easy to highly control the average ratio of the binding between the antibody and the affinity peptide-containing group within a desired range (1.5 to 2.5). In the present invention, the average ratio of the binding between the antibody and a predetermined group (e.g., an affinity peptide-containing group) can be confirmed by analyzing the MS analysis data with DAR calculator (software of Agilent).

[0074] In view of the fact that the total number of atoms constituting the main chain in the first linker and the total number of atoms constituting the main chain in the second linker are 5 to 7, the number of atoms constituting the main chain in the first linker is 1 to 6, and the number of atoms constituting the main chain in the second linker is 1 to 6. More specifically, the relationship between the number of atoms constituting the main chains in the first linker and the second linker is as follows.

[0075]

Table 1

[0076] The main chains in the first linker and the second linker are composed of a structure including a chain structure, a cyclic structure, or a combination thereof. When the main chain is a chain structure not including 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 including a cyclic structure, the number of predetermined atoms constituting the cyclic structure can be determined by counting as the number of atoms in the main chain. Specifically, the number of atoms in the main chain in the cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (for example, refer to the bold paths in (a) to (d) below). When the main chain is a structure including a combination of a chain structure and a cyclic structure, the number of atoms in the main chain can be determined by adding up the number of atoms in the chain structure not including the cyclic structure and the number of atoms in the shortest path connecting two bonds in the cyclic structure.

Chemical formula

[0077] The main chains in the first linker and the second linker are set such that the number of atoms constituting the main chain is 1 to 6 as described above. Therefore, the main chains in the first linker and the second linker are a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR d -(R d (wherein R represents a hydrogen atom or a substituent), -O-, -S-, or a group composed of a combination of two or more (e.g., 2 or 3) thereof may be used.

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

[0079] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. By appropriately setting 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 (hereinafter the same applies to groups having a cyclic structure). As the arylene, an arylene having 6 to 14 carbon atoms is preferable, an arylene having 6 to 10 carbon atoms is more preferable, and an arylene having 6 carbon atoms is particularly preferable. Examples of the arylene include phenylene, naphthylene, and anthracenylene. As the divalent non-aromatic cyclic hydrocarbon group, a divalent non-aromatic cyclic hydrocarbon group that is monocyclic or polycyclic and has 3 to 12 carbon atoms is preferable, a divalent non-aromatic cyclic hydrocarbon group that is monocyclic or polycyclic and has 4 to 10 carbon atoms is more preferable, and a divalent non-aromatic cyclic hydrocarbon group that is monocyclic and has 5 to 8 carbon atoms is particularly preferable. Examples of the divalent non-aromatic cyclic hydrocarbon group include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. As the divalent cyclic hydrocarbon group, arylene is preferable.

[0080] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic ring preferably contains at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably contains at least one selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom. 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 the divalent aromatic heterocyclic group 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. The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a non-aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent non-aromatic heterocyclic group include pyrroledionediyl, pyrrolinedionediyl, oxirandiyl, aziridinediyl, azetidinediyl, oxetandiyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolandiyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinodiyl, thiomorpholinodiyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

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

[0082] La and Lb represent a first linker and a second linker, respectively. The main chain in 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 atom, nitrogen atom, sulfur atom). However, from the viewpoints of ease of organic synthesis and improvement of stability, etc., it may be composed of only carbon atoms. In a specific case, the main chain in 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 composed of a combination of two or more (e.g., 2 to 4, preferably 2 or 3) of these.

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

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

[0085] The main chain in 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. Since La is a part contained in a thiol group-introduced antibody derivative and a conjugate of an antibody and a functional substance, the thiol group-introduced antibody derivative and the conjugate preferably do not contain -C(=O)- and -C(=S)- which are relatively less stable (i.e., more reactive) compared to a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, and a divalent heterocyclic group. Since La is a part contained in the thiol group-introduced antibody derivative and is preferably a group with low 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 considering that organic synthesis is easy, the main chain in 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 in the first linker represented by La is even more preferably an ethylene group, a propylene group, or a butylene group, and particularly preferably an ethylene group.

[0086] On the other hand, the main chain in the second linker represented by Lb is a part that is contained in the antibody intermediate but not in the thiol group-introduced antibody derivative produced from the antibody intermediate and the conjugate of the antibody and the functional substance. Therefore, the stability of the main chain is less likely to be a problem. Also, when producing a thiol group-introduced antibody derivative from an antibody intermediate, although the thiol group generated by cleavage between the thiol group and the carbonyl group in the thiocarbonyl group (S-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. Therefore, this second linker may be decomposed in the production without any problem. That is, 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 is 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.), -O-, -S-, or a group composed of a combination of two or more (e.g., 2 or 3) of these can be preferably constituted. The number of atoms constituting the main chain in the second linker is preferably 1 to 5, more preferably 3 to 5.

[0087] Preferably, from the viewpoint of ease of synthesis and the like, the main chain in 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.), -O-, -S-, or a group composed of a combination of two or more (e.g., 2 or 3) of these may be constituted. The number of atoms constituting the main chain in the second linker is preferably 1 to 5, more preferably 3 to 5.

[0088] More preferably, the main chain in 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., 2 or 3) of these. The number of atoms constituting the main chain in the second linker is preferably 1 to 5, more preferably 3 to 5.

[0089] Even more preferably, the main chain in the second linker represented by Lb may be composed of a linear alkylene, arylene, -C(=O)-, -C(=S)-, -O-, -S-, or a combination of two or more (e.g., 2 or 3) of these. The number of atoms constituting the main chain in the second linker is preferably 1 to 5, 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 each have, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents. Further, as described above, Rd, which is one of the groups constituting the main chain in the first linker and the second linker, may represent a substituent. Examples of such substituents include, for example, the following: (i’) A halogen atom; (ii’) A monovalent hydrocarbon group; (iii’) An aralkyl; (iv’) A monovalent heterocyclic group; (v’) R e -O-, R e -C(=O)-, R e -O-C(=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 each represents a hydrogen atom or a monovalent hydrocarbon group.); (vii’) A nitro group, a sulfuric acid group, a sulfonic acid group, a cyano group, and a carboxyl group.

[0092] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, aralkyl, and monovalent heterocyclic group in the above substituents are the same as those of the halogen atom, monovalent hydrocarbon group, aralkyl, and monovalent heterocyclic group described in the above R, R A , and R B and in the above (i) to (iv).

[0093] Preferably, the substituent may be the following: (i’) A halogen atom; (ii’) alkyl, phenyl, or naphthyl having 1 to 12 carbon atoms; (iii’) aralkyl having 3 to 15 carbon atoms; (iv’) 5- or 6-membered heterocyclic ring; (v’) R e -O-, R e -C(=O)-, R e -O-C(=O)-, or R e -C(=O)-O-(R e represents a hydrogen atom or alkyl 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 group as those listed in (vii’) above.

[0094] More preferably, the substituent may be as follows: (i’) halogen atom; (ii’) alkyl having 1 to 12 carbon atoms; (iii’) R e -O-, R e -C(=O)-, R e -O-C(=O)-, or R e -C(=O)-O-(R e represents a hydrogen atom or alkyl 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 f and Rg is the same or different and represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.); or (v’) the same group as those listed in (vii’) above.

[0095] More preferably, the substituent may be as follows: (i’) a halogen atom; (ii’) an alkyl group having 1 to 6 carbon atoms; (iii’) R e -O-, R e -C(=O)-, R e -O-C(=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 an alkyl group having 1 to 6 carbon atoms.); or (v’) the same group as those listed in (vii’) above.

[0096] Particularly preferably, the substituent may be as follows: (i’) a halogen atom; (ii’) an alkyl group having 1 to 4 carbon atoms; (iii’) R e -O-, R e -C(=O)-, R e -O-C(=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 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 each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.); or (v’) The same groups as those listed in (vii’).

[0097] Preferably, the compound represented by formula (I) may be a compound represented by the following formula (I’).

Chemical formula

[0098] In formula (I’), the definitions, examples and preferred examples of the substituents represented by R 1 , R 2 , R 3 , and R 4 are the same as those of the above substituents which the group constituting the main chain in the first linker may have.

[0099] More preferably, the compound represented by formula (I’) may be a compound represented by the following formula (I’’).

Chemical formula

[0100] The compound of the present invention represented by the formula (I) to (I'') or a salt thereof can be obtained, for example, by synthesizing a compound in which the moiety Y in the compound represented by the formula (I) to (I'') or a salt thereof is substituted with a leaving group (a leaving group having a higher leaving ability than X) or a salt thereof, and then reacting the synthesized compound or a salt thereof with an affinity peptide. For example, such a reaction can be carried out at an appropriate temperature (e.g., about 15 to 200 °C) in an appropriate reaction system (e.g., an organic solvent, an aqueous solution, or a mixed solvent thereof). 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, the compound in which the moiety Y in the compound represented by the formula (I) to (I'') or a salt thereof is 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. [Chemical formula] [In the formula, X represents a leaving group, X' represents a leaving group having a higher leaving ability 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 a first linker, Lb represents a second linker, The total number of atoms constituting the main chain in the first linker and the total number of atoms constituting the main chain in the second linker are 5 to 7. ]

[0102] The leaving group having a higher leaving ability than the leaving group X represented by X' is not particularly limited as long as it is a leaving group having 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 the symbols, terms, and expressions such as X (leaving group), W (oxygen atom or sulfur atom), La (first linker), Lb (second linker), etc. 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’).

Chemical formula

[0106] In formula (VI’), the definitions, examples, and preferred examples of the substituents represented by R 1 , R 2 , R 3 , and R 4 are the same as those of the above substituents that the group constituting the main chain in the first linker may have.

[0107] Even more preferably, the compound represented by formula (VI’) may be a compound represented by the following formula (VI’’).

Chemical formula

[0108] Particularly preferably, the compound represented by formula (VI'') may be a compound represented by the following formula (VI''-1) or (VI''-2).

Chemical formula

[0109] The compound represented by formula (VI) or a salt thereof can be produced from the compound represented by the following formula (V) or a salt thereof.

Chemical formula

[0110] The definitions, examples, and preferred examples of the symbols, terms, and expressions such as X (leaving group), W (oxygen atom or sulfur atom), La (first linker), Lb (second linker), etc. 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 a suitable organic solvent system (e.g., an organic solvent containing an alkyl halide such as CH 2 Cl 2 etc. (e.g., methyl halide), 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').

Chemical formula

[0114] In formula (V'), R 1 、R 2 、R 3 、and R 4The definitions, examples and preferred examples of the substituents shown by [description] are the same as those of the above substituents which the groups constituting the main chain in the first linker may have.

[0115] More preferably, the compound represented by formula (V') may be a compound represented by the following formula (V'').

Chemical formula

[0116] Particularly preferably, the compound represented by formula (V'') may be a compound represented by the following formula (V''-1) or (V''-2).

Chemical formula

[0117] The compound represented by formula (V) can be obtained by reacting a compound represented by X-C(=O)-La-SH with a dicarboxylic acid compound represented by HO-C(=W)-Lb-C(=O)-OH or a cyclic compound formed 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 a suitable organic solvent system 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] Confirmation of the formation of a series of compounds or salts thereof as described above depends on the specific raw materials and molecular weights of the products. For example, it can be carried out by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), NMR, or mass spectrometry. Such compounds or salts thereof can be appropriately purified by any method such as chromatography (e.g., the chromatography described above, and affinity chromatography).

[0119] 3. Antibody Intermediate or Salt Thereof The present invention provides an antibody intermediate or a salt thereof containing a structural unit represented by the following formula (II). [Chemical Formula] [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig through 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. Y represents an affinity peptide having a binding region to the CH2 domain in the immunoglobulin unit. 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 in the first linker and the total number of atoms constituting the main chain in the second linker are 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 above formula.

[0121] In formula (II), the average ratio (r) of the above amide bonds per two heavy chains indicates the average ratio of the binding between the immunoglobulin unit and the affinity peptide-containing group (number of affinity peptide-containing groups / immunoglobulin unit). Such an average ratio is 1.5 to 2.5. Such an average ratio may preferably be 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 preferably be 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 may preferably 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.

[0122] Preferably, the structural unit represented by formula (II) may be a structural unit represented by the following formula (II’).

Chemical formula

[0123] In formula (II’), R 1 、R 2 、R 3 、and R 4The definitions, examples, and preferred examples of the substituents shown by [reference] are the same as those of the above-mentioned substituents that the groups constituting the main chain in the first linker may have.

[0124] More preferably, the structural unit represented by formula (II') may be a structural unit represented by the following formula (II''). [Chemical formula] [In the formula, 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 of the present invention or a salt thereof can be obtained by reacting the compound of the present invention or a salt thereof with an antibody containing the above immunoglobulin unit. In the reaction, first, the compound of the present invention or a salt thereof is mixed with the antibody. Thereby, the compound of the present invention or a salt thereof can associate with the antibody via an affinity peptide having an affinity for the antibody. Next, after the association of the antibody, a carbonyl group (X-C=O) having a leaving group (X) can react regioselectively with the amino group in the side chain of the lysine residue present at positions 288 / 290 of the antibody. By such a reaction, the amino group and the carbon atom of the carbonyl group are bonded, and the leaving group (X) is detached from the carbonyl group, thereby obtaining the antibody intermediate of the present invention or a salt thereof. The molar ratio of the compound of the present invention or a salt thereof to the antibody in the reaction (the compound of the present invention or a salt thereof / antibody) is not particularly limited because it varies depending on factors such as the compound of the present invention or a salt thereof and the type of the antibody, but 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 reactions can be appropriately carried out under conditions (mild conditions) that cannot cause denaturation and decomposition of proteins (e.g., cleavage of amide bonds). For example, such reactions can be carried out at room temperature (e.g., about 15 - 30 °C) in a suitable reaction system, such as a buffer solution. The pH of the buffer solution is, for example, 5 - 9, preferably 5.5 - 8.5, and more preferably 6.0 - 8.0. The buffer solution may contain a suitable catalyst. The reaction time is, for example, 1 minute - 20 hours, preferably 10 minutes - 15 hours, more preferably 20 minutes - 10 hours, and even more preferably 30 minutes - 8 hours. For details of such reactions, see, for example, G.J.L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G.J.L. Bernardes et al., Chem. Asian J., 4, 630 (2009); B.G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate Chem., 25, 825 (2014).

[0127] Confirmation of the production of the antibody intermediate or its salt depends on the specific raw materials and the molecular weight of the product. For example, it can be carried out by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry. Confirmation of site selectivity can be carried out, for example, by peptide mapping. Peptide mapping can be carried out, for example, by protease treatment and mass spectrometry. As the protease, endoprotease is preferred. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. Confirmation of the number of introduced affinity peptides can be carried out, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The antibody intermediate or its salt 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 contains a structural unit represented by the following formula (III). [Chemical formula] [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig through 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 in 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 (the first linker) in formula (III) are the same as those of the above formula.

[0130] In formula (III), the average ratio (r) of the above amide bonds per two heavy chains indicates the average ratio of the bond between the immunoglobulin unit and the thiol-containing group (the number of thiol-containing groups / immunoglobulin unit). Such an average ratio is 1.5 to 2.5. Such an average ratio may preferably be 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 preferably be 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 may preferably 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’).

Chemical formula

[0132] In formula (III’), the definitions, examples, and preferred examples of the substituents represented by R 1 、R 2 、R 3 、and R 4 are the same as those of the above substituents that the groups constituting the main chain in the first linker may have.

[0133] More preferably, the structural unit represented by formula (III’) may be a structural unit represented by the following formula (III’’).

Chemical formula

[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 thiol ester cleavage reaction. The thiol ester cleavage reaction can be carried out under conditions that do not cause denaturation and decomposition of proteins (immunoglobulins / antibodies) (such as cleavage of amide bonds) (mild conditions as described above). More specifically, it can be cleaved by stirring in a hydroxylamine hydrochloride solution in the range of pH 4.0 to pH 8.0 and 10 mM to 10 M for 1 hour (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148 - 160.).

[0135] Confirmation of the production of a thiol group-introduced antibody derivative or a salt thereof depends on the specific raw materials and the molecular weight of the product. For example, it can be carried out by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. Confirmation of site selectivity can be carried out, for example, by peptide mapping. Peptide mapping can be carried out, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. As the protease, an endoprotease is preferred. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Confirmation of the number of introduced thiol groups can be carried out, 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 contains a structural unit represented by the following formula (IV). [Chemical Formula] [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and forms an amide bond with a carbonyl group adjacent to Ig through 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 in 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.

[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 (the first linker) in formula (IV) are the same as those of the above formula.

[0138] The functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, and preferably drugs or labeling substances. The functional substance may also be a single functional substance or a substance to which two or more functional substances are linked.

[0139] The drug may be a drug for any disease. Examples of such diseases include cancer (e.g., lung cancer, gastric cancer, colorectal 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), cerebrovascular diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), diseases in the orthopedic field (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, digestive organ diseases). The drug may be a preventive or therapeutic drug for the disease or a drug for alleviating side effects.

[0140] More specifically, the drug is an anti-cancer agent. Examples of anti-cancer agents include, for example, chemotherapeutic agents, toxins, radioisotopes or substances containing them. Examples of chemotherapeutic agents include, for example, DNA-damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, platinum compounds. Examples of toxins include, for example, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin). Examples of radioisotopes include, for example, radioisotopes of hydrogen atoms (e.g., 3 H), radioisotopes of carbon atoms (e.g., 14 C), radioisotopes of phosphorus atoms (e.g., 32 P), radioisotopes of sulfur atoms (e.g., 35 S), radioisotopes of yttrium (e.g., 90 Y), radioisotopes of technetium (e.g., 99m Tc), radioisotopes of indium (e.g., 111 In), radioisotopes of iodine atoms (e.g., 123 I, 125 I, 129 I, 131 I), radioisotopes of samarium (e.g., 153 Sm), radioisotopes of rhenium (e.g., 186 Re), radioisotopes of astatine (e.g., 211 At), radioisotopes of bismuth (e.g., 212 Bi). More specifically, examples of the drug include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracycline, dmDNA31, tubulysin.

[0141] A labeling substance is a substance that enables the 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, aptamer), 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), or substances containing them.

[0142] A stabilizer is a substance that enables the stabilization of an antibody. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0143] Functional substances may also be peptides, proteins, nucleic acids, low-molecular-weight organic compounds, sugar chains, lipids, high-molecular-weight polymers, metals (e.g., gold), or chelators. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide pharmaceuticals. 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 general knowledge in the art (e.g., International Publication No. WO 2004 / 010957, U.S. Patent Application Publication No. US 2006 / 0074008 A1, U.S. Patent Application Publication No. US 2005 / 0238649 A1). For example, derivatization may be carried out using any crosslinking agent. Alternatively, derivatization may be carried out using a specific linker having a desired functional group. For example, such a linker may be one that can separate the functional substance and the antibody by cleavage of the linker in an appropriate environment (e.g., intracellular or extracellular). Examples of such linkers include peptidyl linkers (e.g., U.S. Patent No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)) that are cleaved by specific proteases [e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secreted proteases)], and linkers (e.g., U.S. Patent Nos. 5,622,929, 5,122,368; 5,824,805) that can be cleaved at local acidic sites present in vivo. The linker may be self-immolative (e.g., International Publication Nos. WO 02 / 083180, WO 04 / 043493, WO 05 / 112919). In the present invention, the derivatized functional substance is also simply referred to as the "functional substance".

[0145] In formula (IV), the average ratio (r) of the above amide bonds per two heavy chains represents the average ratio of the binding between the immunoglobulin unit and the functional substance-containing group (the number of functional substance-containing groups / immunoglobulin unit). Such an average ratio is from 1.5 to 2.5. Such an average ratio may preferably be 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 preferably be 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 may preferably be from 1.6 to 2.4, more preferably from 1.7 to 2.3, even more preferably from 1.8 to 2.2, and particularly preferably from 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’).

Chemical formula

[0147] In formula (IV’), the definitions, examples and preferred examples of the substituents represented by R 1 、R 2 、R 3 、and R 4 are the same as those of the above substituents that the group constituting the main chain in the first linker may have.

[0148] Preferably, the structural unit represented by formula (IV’) may be a structural unit represented by the following formula (IV’’).

Chemical formula

[0149] The conjugate of the present invention or a salt thereof can be obtained by reacting the thiol group-introduced antibody derivative of the present invention or a salt thereof with a functional substance. Such a reaction can be carried out under conditions that do not cause denaturation and decomposition of proteins (immunoglobulins / antibodies) (e.g., cleavage of amide bonds) (mild conditions as described above). As the functional substance, those having any functional group capable of reacting with a thiol group under mild conditions can be used, but it is preferable to use a functional group that easily reacts with a thiol group. Examples of such functional groups include a maleimide group, a disulfide group, an α-haloketone, an α-haloamide, a benzyl bromide, and an iodoalkyl. Alternatively, when the functional substance does not have a functional group that easily reacts with a bioorthogonal functional group, a derivative derivatized as described above can be used as the functional substance. In the reaction, the molar ratio of the functional substance to the thiol group-introduced antibody derivative or a salt thereof (functional substance / thiol group-introduced antibody derivative or a salt thereof) is not particularly limited because it varies depending on factors such as the thiol group-introduced antibody derivative or a salt thereof, the type of the functional substance, and the reaction time, etc., but is, for example, 2 or more, preferably 3 or more, more preferably 5 or more. In order to sufficiently react the functional substance 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 with respect to the thiol group-introduced antibody derivative or a salt thereof.

[0150] Confirmation of the production of the conjugate or its salt depends on the specific raw materials and molecular weights of the products. For example, it can be carried out by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. Confirmation of site selectivity can be carried out, for example, by peptide mapping. Peptide mapping can be carried out, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. As the protease, endoprotease is preferred. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Confirmation of the number of introduced functional substances can be carried out, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The conjugate or its salt can be appropriately purified by any method such as chromatography (e.g., the chromatography described above, and affinity chromatography).

[0151] 6. Use The compound of the present invention or its salt can, for example, site-selectively modify the lysine residues at positions 288 / 29 of an antibody. Therefore, the present invention provides a reagent for antibody derivatization containing the compound of the present invention or its salt.

[0152] The reagent of the present invention may be provided in the form of a composition further containing other components. Examples of such other components include solutions and stabilizers (e.g., antioxidants, preservatives). As the solution, an aqueous solution is preferred. Examples of the aqueous solution include water (e.g., distilled water, sterilized distilled water, purified water, physiological saline), buffer solutions (e.g., aqueous phosphoric acid solution, Tris-hydrochloric acid buffer solution, carbonic acid-bicarbonate buffer solution, aqueous boric acid solution, glycine-sodium hydroxide buffer solution, citric acid buffer solution), 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 a liquid or powder form (e.g., freeze-dried powder).

[0153] The antibody intermediate of the present invention or a salt thereof, and the thiol group-introduced antibody derivative of the present invention or a salt thereof are useful, for example, as an intermediate for the preparation of a conjugate of an antibody and a functional substance or a salt thereof.

[0154] The conjugate of the present invention or a salt thereof is useful, for example, as a medicine or a reagent (e.g., diagnostic agent, research reagent). In particular, the conjugate of the present invention or a salt thereof, which is site-selectively modified with a functional substance and in which the average ratio of the binding between the antibody and the functional substance is highly controlled within a desired range (1.5 to 2.5), is useful as a medicine. It has been reported that when the number and binding position of the drug in an antibody-drug conjugate (ADC) are changed, the pharmacokinetics, drug release rate, and effects change. From these facts, it is required to control the number and position of the drug to be conjugated in the next-generation ADC. If the number and position are constant, it is considered that problems such as expected efficacy, variations in the conjugated agent, and lot-to-lot differences, so-called regulation problems, can be solved. Therefore, the conjugate of the present invention or a salt thereof can solve 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 excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc., binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc., disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, etc., lubricants such as magnesium stearate, aerosil, talc, sodium lauryl sulfate, etc., fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, orange powder, etc., preservatives such as sodium benzoate, sodium bisulfite, methyl paraben, propyl paraben, etc., stabilizers such as citric acid, sodium citrate, acetic acid, etc., suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc., dispersing agents such as surfactants, diluents such as water, physiological saline, orange juice, etc., base waxes such as cacao butter, polyethylene glycol, white kerosene, etc., but are not limited thereto. The conjugate of the present invention or a salt thereof may also have any modification (e.g., PEGylation) to achieve stability.

[0156] Formulations suitable 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, emulsions in which a solution of an effective amount of the active ingredient is dispersed and emulsified in a suitable dispersion medium, and the like.

[0157] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Examples of pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, tonicity agents, and the like. Also included are aqueous and non-aqueous sterile suspension solutions, which may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, preservatives, and the like.

[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 receptivity of the administration subject, body weight, age, etc., but can be appropriately set.

Example

[0159] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0160] [Example 1: Synthesis of a compound (peptide thioester linker conjugate - thiophenol activator) having an affinity substance, a cleavable moiety, and a reactive group for a soluble protein, and modification and analysis of the anti-HER2 antibody trastuzumab using the compound] (1-1) Synthesis of IgG1 Fc-binding peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH, an affinity substance for a soluble protein 2The peptide of SEQ ID NO: 2 was synthesized by the Fmoc solid-phase synthesis method. The peptide synthesizer used was Liberty Blue manufactured by CEM Corporation. All reagents used were those manufactured by Watanabe Chemical Industries, Ltd. Double coupling was performed for Resin, Fmoc-NH-SAL-PEG Resin, HL, arginine (R), cysteine (C), and histidine (H). Cleavage from the resin was carried out under stirring conditions for 3 hours in a solution of trifluoroacetic acid: water: triisopropylsilane: ethanedithiol = 94:2.5:1.0:2.5. After cleavage, the resin was removed by filtration and trifluoroacetic acid was removed. Diethyl ether was added to the generated crystals for ether precipitation, and the generated white crystals were recovered by filtration. This was dissolved in a 0.1% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as a filler, eluted with a mixed solution of water and acetonitrile containing 0.1% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was recovered, concentrated under reduced pressure to remove only acetonitrile, and then freeze-dried.

[0161] (1-2) Formation of an intramolecular disulfide bond at Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 2) (1-1) The synthesized peptide was dissolved in DMSO, and 0.1 M Tris-HCl pH 8.0 was added. Oxidized glutathione was added to this solution and stirred at room temperature for 20 hours. 2 M aqueous trifluoroacetic acid solution was added to the reaction solution to stop the reaction, which was dissolved in a 0.05% aqueous trifluoroacetic acid solution, subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as a filler, eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was recovered, concentrated under reduced pressure to remove acetonitrile, and then freeze-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)

Chemical formula

[0164] (1 - 3 - 2)

Chemical formula

[0165] (1 - 3 - 1) The compound synthesized (1.2 g, 3.04 mmol) in DMF / H 2It was dissolved in a mixed solvent of O = 5 / 1, and TCEP·HCl (1.74 g, 6.08 mmol) was added, followed by stirring at room temperature for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), it was extracted with ethyl acetate and water, and the organic layer was concentrated. It was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fraction containing the product was collected, the organic solvent was removed by concentration under reduced pressure, and then vacuum drying was performed to obtain the above compound (1.18 g, 6.5 mmol).

[0166] (1-3-3)

Chemical formula

[0167] (1-3-4)

Chemical formula

[0168] (134 mg, 0.45 mmol) of the compound synthesized in (1-3-3) was added with CH 2 Cl 2(2.25 mL), triethylamine (157 μL, 1.13 mmol) was added and dissolved. Pentafluorophenyl trifluoroacetate (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 carried out with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected, the organic solvent was removed by concentration under reduced pressure, and then vacuum drying was performed 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 the peptide and the linker

Chemical Structure

[0170] Ac - FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC - NH synthesized in (1 - 2) 2(SEQ ID NO:2) (30.0 mg, 7.06 μmol, where the 5th and 34th cysteines each form an intramolecular disulfide bond) was dissolved in N,N-dimethylformamide (1.00 mL), and the thioester linker synthesized in (1-3) (96.0 mg, 201 μmol) was added, followed by stirring at room temperature for 24 hours. This was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as the filler, eluting with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above peptide thioester linker conjugate - thiophenol activator (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 analysis by ESI-TOFMS (1-4) The peptide linker conjugate synthesized was dissolved in dimethyl sulfoxide to make it 10 mM. 500 μg of anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and when the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks of 152660 with one conjugated peptide introduced, 157093 with two conjugated peptides introduced, and 161528 with three conjugated peptides introduced were confirmed for the product (Figure 7).

[0173] (1-6) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To 2 μL of a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equal volume to the antibody) was added to the antibody-peptide conjugate generated in (1-5), and the mixture was stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the heavy chain peak of the raw material trastuzumab was observed at 50596 and the light chain peak was observed at 23439, and for the product, a peak was observed at 55033 with one linker introduced into the heavy chain and at 23439, the same as the raw material, for the light chain (Figure 8).

[0174] (1-7) Confirmation of the peptide / antibody binding ratio of the specific modified form of trastuzumab by DAR calculator Regarding the MS data analyzed in (1-5), the results of confirming the peptide / antibody binding ratio by DAR calculator (Agilent software) 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 generation of an antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0175]

Chemical formula

[0176]

Table 2

[0177] (1-8) Production of a thiol group-introduced antibody derivative by cleavage of the thioester group To the antibody intermediate obtained in (1-7), a hydroxylamine solution was added according to the previously reported (WO2019 / 240287A1), and the mixture was allowed to stand at room temperature for 1 hour. After 2 hours, it was replaced with 20 mM PBS buffer, 10 mM EDTA (pH 7.4) to obtain a thiol group-introduced antibody derivative. When the mass was measured by ESI-TOFMS, a peak was confirmed at 148409 where the cleavage reaction had proceeded.

[0178] [Chemical formula] [Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and through the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering, an amide bond is formed with the carbonyl group adjacent to Ig, The average ratio r of the above amide bond per two heavy chains is 2.0. ]

[0179] (1-9) Peptide mapping by trypsin treatment For the trastuzumab-thiol introduced product obtained in (1-8), peptide mapping was performed in the following steps.

[0180] (1-9-1) Trypsin treatment of trastuzumab-thiol introduced product To a 1.5 mL low adsorption microtest tube, 10 μL of the sample solution, 10 μL of a 20 mM aqueous solution of dithiothreitol dissolved in 50 mM ammonium bicarbonate buffer, 40% trifluoroethanol were added, and the mixture was heated at 65 °C for 1 hour. Then, 10 μL of a 50 mM aqueous solution of iodoacetamide was added, and the reaction was carried out at room temperature for 30 minutes in the dark. After the reaction, 40 μL of 50 mM ammonium bicarbonate buffer was added and stirred, 10 μL of a 20 ng / μL aqueous solution of trypsin was added, and enzymatic digestion was carried out at 37 °C for 16 hours. After digestion, 2 μL of a 20% aqueous solution of trifluoroacetic acid was added to stop the reaction, and LC-MS / MS measurement was performed.

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

[0182] (HPLC analysis conditions) Trap column: Acclaim PepMap (registered trademark) 100, 75μm x 2cm (Thermo Fisher Scientific) Analysis column: ESI-column (NTCC-360 / 75-3-125, 75μm × 12.5cm, 3μm (Nikkyo Technos Co., Ltd.)) Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300 nL / min Sample injection volume: 1 μL Gradient condition (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 Aquisition Activation Type: Collision Induced Dissociation (CID) Data acquisition was performed using the attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

[0184] (Analysis of the modification site of trastuzumab (1-9-3)) For the analysis of modification sites with respect to the LC-MS / MS measurement results, it was performed using BioPharma Finder 3.0 (Thermo Fisher Scientific). The analysis with BioPharma Finder was carried out by setting the S / N Threshold to 1 and the MS Noise Level to 0.01% of the peak top intensity. Also, the digestive enzyme was set to Trypsin and the Specificity was set to High. For Static Modification, Carbamidomethyl (+57.021Da) was set as the modification of cysteine residues by iodoacetamide. For Dynamic Modifications, oxidation of methionine residues (+15.995Da) and a modified form on lysine residues (a thiol-introduced form (+145.019Da) that underwent Carbamidomethylation by iodoacetamide) were set. In addition, a filter was set so that only those with a Confidence Score of 80 or more, a Mass Accuracy within 5ppm at the time of peptide identification, and observable MS / MS were included. Regarding the residue numbers of lysine residues, for the heavy chain VH domain and the light chain, the numbers in the sequence (i.e., the N-terminal amino acid is numbered 1. The same applies hereinafter) were used, and for the heavy chain CH1, CH2, and CH3 domains, EU numbering was used for notation. Also, (1) and (2) shown in Figure 9 were used as the data of the amino acid sequence for the search of modification sites.

[0185] (1-9-4) Analysis results of modification sites of trastuzumab by LC-MS / MS As a result of the analysis using LC-MS / MS, a peptide consisting of 18 amino acids containing a modification site on a lysine residue by trypsin digestion of trastuzumab (a thiol-introduced product (+145.019 Da) that underwent carbamidomethylation with iodoacetamide), the MS spectrum of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 10) (measured value: m / z 577.03606, theoretical value: 577.03557, tetravalent) was observed (Figure 10), and a product ion with m / z 682.13 (theoretical value: 682.01) corresponding to trivalent y16, which indicates modification of the lysine residue at position 288 or 290 in the EU numbering of the heavy chain, was confirmed from the CID spectrum (Figure 11). Also, analysis with BioPharma Finder showed that modification of the lysine residue at position 288 or 290 occurred highly selectively (Figure 12). From this result, it was found that in the trastuzumab thiol-introduced product obtained in the above (1-8), conjugation proceeded selectively at Lys288 and Lys290 in the EU numbering on the heavy chain of the antibody.

[0186] [Example 2: Synthesis of a compound having an affinity substance, a cleavable moiety, and a reactive group for a soluble protein (peptide thioester linker conjugate - thiophenol activator), modification of the anti-HER2 antibody trastuzumab using the compound, and analysis thereof] (2-1) Synthesis of thioester linker (2-1-1) [Chemical formula]

[0187] (295 mg, 1.62 mmol) of the compound synthesized in (1-3-2) was added to CH 2 Cl 2(13.5 mL) was dissolved, 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, followed by stirring 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 mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fraction containing the product was collected, and after removing the organic solvent by concentration under reduced pressure, vacuum drying was carried out to obtain the above compound (259 mg, 0.64 mmol).

[0188] (2-1-2)

Chemical formula

[0189] (259 mg, 0.64 mmol) of the compound synthesized in (2-1-1) was dissolved in a mixed solution of CH 2 Cl 2 / TFA = 1 / 1, and stirred at room temperature for 1 hour. After confirming that it dropped 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)

Chemical formula

[0191] (227 mg, 0.66 mmol) of the compound synthesized in (2-1-2) was added with CH 2 Cl 2(3.3 mL), triethylamine (230 μL, 1.65 mmol) was added and dissolved. Pentafluorophenyl trifluoroacetate (225 μL, 1.32 mmol) was added at 0 °C, and the mixture was stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was carried out with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected, the organic solvent was removed by concentration under reduced pressure, and then vacuum drying was performed 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 the peptide and the linker

Chemical Structure

[0193] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH synthesized in (1-2) 2(SEQ ID NO:2) (30.0 mg, 7.06 μmol, provided that the 5th and 34th cysteines each form an intramolecular disulfide bond) was dissolved in N,N-dimethylformamide (1.00 mL), the linker (72.0 mg, 141 μmol) was added, and the mixture was stirred at room temperature for 24 hours. This was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reversed-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as the filler, eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above peptide thioester linker conjugate - 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 analysis by ESI-TOFMS (2 - 2) The peptide linker conjugate synthesized was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks of 152691 with one conjugated peptide introduced, 157163 with two conjugated peptides introduced, and 161634 with three conjugated peptides introduced were confirmed (Figure 13).

[0196] (2 - 4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To 2 μL of a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent amount relative to the antibody) was added to the antibody-peptide conjugate generated in (2-3), and the mixture was stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the heavy chain peak of the raw material trastuzumab was observed at 50596, and the light chain peak was observed at 23439. For the reactant, a peak was observed at 55067 where one linker was introduced into the heavy chain and at 23439, the same as the raw material, for the light chain (Figure 14).

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

[0198] [Chemical formula] Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig via the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering. 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) Production of a thiol group-introduced antibody derivative by cleavage of the thioester group The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (2-5) to the cleavage reaction of the thioester group described in (1-8).

[0201] (2-7) Peptide mapping by trypsin treatment For the trastuzumab-thiol introduced product obtained in (2-6), peptide mapping was performed in the following steps.

[0202] (2-7-1) Trypsin treatment of the trastuzumab-thiol introduced product Trypsin treatment of the trastuzumab-thiol introduced product obtained in (2-6) was performed in the same manner as in (1-9-1).

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

[0204] (2-7-3) Analysis of the modification site of trastuzumab Analysis was performed in the same manner as in (1-9-3).

[0205] (2-7-4) Analysis result of the modification site of trastuzumab by LC-MS / MS As a result of the analysis using LC-MS / MS, a peptide consisting of 18 amino acids containing a modification site on the lysine residue by trypsin digestion of trastuzumab (a thiol derivative (+145.019 Da) that has undergone carbamidomethylation with iodoacetamide), the MS spectrum of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 10) (measured value: m / z 577.03571, theoretical value: 577.03557, tetravalent) was observed (Figure 15), and a product ion with m / z 682.41 (theoretical value: 682.01) corresponding to trivalent y16, which indicates the modification of the lysine residue at position 288 or 290 in the EU numbering of the heavy chain, was confirmed from the CID spectrum (Figure 16). In addition, analysis with BioPharma Finder showed that the modification of the lysine residue at position 288 or 290 occurred highly selectively (Figure 17). From this result, it was found that in the trastuzumab thiol derivative obtained in the above (2-6), conjugation proceeded selectively at Lys288 and Lys290 in the EU numbering on the heavy chain of the antibody.

[0206] [Example 3: Synthesis of a compound having an affinity substance, a cleavable moiety, and a reactive group for a soluble protein (peptide thioester linker conjugate - thiophenol activator), modification of the anti-HER2 antibody trastuzumab using the compound, and analysis thereof] (3-1) Synthesis of thioester linker (3-1-1) [Chemical formula]

[0207] (1-3-2) The compound synthesized in (1-3-2) (220 mg, 1.21 mmol) was dissolved in CH 2 Cl 2(12.0 mL) was dissolved, and Adipic Acid (530 mg, 3.63 mmol), DIPEA (314 μL, 1.82 mmol), and PyBOP (755 mg, 1.45 mmol) were added, followed by stirring 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 after removing the organic solvent by concentration under reduced pressure, vacuum drying was carried out to obtain the above compound (205 mg, 0.63 mmol).

[0208] (3-1-2)

Chemical Structure

[0209] (205 mg, 0.63 mmol) of the compound synthesized in (3-1-1) was dissolved by adding CH 2 Cl 2 (3.15 mL) and triethylamine (220 μL, 1.58 mmol). Pentafluorophenyl trifluoroacetate (215 μL, 1.26 mmol) was added at 0 °C, and the mixture was 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 after removing the organic solvent by concentration under reduced pressure, vacuum drying was carried out 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 [Chemical formula] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO: 2.

[0211] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH synthesized in (1-2) 2 (SEQ ID NO: 2) (30.0 mg, 7.06 μmol, provided that the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) was dissolved in N,N-dimethylformamide (1.00 mL), the linker (69.0 mg, 141 μmol) was added, and the mixture was stirred at room temperature for 24 hours. This was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as the filler, and eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid. Each fraction was confirmed by LC-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the above peptide thioester linker conjugate-thiophenol activator (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 analysis by ESI-TOFMS (3-2) The peptide linker conjugate synthesized 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 the 10 mM peptide reagent was added, followed by stirring at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the starting material trastuzumab. Peaks at 152676 with one conjugated peptide introduced, 157126 with two conjugated peptides introduced, and 161572 with three conjugated peptides introduced were confirmed (Figure 18).

[0214] (3-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specific modified form of trastuzumab under reducing conditions (3-3) To the antibody-peptide conjugate generated in (3-3), 2 μL (equivalent amount relative to the antibody) of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, and the mixture was 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 at 23439 for the starting material trastuzumab. For the reaction product, peaks were observed at 55048 with one linker introduced into the heavy chain and 23439, the same as the starting material, for the light chain (Figure 19).

[0215] (3-5) Confirmation of the peptide / antibody binding ratio of the specific modified form of trastuzumab using a DAR calculator (3-3) The results of confirming the peptide / antibody binding ratio for the MS data analyzed in (3-3) using a DAR calculator (Agilent software) 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 generation of the antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio 1.9) was confirmed.

[0216]

Chemical formula

[0217] [Table 4]

[0218] (3 - 6) Production of a thiol - group - introduced antibody derivative by cleavage of a thioester group The thiol - group - introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (3 - 5) to the thioester group cleavage reaction described in (1 - 8).

[0219] [Example 4: Synthesis of a compound (peptide thioester linker - thiophenol activator) having an affinity substance, a cleavable moiety, and a reactive group for a soluble protein, modification of the anti - HER2 antibody trastuzumab using the compound, and its analysis] (4 - 1) Synthesis of a thioester linker (4 - 1 - 1) [Chemical formula]

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

[0221] (4-1-2)

Chemical formula

[0222] (104 mg, 0.26 mmol) of the compound synthesized in (4-1-1) was dissolved in a mixed solution of CH 2 Cl 2 / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming that it dropped 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)

Chemical formula

[0224] (104 mg, 0.37 mmol) of the compound synthesized in (4-1-2) was added with CH 2 Cl 2(1.85 mL), triethylamine (130 μL, 0.93 mmol) was added and dissolved. At 0 °C, N-Succinimidyl Trifluoroacetate (156 mg, 0.74 mmol) was added and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 1 / 1), the reaction solution was concentrated. Elution was carried out with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 1 / 1). The fraction containing the product was collected, the organic solvent was removed by concentration under reduced pressure, and then vacuum drying was performed 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 the peptide and the linker

Chemical Structure

[0226] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH synthesized in (1-2) 2(SEQ ID NO: 2) (29.7 mg, 7.00 μmol, provided that the 5th and 34th cysteines form intramolecular disulfide bonds respectively) was dissolved in N,N-dimethylformamide (1.00 mL), the linker (66.8 mg, 0.14 mmol) was added, and the mixture was stirred at room temperature for 4 hours. This was dissolved in a 0.05% aqueous trifluoroacetic acid solution and subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as the filler, and eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid. Each fraction was confirmed by LC-MS. The fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and then lyophilized to obtain the above-mentioned peptide thioester linker conjugate-NHS activator (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 analysis by ESI-TOFMS (4-2) The peptide linker conjugate synthesized was dissolved in dimethyl sulfoxide to make it 10 mM. 500 μg of anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks of 152722 with one introduced binding peptide, 157215 with two introduced binding peptides, and 161708 with three introduced binding peptides were confirmed (Figure 20).

[0229] (4-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To 2 μL of a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent amount relative to the antibody) was added to the antibody-peptide conjugate produced in (4-3), and the mixture was stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the heavy chain peak of the raw material trastuzumab was observed at 50594, and the light chain peak was observed at 23439. For the reactant, a peak was observed at 55091 where one linker was introduced into the heavy chain and at 23439, the same as the raw material, for the light chain (Figure 21).

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

[0231] [Chemical formula] Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig through the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering. 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 1.8.)

[0232] [Table 5]

[0233] (4-6) Production of a thiol group-introduced antibody derivative by cleavage of the thioester group The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (4-5) to the cleavage reaction of the thioester group described in (1-8).

[0234] [Example 5: Synthesis of a compound (peptide thioester linker conjugate-thiophenol activator) having an affinity substance, a cleavable moiety, and a reactive group for a soluble protein, modification of the anti-HER2 antibody trastuzumab using the compound, and analysis thereof] (5-1) Binding of a peptide and a linker

Chemical formula

[0235] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC-NH synthesized by the method described in (1-1) 2 (SEQ ID NO: 3) (30.0 mg, 7.06 μmol, provided that the two cysteines at the 5th and 34th positions form an intramolecular disulfide bond respectively) was conjugated with a linker in the same manner as in Example 2 (2-2) to obtain the above peptide thioester linker conjugate-thiophenol activator (10.0 mg, 2.19 μmol).

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

[0237] (5-2) Specific modification of the 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), 3.38 μL (10 equivalents relative to the antibody) of the 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the starting material trastuzumab. Peaks at 152707 with one conjugated peptide, 157188 with two conjugated peptides, and 161676 with three conjugated peptides were confirmed (Figure 22).

[0238] (5-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specific modification of trastuzumab under reducing conditions To the antibody-peptide conjugate produced in (5-2), 2 μL (equivalent amount relative to the antibody) of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, and the mixture was 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 starting material trastuzumab. For the reaction product, a peak was observed at 55077 with one linker introduced into the heavy chain and at 23439, the same as the starting material, for the light chain (Figure 23).

[0239] (5-4) Confirmation of the peptide / antibody binding ratio of the specific modification of trastuzumab using a DAR calculator Table 6 shows the results of confirming the peptide / antibody binding ratio for the MS data analyzed in (5-2) using a DAR calculator (software from Agilent). 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 represented by the following structural formula (average peptide / antibody binding ratio 2.0) was confirmed.

[0240]

Chemical formula

[0241]

Table 6

[0242] (5-5) Production of a thiol group-introduced antibody derivative by cleavage of a thioester group The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (5-4) to the cleavage reaction of the thioester group described in (1-8) to obtain the thiol group-introduced antibody derivative described in (1-8).

[0243] [Example 6: Synthesis of a compound (peptide thioester linker conjugate-thiophenol activator) having an affinity substance, a cleavable moiety, and a reactive group for a soluble protein, modification of the anti-HER2 antibody trastuzumab using the compound, and analysis thereof] (6-1) Binding of a peptide and a linker

Chemical formula

[0244] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH synthesized by the method described in (1-1) 2(SEQ ID NO: 4) (30.0 mg, 7.06 μmol, provided that the 5th and 34th cysteines each form an intramolecular disulfide bond) was linked with a linker in the same manner as in Example 2 (2-2), and the above peptide thioester linker conjugate-thiophenol activator (22.2 mg, 4.82 μmol) was obtained.

[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 anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the starting material trastuzumab. Peaks of 152720 with one conjugated peptide introduced, 157216 with two conjugated peptides introduced, and 161716 with three conjugated peptides introduced were confirmed (Figure 24).

[0247] (6-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To the antibody-peptide conjugate generated in (6-2), 2 μL (equivalent amount relative to the antibody) of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, and the mixture was 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 starting material trastuzumab. For the reaction product, peaks were observed at 55091 with one linker introduced into the heavy chain and 23439 same as the starting material in the light chain (Figure 25).

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

[0249] [Chemical formula] [Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig through the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering. Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 4. The average ratio r of the above amide bond per two heavy chains is 2.0. ]

[0250] [Table 7]

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

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

[0253] Ac-NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH synthesized by the method described in (1-1) 2 (SEQ ID NO: 5) (30.0 mg, 7.06 μmol, provided that the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) was conjugated with a linker in the same manner as in Example 2 (2-2), and the above peptide thioester linker conjugate - thiophenol activator (12.0 mg, 2.69 μmol) was obtained.

[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 anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks at 152573 with one conjugated peptide introduced and 156927 with two conjugated peptides introduced were confirmed (Figure 26).

[0256] (7-3) Confirmation of Heavy Chain Selectivity by ESI-TOFMS Analysis of the Specifically Modified Trastuzumab under Reducing Conditions To 2 μL of a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent amount relative to the antibody) was added to the antibody-peptide conjugate produced in (7-2), and the mixture was stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the heavy chain peak of the raw material trastuzumab was observed at 50596 and the light chain peak at 23439. For the reactant, peaks were observed at 54942 with one linker introduced into the heavy chain and 23439, the same as the raw material, in the light chain (Figure 27).

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

[0258] [Chemical formula] Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig via the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering, Y represents an affinity peptide represented by the amino acid sequence of SEQ ID NO: 5, and the average ratio r of the above amide bond per two heavy chains is 2.0.)

[0259] [Table 8]

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

[0261] (7-6) Peptide mapping by trypsin treatment For the trastuzumab thiol-introduced product obtained in (7-5), peptide mapping was performed in the following steps.

[0262] (7-6-1) Trypsin treatment of the trastuzumab thiol-introduced product Trypsin treatment of the trastuzumab thiol-introduced product obtained in (7-5) was performed in the same manner as in (1-9-1).

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

[0264] (7-6-3) Analysis of the modification site of trastuzumab Analysis was performed in the same manner as in (1-9-3).

[0265] (7-6-4) Analysis result of the modification site of trastuzumab by LC-MS / MS As a result of the analysis using LC-MS / MS, a peptide consisting of 18 amino acids containing a modification site at a lysine residue by trypsin digestion of trastuzumab (a thiol derivative (+145.019 Da) that has undergone carbamidomethylation with iodoacetamide), the MS spectrum of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 10) (measured value: m / z 769.04506, theoretical value: 769.04482, trivalent) was observed (Figure 28), and a product ion of m / z 1022.71 (theoretical value: 1022.51) corresponding to bivalent y16, indicating modification of the lysine residue at position 288 or 290 in the EU numbering of the heavy chain, was confirmed from the CID spectrum (Figure 29). Also, analysis with BioPharma Finder showed that modification of the lysine residue at position 288 or 290 occurred highly selectively (Figure 30). From this result, it was found that in the trastuzumab thiol derivative obtained in the above (7-5), site-selective conjugation proceeded at Lys288 and Lys290 in the EU numbering on the heavy chain of the antibody.

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

[0267] Ac-MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH synthesized by the method described in (1-1) 2(SEQ ID NO: 6) (30.0 mg, 7.06 μmol, provided that the 5th and 34th cysteines form intramolecular disulfide bonds respectively) was linked with a linker in the same manner as in Example 2 (2-2), and the above peptide thioester linker conjugate-thiophenol activator (16.8 mg, 3.87 μmol) was 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 anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks of 152457 with one conjugated peptide introduced, 156692 with two conjugated peptides introduced, and 160929 with three conjugated peptides introduced were confirmed (Figure 31).

[0270] (8-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To the antibody-peptide conjugate generated in (8-2), 2 μL (equivalent amount relative to the antibody) of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, and the mixture was 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. Peaks were observed at 54829 with one linker introduced into the heavy chain and 23439 same as the raw material in the light chain for the reaction product (Figure 32).

[0271] (8-4) Confirmation of Peptide / antibody Binding Ratio of a Specific Modifier of Trastuzumab by DAR Calculator Table 9 shows the results of confirming the peptide / antibody binding ratio for the MS data analyzed in (8-2) using a DAR calculator (Agilent software). The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 9 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0272] [Chemical formula] [Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig through the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering. 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) Production of Thiol Group-introduced Antibody Derivative by Cleavage of Thioester Group The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (8-4) to the cleavage reaction of the thioester group described in (1-8) to obtain the thiol group-introduced antibody derivative described in (1-8).

[0275] [Example 9: Synthesis of a Compound (Peptide Thioester Linker Conjugate-Thiophenol Activator) Having an Affinity Substance, Cleavable Moiety, and Reactive Group for a Soluble Protein, and Modification of Anti-HER2 Antibody Trastuzumab Using the Compound and Its Analysis] (9-1) Binding of Peptide and Linker

Chem.

[0276] Ac-QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH synthesized by the method described in (9-1) 2 (SEQ ID NO: 7) (30.0 mg, 7.06 μmol, provided that the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) was conjugated with a linker in the same manner as in Example 2 (2-2), and the above peptide thioester linker conjugate-thiophenol activator (14.1 mg, 3.35 μmol) was obtained.

[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 anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer. When the mass was measured by ESI-TOFMS, a peak was observed at 148223 for the raw material trastuzumab. Peaks of 152236 with one conjugated peptide introduced, 156430 with two conjugated peptides introduced, and 160541 with three conjugated peptides introduced were confirmed (Figure 33).

[0279] (9-3) Confirmation of Heavy Chain Selectivity by ESI-TOFMS Analysis of the Specifically Modified Trastuzumab under Reducing Conditions To 2 μL of a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent amount relative to the antibody) was added to the antibody-peptide conjugate generated in (9-2), and the mixture was stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the heavy chain peak of the raw material trastuzumab was observed at 50596, and the light chain peak was observed at 23439. For the reactant, a peak was observed at 54698 where one linker was introduced into the heavy chain and at 23439, the same as the raw material, for the light chain (Figure 34).

[0280] (9-4) Confirmation of the peptide / antibody binding ratio by the DAR calculator for the specific modified form of trastuzumab Regarding the MS data analyzed in (9-2), the results of confirming the peptide / antibody binding ratio by the DAR calculator (Agilent software) 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 generation of an antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0281] [Chemical formula] Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig via the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering. 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) Production of a thiol group-introduced antibody derivative by cleavage of the thioester group The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (9-4) to the cleavage reaction of the thioester group described in (1-8).

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

[0285] [Table 11] 1) Linker length: The number of atoms constituting the main chain that links the reactive moiety [X (leaving group)-C=O] with the lysine residue at positions 288 / 290 of the heavy chain in the immunoglobulin unit and the binding moiety [O=C-Y (affinity peptide)] with the affinity peptide 2) Binding ratio: The binding ratio of the peptide to the antibody (peptide / antibody) (The same applies hereinafter)

[0286] [Table 12]

[0287] [Reference Examples 1 to 5] With reference to the above examples, the following peptide thioester linker conjugates-NHS activators or peptide thioester linker conjugates-thiophenol activators were synthesized, and specific modification of the anti-HER2 antibody trastuzumab and analysis by ESI-TOFMS were performed. Also, in the same manner as in the examples, when the peptide / antibody binding ratio (PAR) of the specifically modified trastuzumab was confirmed using a DAR calculator, compounds with a linker length of 6 or fewer or 11 or more atoms (in the compound represented by formula (I), corresponding to a total of fewer than 4 or 9 or more atoms in the main chain of the first linker and the main chain of the second linker) showed an average PAR of 0.5 or less.

[0288]

Table 13

[0289] [Example 10: Site-selective modification of multiple target regions of IgG1 Fc with an IgG1 Fc affinity peptide reagent and synthesis of an antibody-drug conjugate] (10-1) Production of a thiol group-introduced antibody derivative by cleavage of a thioester group following site-selective modification of the anti-HER2 antibody trastuzumab The following peptide reagent described in the prior report (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), 3.38 μL (10 equivalents relative to the antibody) of the 10 mM above peptide reagent was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, a hydroxylamine solution was added according to the prior report (WO2019 / 240287A1), and the mixture was allowed to stand at room temperature for 1 hour. When the mass of the obtained thiol group-introduced antibody was measured by ESI-TOFMS, a peak was confirmed at 148760 where the cleavage reaction with hydroxylamine had proceeded. Considering that the following peptide reagent 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 following peptide reagent.

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

[0291] (10-2) Positional selective modification of multiple target regions of anti-HER2 antibody trastuzumab The thiol-introduced antibody solution synthesized in (10-1) was replaced with 50 mM HEPES buffer (pH 8.2). To this solution, a dimethylformamide solution of the peptide linker conjugate synthesized in (3-2) was added in an amount of 10 equivalents 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. When the mass of the obtained antibody was measured by ESI-TOFMS, a peak of 157531 in which two binding peptides were introduced was confirmed for the thiol-introduced antibody synthesized in (3-2).

[0292] (10-3) Production of thiol group-introduced antibody derivative by cleavage of thioester group To the antibody intermediate obtained in (10-2), a hydroxylamine solution was added according to the previously reported method (WO2019 / 240287A1), and the mixture was allowed to stand at room temperature for 1 hour. After 2 hours, it was replaced with 20 mM PBS buffer and 10 mM EDTA (pH 7.4) to obtain a thiol group-introduced antibody derivative. When the mass was measured by ESI-TOFMS, a peak was confirmed at 148760 where the cleavage reaction had proceeded. Therefore, it was confirmed that the obtained thiol group-introduced antibody derivative had the following structure.

[0293] [Chemical formula] 〔Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with the carbonyl group adjacent to Ig via the amino group in the side chain of the lysine residue present at positions 288 / 290 in the two heavy chains according to Eu numbering. Similarly, an amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the lysine residue present at positions 246 / 248 in the two heavy chains according to Eu numbering. The average ratios r (at positions 288 / 290) and n (at positions 246 / 248) of the above amide bonds per two heavy chains were 1.9 and 2.0, respectively, as a result of the evaluation.〕

[0294] (10-4) Production of Thiol Group-Introduced Antibody Derivative by Cleavage of Drug Mimic Thioester Group (10-3) To a buffer (pH 7.4 PBS buffer) solution (20 μM) of the thiol group-introduced antibody obtained in (10-3), a DMF solution (1.25 mM) of MC-VC-PAB-MMAE reported previously (Org. Process Res. Dev. 2019, 23, 12, 2647-2654) was added in an amount of 10 equivalents, and after allowing to stand at room temperature for 2 hours, purification was carried out using NAP-5 Columns (manufactured by GE Healthcare) to obtain an ADC. When the mass was measured by ESI-TOFMS, a peak was confirmed at 154029 into which 4 MC-VC-PAB-MMAE were introduced.

[0295]

Chemical Structure

Claims

1. The following formula (I''): 【Chemical 1】 〔In the formula, X is a leaving group selected from the following: (a) R-S (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and S represents a sulfur atom.); (b) R-O (wherein R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and O represents an oxygen atom.); or (d) a halogen atom, is shown, Y is an affinity peptide selected from the group consisting of the following, which has a binding region to the CH2 domain in human IgG containing two heavy chains and two light chains: (A) an affinity peptide containing the amino acid sequence of CQRRFYERALHDPNLNEERNRIRSIKDDC (SEQ ID NO: 1); (a) an affinity peptide containing the amino acid sequence of FNMQCQRRFYERALHDPNLNEERNRIRSIKDDC (SEQ ID NO: 2); (b) an affinity peptide containing the amino acid sequence of FNMQCQRRFYERALHDPNLNEERNRIRSIKEDC (SEQ ID NO: 3); (c) an affinity peptide containing the amino acid sequence of FNMQCQRRFYERALHDPNLNEERNRIRSIKEEEC (SEQ ID NO: 4); (d) an affinity peptide containing the amino acid sequence of NMQCQRRFYERALHDPNLNEERNRIRSIKEEEC (SEQ ID NO: 5); (e) an affinity peptide containing the amino acid sequence of MQCQRRFYERALHDPNLNEERNRIRSIKEEEC (SEQ ID NO: 6); and (f) an affinity peptide containing the amino acid sequence of QCQRRFYERALHDPNLNEERNRIRSIKEEEC (SEQ ID NO: 7 ) is shown, is shown, the N-terminal and C-terminal amino acid residues in the affinity peptide may be protected, and 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, an amide bond is formed with the carbonyl group (C=O) adjacent to Y via the amino group in the side chain of the lysine residue of the affinity peptide, O represents an oxygen atom, S represents a sulfur atom, W represents an oxygen atom or a sulfur atom, Lb represents a second linker, The number of atoms constituting the main chain in the second linker is 3 to 5.〕 A compound represented by or a salt thereof.

2. The following formula (V''): 【Chemical Formula 2】 [In the formula, 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, Lb represents a second linker, The number of atoms constituting the main chain in the second linker is 3 to 5.] A compound represented by the formula or a salt thereof.

3. The compound represented by the formula (V'') is the following formula (V''-1) or (V''-2): [Chemical Formula 3] [In the formula, O, OH, S, and W are the same as those in the formula (V'').] The compound or a salt thereof according to claim 2, represented by the formula.

4. The following formula (VI''): 【Chemical Formula 4】 [In the formula, X represents a leaving group, X' represents a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyloxy group, O represents an oxygen atom, S represents a sulfur atom, W represents an oxygen atom or a sulfur atom, Lb represents a second linker, The number of atoms constituting the main chain in the second linker is 3 to 5.] A compound represented by the formula or a salt thereof.

5. The compound represented by the formula (VI'') is the following formula (VI''-1) or (VI''-2): 【Chemical Formula 5】 [In the formula, O, S, and W are the same as those in the formula (VI''), F is a fluorine atom.] The compound or a salt thereof according to claim 4, represented by the formula.

6. The following formula (VI''): 【Chemical Formula 6】 [In the formula, X represents a leaving group, X' represents a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyloxy group, O represents an oxygen atom, S represents a sulfur atom, W represents an oxygen atom or a sulfur atom, Lb represents a second linker, The number of atoms constituting the main chain in the second linker is 3 to 5.] A compound represented by the formula or a salt thereof, which has a binding region in the CH2 domain of human IgG containing two heavy chains and two light chains, and is an affinity peptide selected from the following group: (A) An affinity peptide containing the amino acid sequence of CQRRFYEA LHDPNLNEE QRNAIRSIKDDC (SEQ ID NO: 1); (a) An affinity peptide containing the amino acid sequence of FNMQCQRRFYEA LHDPNLNEE QRNAIRSIKDDC (SEQ ID NO: 2); (b) An affinity peptide comprising the amino acid sequence of FNMQCQRRFYERALHDPNLNE EQRNA RIRSIKE DC (SEQ ID NO: 3); (c) An affinity peptide comprising the amino acid sequence of FNMQCQRRFYERALHDPNLNE EQRNA RIRSIKEE C (SEQ ID NO: 4); (d) An affinity peptide comprising the amino acid sequence of NMQCQRRFYERALHDPNLNE EQRNA RIRSIKEE C (SEQ ID NO: 5); (e) An affinity peptide comprising the amino acid sequence of MQCQRRFYERALHDPNLNE EQRNA RIRSIKEE C (SEQ ID NO: 6); and (f) An affinity peptide comprising the amino acid sequence of QCQRRFYERALHDPNLNE EQRNA RIRSIKEE C (SEQ ID NO: 7), wherein the N-terminal and C-terminal amino acid residues in the affinity peptide may be protected, and the two thiol groups in the side chains of the two cysteine residues (C) in the affinity peptide may be reacted with the amino group in the side chain of the lysine residue of the affinity peptide, which may be linked by a disulfide bond or via a linker, to produce a compound represented by the following formula (I''): 【Chemical Formula 7】 [In the formula, X, O, S, W, and Lb are the same as those in the formula (VI''), Y represents the affinity peptide. ] or a salt thereof, a method for producing a compound represented by the formula (I'') or a salt thereof.

7. (1') The following formula (V''): [Chemical Formula 8] [In the formula, 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, Lb represents a second linker, The number of atoms constituting the main chain in the second linker is 3 to 5. ] or a salt thereof is reacted with a carboxyl group modifying reagent to further produce a compound represented by the following formula (VI''): 【Chemical Formula 9】 [In the formula, X, O, S, W, and Lb are the same as those in the formula (V''), X' represents a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyl oxy group. ] or a salt thereof, the method according to claim 6.

8. The following formula (V''): 【Chemical 10】 [In the formula, 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, Lb represents a second linker, reacting a compound represented by [wherein the number of atoms constituting the main chain in the second linker is 3 to 5] or a salt thereof with a carboxyl group-modifying reagent to obtain a compound represented by the following formula (VI''): 【Chemical Formula 11】 [wherein, X, O, S, W, and Lb are the same as those in the formula (V''), X' represents a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyl oxy group], which comprises producing a compound represented by the formula (VI'') or a salt thereof.

Images