Compound or salt thereof, and antibody obtained therefrom

Abstract

To provide a desired antibody or antibody composition by modifying a thiol group chemically introduced to an antibody.SOLUTION: The present invention provides an antibody composition comprising (A) an antibody intermediate or a salt thereof having a bioorthogonal functional group which may be protected, which is represented by the following formula (A), where Ab is a predetermined antibody, S is a sulfur atom, L is a divalent group, R is a bioorthogonal functional group which may be protected, and n is an integer from 1 to 8, and (B) a thiol group-introduced antibody or a salt thereof, which is represented by the following formula (B), where Ab and n are the same as in formula (A), and SH is a thiol group, the antibody composition having desired properties.SELECTED DRAWING: None

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. 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] An ADC is produced by binding a drug to a functional group in the side chain of a specific amino acid residue present in the antibody. An example of such a functional group used for the production of an ADC is a thiol group in the side chain of a cysteine residue present in the antibody. As techniques for modifying a thiol group in an antibody, a technique using a 3-arylpropionitrile compound (Patent Document 1) and a technique using an aryl / heteroaryl sulfone / sulfoxide compound (Patent Document 2) are known. The above prior art for modifying a thiol group in an antibody modifies a thiol group in the side chain of a cysteine residue in the antibody (e.g., a thiol group in the side chain of a cysteine residue naturally present in the antibody and a thiol group in the side chain of a cysteine residue genetically introduced into the antibody).

[0004] However, as far as the present inventors are aware, the prior art does not teach or suggest modifying a thiol group chemically introduced into an antibody (e.g., a thiol group introduced into the antibody via the side chain of an amino acid residue other than a cysteine residue in the antibody).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] An object of the present invention is to provide a desired antibody or antibody composition by modifying a thiol group chemically introduced into an antibody. [Means for Solving the Problems]

[0007] As a result of intensive studies, the present inventors have found that a specific compound can modify a thiol group chemically introduced into an antibody with high efficiency. Since such a specific compound is excellent in the efficiency of the reaction, it is considered useful not only for modifying a thiol group chemically introduced into an antibody but also for modifying a thiol group in a cysteine residue in an antibody. Further, according to such a specific compound, a desired antibody in which a thiol group in the antibody is modified with high efficiency can be produced.

[0008] The present inventors have also found that an antibody modified with the specific compound is excellent in desired properties such as low antibody aggregability.

[0009] The present inventors have further found that, according to the present invention, a special antibody composition containing a desired antibody having the above characteristics can be obtained, and thus the present invention has been completed.

[0010] That is, the present invention is as follows. [1] (A) An antibody intermediate having a bioorthogonal functional group which may be protected, represented by formula (A), or a salt thereof, and (B) an antibody having a thiol group introduced, represented by formula (B), or a salt thereof, The sulfur atom (S) adjacent to the antibody (Ab) in formula (A) and the thiol group (SH) adjacent to the antibody (Ab) in formula (B) are directly bonded to, or bonded via a linker to, atoms in the side chain of the same amino acid residue present at the same position in the constant region of the antibody heavy chain, The molecular weight of the partial structure represented by L-R is 700 or less, The percentage of the amount of the antibody intermediate or a salt thereof with respect to the total amount of the antibody intermediate or a salt thereof and the thiol group-introduced antibody or a salt thereof [100(%)×(the amount of the antibody intermediate or a salt thereof) / (the total amount of the antibody intermediate or a salt thereof and the thiol group-introduced antibody or a salt thereof)] is 80% or more, An antibody composition in which the aggregation rate of the antibody intermediate or a salt thereof and the thiol group-introduced antibody or a salt thereof is 5% or less. 〔2〕The antibody composition according to 〔1〕, wherein the antibody intermediate is represented by formula (I’), (II’), (III’), or (IV’). 〔3〕The antibody composition according to 〔1〕 or 〔2〕, wherein the amino acid residue is an amino acid residue other than a cysteine residue. 〔4〕The antibody composition according to any one of 〔1〕 to 〔3〕, wherein the sulfur atom (S) adjacent to the antibody (Ab) in formula (A) and the thiol group (SH) adjacent to the antibody (Ab) in formula (B) are site-specifically bonded via a peptide-free linker to the nitrogen atom in the side chain of a lysine residue present at the same position in the constant region of the antibody (Ab) heavy chain. 〔5〕The antibody composition according to any one of 〔1〕 to 〔4〕, wherein the antibody is an IgG antibody. 〔6〕The antibody composition according to 〔4〕 or 〔5〕, wherein the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain in EU numbering. 〔7〕The antibody composition according to any one of 〔1〕 to 〔6〕, wherein the antibody intermediate is selected from the group consisting of antibody intermediates represented by formula (1’) to (10’). 〔8〕The antibody composition according to any one of 〔1〕 to 〔7〕, wherein the optionally protected bioorthogonal functional group is an alkyne residue or an azide. The antibody composition of [8], wherein the alkyne residue is a cyclic group having a triple bond between carbon atoms, which may be substituted. A reagent for derivatizing a thiol group-introduced antibody or a salt thereof, comprising a compound represented by formula (I), (II), (III), or (IV) or a salt thereof. The reagent of

[10] , wherein the reagent reacts with a thiol group introduced via a side chain of an amino acid residue other than a cysteine residue in the thiol group-introduced antibody or a salt thereof to derivatize the thiol group-introduced antibody or a salt thereof. The reagent of

[10] or

[11] , wherein the thiol group in the thiol group-introduced antibody is selectively bound via a peptide-free linker to a nitrogen atom in the side chain of a lysine residue in the constant region of the antibody heavy chain. The reagent of any one of

[10] to

[12] , wherein the thiol group-introduced antibody is an IgG antibody. The reagent of

[12] or

[13] , wherein the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain in EU numbering. 〔15〕The divalent group is one group selected from the group consisting of alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, and -(O-R3) m - and consisting of or the divalent group is a divalent group in which two or more groups selected from the group consisting of alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, and -(O-R3) - are linked, m R2 is a hydrogen atom or alkyl, R3 is alkylene or arylene, m is an integer of 1 to 5, and the reagent of any one of

[10] to

[14] . The reagent of any one of

[10] to

[15] , wherein the compound is selected from the group consisting of compounds represented by formulas (1) to (10). The reagent of any one of

[10] to

[15] , wherein the compound is selected from the group consisting of compounds represented by formulas (1) to (10). 〔17〕A compound represented by formula (I), (II), (III), or (IV) or a salt thereof. 〔18〕The compound according to 〔17〕, or a salt thereof, wherein the compound is selected from the group consisting of compounds represented by formulae (2) to (10). 〔19〕An antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (I’), (II’), (III’), or (IV’), or a salt thereof. 〔20〕The sulfur atom (S) adjacent to the antibody (Ab) in formula (I’), (II’), (III’), or (IV’) is directly bonded to an atom in the side chain of an amino acid residue other than a cysteine residue in the constant region of the antibody (Ab) heavy chain, or is bonded via a linker. The antibody intermediate according to 〔19〕, or a salt thereof. 〔21〕The sulfur atom (S) adjacent to the antibody (Ab) in formula (I’), (II’), (III’), or (IV’) is regioselectively bonded via a peptide-free linker to the nitrogen atom in the side chain of a lysine residue in the constant region of the antibody (Ab) heavy chain. The antibody intermediate according to 〔20〕, or a salt thereof. 〔22〕The antibody intermediate according to any one of 〔19〕 to 〔21〕, or a salt thereof, wherein the antibody intermediate is an IgG antibody. 〔23〕The antibody intermediate according to 〔21〕 or 〔22〕, or a salt thereof, wherein the lysine residue is present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain in EU numbering. 〔24〕The antibody intermediate according to any one of 〔19〕 to 〔23〕, or a salt thereof, wherein the antibody intermediate is selected from the group consisting of antibody intermediates represented by formulae (1’) to (12’). 〔25〕A method for producing an antibody intermediate having an optionally protected bioorthogonal functional group or a salt thereof, comprising reacting a compound represented by formula (I), (II), (III), or (IV) or a salt thereof with a thiol group-introduced antibody or a salt thereof to produce an antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (I’), (II’), (III’), or (IV’) or a salt thereof. 〔26〕A conjugate of an antibody and a functional substance represented by formula (I''), (II''), (III''), or (IV''), or a salt thereof. 〔27〕The conjugate according to 〔26〕, or a salt thereof, wherein the conjugate is selected from the group consisting of conjugates represented by formulas (1'') to (12''). 〔28〕Reacting an antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (I'), (II'), (III'), or (IV'), or a salt thereof, with a functional substance having a bioorthogonal functional group capable of reacting with the bioorthogonal functional group carried by the antibody intermediate, to produce a conjugate of an antibody and a functional substance represented by formula (I''), (II''), (III''), or (IV''), or a salt thereof, When the antibody intermediate or a salt thereof has an alkyne residue, the antibody intermediate or a salt thereof is reacted with a functional substance having an azide. A method for producing a conjugate of an antibody and a functional substance, or a salt thereof, wherein when the antibody intermediate or a salt thereof has an azide, the antibody intermediate or a salt thereof is reacted with a functional substance having an alkyne residue. 〔29〕Reacting a compound represented by formula (I), (II), (III), or (IV), or a salt thereof, with a thiol group-introduced antibody or a salt thereof to further produce an antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (I'), (II'), (III'), or (IV'), or a salt thereof. The method according to 〔28〕.

Advantages of the Invention

[0011] The antibody composition of the present invention can efficiently produce a conjugate of an antibody and a functional substance, or a salt thereof, in a state with a low aggregation rate. The reagent of the present invention can derivatize a thiol group-introduced antibody or a salt thereof. The compound of the present invention, or a salt thereof, can be used for derivatization of a thiol group-introduced antibody or a salt thereof. ​The antibody intermediate of the present invention or a salt thereof can be used for the production of a conjugate of an antibody and a functional substance or a salt thereof. The conjugate of the present invention or a salt thereof can be used as a medicine or a reagent (e.g., diagnostic agent, research reagent).

Brief Description of Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] 1. Definition of General Terms (Antibodies and Related Terms) In the present invention, the term "antibody" is as follows. 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, its origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of lysine residues, and definitions of position selectivity, examples, and preferred examples are the same as those of the antibodies described below.

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

[0015] The type of the antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a divalent antibody (e.g., IgG, IgD, IgE), or an antibody with a valence of 4 or more (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a predetermined sugar chain is 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, for example, 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 a variable region Domains and antibody fragments (e.g., antibody fragments containing CH1 domain and CH2 domain, and antibody fragments without constant region) can be used, but full-length antibodies are 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. Proteins modified with biomolecules such as sugars (e.g., glycoproteins) may also be used], sugar chains, nucleic acids, and low-molecular-weight compounds. Preferably, the antibody may be an antibody using a protein as the antigen. Examples of the protein 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 the disease target protein 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 · 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 specific chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, olar Tatoximab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab, ikesekiizumab (IgG4), reslizumab (IgG4), atezolizumab), certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesvacumab, brodalumab (IgG2), orlaratumumab) (when not referring to the IgG subtype, it indicates IgG1).

[0028] For the positions of amino acid residues in the antibody and the positions in the constant region of the heavy chain (e.g., CH2 domain), follow the EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, when targeting human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the human IgG CH2 region, the lysine residue at position 248 corresponds to the 18th amino acid residue in the human IgG CH2 region, the lysine residue at position 288 corresponds to the 58th amino acid residue in the human IgG CH2 region, the lysine residue at position 290 corresponds to the 60th amino acid residue in the human IgG CH2 region, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the human IgG CH2 region. The notation of 246 / 248 indicates that the lysine residue at position 246 or 248 is the target. The notation of 288 / 290 indicates that the lysine residue at position 288 or 290 is the target.

[0029] (halogen atom) Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0030] (Monovalent hydrocarbon group and related terms) Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0031] 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.

[0032] As alkyl, alkyl having 1 to 12 carbon atoms is preferable, alkyl having 1 to 6 carbon atoms is more preferable, and alkyl having 1 to 4 carbon atoms is even more preferable. When alkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0033] As alkenyl, alkenyl having 2 to 12 carbon atoms is preferable, alkenyl having 2 to 6 carbon atoms is more preferable, and alkenyl having 2 to 4 carbon atoms is even more preferable. When alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0034] As alkynyl, alkynyl having 2 to 12 carbon atoms is preferable, alkynyl having 2 to 6 carbon atoms is more preferable, and alkynyl having 2 to 4 carbon atoms is even more preferable. When alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0035] As the monovalent chain hydrocarbon group, alkyl is preferred.

[0036] 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 does not necessarily have to be composed only of alicyclic hydrocarbons, and a part of it may contain a chain structure. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, and these may be either monocyclic or polycyclic.

[0037] As the cycloalkyl, cycloalkyl having 3 to 12 carbon atoms is preferred, cycloalkyl having 3 to 6 carbon atoms is more preferred, and cycloalkyl having 5 to 6 carbon atoms is even more preferred. When the cycloalkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0038] As the cycloalkenyl, cycloalkenyl having 3 to 12 carbon atoms is preferred, cycloalkenyl having 3 to 6 carbon atoms is more preferred, and cycloalkenyl having 5 to 6 carbon atoms is even more preferred. When the cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0039] As the cycloalkynyl, cycloalkynyl having 3 to 12 carbon atoms is preferred, cycloalkynyl having 3 to 6 carbon atoms is more preferred, and cycloalkynyl having 5 to 6 carbon atoms is even more preferred. When the cycloalkynyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutinyl, cyclopentynyl, and cyclohexynyl.

[0040] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0041] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it does not necessarily consist only of an aromatic ring, and a chain structure or an alicyclic hydrocarbon may be included in a part thereof, 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 preferred, aryl having 6 to 10 carbon atoms is more preferred, and aryl having 6 carbon atoms is even more preferred. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

[0042] As the monovalent aromatic hydrocarbon group, phenyl is preferred.

[0043] Among these, as the monovalent hydrocarbon group, alkyl, cycloalkyl, and aryl are preferred.

[0044] (Monovalent heterocyclic group and related terms) The monovalent heterocyclic group means a group obtained by removing one hydrogen atom from the heterocyclic ring of a heterocyclic compound. 1 The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocyclic group, it 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.

[0045] 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 still more preferable. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. 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.

[0046] As the monovalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is still more preferable. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above 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.

[0047] Among these, as the monovalent heterocyclic group, a 5-membered or 6-membered heterocyclic group is preferable.

[0048] (Divalent group) The divalent group is one group selected from the group consisting of a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -C(=S)-NR2-, -NR2-C(=S)-, -O-, -S-, -(O-R3) m -, and -(S-R3) m - and is a group having a main chain structure containing two or more (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, particularly preferably 2 or 3) of these groups. R2 represents a hydrogen atom or a substituent described later. R3 represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. m is an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, and particularly preferably an integer of 1 to 3.

[0049] 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.

[0050] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. 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 which is monocyclic or polycyclic and has 3 to 12 carbon atoms is preferable, a divalent non-aromatic cyclic hydrocarbon group which is monocyclic or polycyclic and has 4 to 10 carbon atoms is more preferable, and a divalent non-aromatic cyclic hydrocarbon group which 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.

[0051] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The hetero atom constituting the heterocyclic ring 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. 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 pyrrolidionediy, pyrrolinedionediy, oxirandiy, aziridinediy, azetidinediy, oxetandiy, thietandiy, pyrrolidinediy, dihydrofurandiy, tetrahydrofurandiy, dioxolanediy, tetrahydrothiophenediy, pyrrolinediy, imidazolidinediy, oxazolidinediy, piperidinediy, dihydropyrandiy, tetrahydropyrandiy, tetrahydrothiopyrandiy, morpholinediy, thiomorpholinediy, piperazinediy, dihydrooxazinediy, tetrahydrooxazinediy, dihydropyrimidinediy, and tetrahydropyrimidinediy. As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

[0052] Preferably, the divalent group is a divalent group having a main chain structure containing one group selected from the group consisting of alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, and -(O-R3) m - or Two or more groups selected from the group consisting of alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, and -(O-R3) m A divalent group having a main chain structure containing two or more groups selected from the group consisting of: R2 is a hydrogen atom or alkyl; R3 is alkylene or arylene; m may be an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5). Alkylene, arylene, and alkyl are the same as those described above.

[0053] The main chain structure in the divalent group may be substituted by one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to 3) of the substituents described below.

[0054] (Substituent) Examples of the substituent include: (i) A halogen atom; (ii) A monovalent hydrocarbon group; (iii) A monovalent heterocyclic group; (iv) An 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 each independently represent a hydrogen atom or a monovalent hydrocarbon group.); (vii) nitro group, sulfuric acid group, sulfonic acid group, cyano group, and carboxyl group.

[0055] 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 described above, respectively.

[0056] Aralkyl refers to 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, for example, benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0057] 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-membered 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 alkyl having 1 to 12 carbon atoms.); (vi) NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O-, or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms.); or (vii) the same group as that listed in (vii) above.

[0058] More preferably, the substituent may be as follows: (i) A halogen atom; (ii) An alkyl 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 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 each represents a hydrogen atom or an alkyl having 1 to 12 carbon atoms.); or (v) The same group as that listed in (vii) above.

[0059] Even more preferably, the substituent may be as follows: (i) A halogen atom; (ii) An alkyl 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[[ID=W59]] a represents a hydrogen atom or an alkyl 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 cis the same as or different from, a hydrogen atom, or an alkyl group having 1 to 6 carbon atoms.); or (v) a group same as those listed in the above (vii).

[0060] 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 as or different from, a hydrogen atom, or an alkyl group having 1 to 4 carbon atoms.); or (v) a group same as those listed in the above (vii).

[0061] (Bioorthogonal functional group) A bioorthogonal functional group refers to a group that does not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphoric acid) or reacts with biological components at a slow rate, but selectively reacts with components other than biological components. Bioorthogonal functional groups are well known in the art (see, for example, Sharpless K.B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

[0062] In the present invention, a bioorthogonal functional group for a protein is used as the bioorthogonal functional group. This is because the thiol group-introducing antibody to be derivatized with the reagent of the present invention is a protein. The bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 natural amino acid residues constituting the protein or reacts with the side chains at a slow rate, but reacts with the target functional group. The 20 natural amino acids constituting the protein are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Among these 20 natural amino acids, glycine having no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine whose side chains are hydrocarbon groups (i.e., do not contain a heteroatom selected from the group consisting of a sulfur atom, a nitrogen atom, and an oxygen atom in the side chain) are inert to normal reactions. Therefore, the bioorthogonal functional group for a protein is a group that does not react with the side chains of these amino acids having side chains that are inert to normal reactions, or reacts with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine at a slow rate, but reacts with the target functional group.

[0063] Examples of such bioorthogonal functional groups include an azide residue, an aldehyde residue, a thiol residue, an alkene residue (in other words, it suffices to have a vinylene (ethenylene) moiety which is the minimum unit having a carbon-carbon double bond; the same applies hereinafter), an alkyne residue (in other words, it suffices to have an ethynylene moiety which is the minimum unit having a carbon-carbon triple bond; the same applies hereinafter), a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an α-halocarbonyl residue (e.g., a carbonyl residue having a fluorine atom, a chlorine atom, a bromine atom or an iodine atom at the α-position; the same applies hereinafter), an isonitrile residue, a sydnone residue, and a selenium residue.

[0064] More specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following. [Chemical formula] [Here, R 1a , single or plural R 1b , and single or plural R 1c are the same or different and are the above-described substituents or electron-withdrawing groups, and · represents a bond. ]

[0065] Examples of the electron-withdrawing group include a halogen atom, an alkyl substituted with a halogen atom (e.g., trifluoromethyl), a boronic acid residue, mesyl, tosyl, triflate, nitro, cyano, a phenyl group, and a keto group (e.g., acyl). A halogen atom, a boronic acid residue, mesyl, tosyl, and triflate are preferable.

[0066] Preferably, the bioorthogonal functional group may be an alkyne residue or an azide. The alkyne residue is more preferably a cyclic group having a triple bond between carbon atoms, which may be substituted with the above-described substituents.

[0067] The bioorthogonal functional group may be protected. The bioorthogonal functional group that may be protected refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. The unprotected bioorthogonal functional group corresponds to the above-described bioorthogonal functional group. The protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of a protecting group. Cleavage of the protecting group can be carried out by a specific treatment under conditions (mild conditions) that do not cause protein denaturation or decomposition (e.g., cleavage of amide bonds). Such specific treatments include, for example, (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) standing when using a cleavable linker containing a self-degradable cleavable moiety. Such protecting groups and their cleavage conditions are common general knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Jounal of American Chemical Society. 132, 1500 (2010).; Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004).; DeSimone, J.M., Journal of American Chemical Society. 132, 17928 (2010); Thompson, D.H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R.G., Journal of Controlled Release, 95, 291 (2004)).

[0068] Examples of the protected bioorthogonal functional group include a disulfide residue, an ester residue, an acetal residue, a ketal residue, an imine residue, and a vicinal diol residue.

[0069] More specifically, the protected bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following. [Chemical formula] 〔Here, the wavy line orthogonal to the bond indicates the cleavage site, single or multiple Rs 2a are the same or different and are selected from the group consisting of a hydrogen atom or the above-described substituents, · represents a bond.〕

[0070] Preferably, the optionally protected bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0071] (Functional substance) The functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to an antibody. Examples include drugs, labeling substances, and stabilizers, and preferably drugs or labeling substances. The function The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked.

[0072] The drug may be a drug for any disease. Such diseases include, for example, 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.

[0073] More specifically, the drug may be an anti-cancer agent. Examples of anti-cancer agents include chemotherapeutic agents, toxins, radioisotopes, or substances containing them. Examples of chemotherapeutic agents include DNA-damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include 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, and tubulysin.

[0074] 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 mentioned above), or substances containing them.

[0075] 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.

[0076] 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.

[0077] When the functional substance does not have a bioorthogonal functional group that readily reacts with the bioorthogonal functional group carried by the antibody intermediate, the functional substance may be derivatized to have such a bioorthogonal functional group. The bioorthogonal functional group carried by the functional substance or the derivatized functional substance is selected from the bioorthogonal functional groups as described above while considering the bioorthogonal functional group carried by the antibody intermediate. For example, when the bioorthogonal functional group carried by the antibody intermediate is an alkyne residue (preferably a cyclic group having a triple bond between carbon atoms which may be substituted by a substituent as described above), the bioorthogonal functional group carried by the functional substance or the derivatized functional substance may be an azide. Also, when the bioorthogonal functional group carried by the antibody intermediate is an azide, the bioorthogonal functional group carried by the functional substance or the derivatized functional substance may be an alkyne residue (preferably a cyclic group having a triple bond between carbon atoms which may be substituted by a substituent as described above). Derivatization is common general knowledge in the art (e.g., WO 2004 / 010957, US 2006 / 0074008 A1, 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 a suitable environment (e.g., intracellular or extracellular). Examples of such linkers include peptidyl linkers (e.g., US Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)) that are degraded 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., US Pat. Nos. 5,622,929, 5,122,368; 5,824,805) that can be cleaved at local acidic sites present in the living body.The linker may be self-immolative (e.g., WO 02 / 083180, WO 04 / 043493, WO 05 / 112919). In the present invention, a derivatized functional substance is also simply referred to as a "functional substance".

[0078] (salt) 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).

[0079] 2. Antibody composition The present invention provides (A) the following formula (A): [Chemical formula] (wherein, Ab is an antibody containing an immunoglobulin unit having two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy and light chains, S is a sulfur atom, L is a divalent group, R is an optionally protected bioorthogonal functional group; and n is an integer of 1 to 8.) An antibody intermediate having an optionally protected bioorthogonal functional group represented by the formula (I) or a salt thereof, (B) Formula (B): [ka] (In the formula, Ab is the same antibody as the antibody in formula (A), SH is a thiol group, wherein n is the same integer as n in formula (A), a sulfur atom (S) adjacent to the antibody (Ab) in formula (A) and a thiol group (SH) adjacent to the antibody (Ab) in formula (B) are directly bonded or linked via a linker to an atom in the side chain of the same amino acid residue located at the same position in the constant region of the antibody heavy chain; The molecular weight of the partial structure represented by LR is 700 or less, the percentage of the amount of the antibody intermediate or its salt relative to the total amount of the antibody intermediate or its salt and the thiol group-incorporated antibody or its salt [100(%) × (amount of the antibody intermediate or its salt) / (total amount of the antibody intermediate or its salt and the thiol group-incorporated antibody or its salt)] is 80% or more; The antibody composition has an aggregation rate of 5% or less for the antibody intermediate or its salt and the thiol group-introduced antibody or its salt.

[0080] The antibody composition of the present invention can be obtained by reacting a thiol group-introduced antibody with a compound represented by "leaving group-L-R" (Figure 1). Therefore, Ab and n, which are common symbols in formulas (A) and (B), are the same between formula (A) and (B). Although such an antibody composition can be obtained according to the present invention, there is no motivation to obtain such an antibody composition according to the prior art.

[0081] Ab in formulas (A) and (B) is an antibody containing two heavy chains and two light chains and including an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy chain and the light chain. Examples of such antibodies include IgG antibodies, IgD antibodies, and IgE antibodies containing two heavy chains and two light chains and including an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy chain and the light chain, IgA antibodies containing four heavy chains and four light chains and including an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy chain and the light chain, and IgM antibodies containing eight heavy chains and eight light chains and including an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy chain and the light chain. Among them, IgG antibodies (e.g., IgG1, IgG2, IgG3, IgG4) are preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a human IgG full-length monoclonal antibody. The divalent group represented by L in formula (A) is the same as those described above.

[0082] The divalent group represented by L in formula (A) is the same as those described above.

[0083] The optionally protected bioorthogonal functional group represented by R in formula (A) is the same as those described above.

[0084] n in formulas (A) and (B) is an integer from 1 to 8. n is preferably an integer from 1 to 6, more preferably an integer from 1 to 4 (i.e., 1, 2, 3, or 4).

[0085] The sulfur atom (S) adjacent to the antibody (Ab) in formula (A) and the thiol group (SH) adjacent to the antibody (Ab) in formula (B) are directly bonded or bonded via a linker to an atom in the side chain of the same amino acid residue present at the same position in the constant region of the antibody heavy chain. Since the antibody composition of the present invention can be obtained by modifying a thiol group-introduced antibody with a compound represented by "leaving group-L-R" (Figure 1), the binding modes of the above sulfur atom (S) and the above thiol group (SH) to the antibody are the same. That is, when the above sulfur atom (S) is directly bonded to the atom in the side chain, the above thiol group (SH) is also directly bonded to the same atom in the same side chain. Also, when the above sulfur atom (S) is bonded to the atom in the side chain via a linker, the above thiol group (SH) is also bonded to the same atom in the same side chain via the same linker.

[0086] In one embodiment, the amino acid residue in the antibody used for the binding of the above sulfur atom (S) and thiol group (SH) may be an amino acid residue other than a cysteine residue. As amino acid residues other than cysteine residues, amino acid residues having side chains that are easy to modify (e.g., amino group, carboxy group, amide group, hydroxy group) (e.g., lysine residue, aspartic acid residue, glutamic acid residue, asparagine residue, glutamine residue, threonine residue, serine residue, tyrosine residue) can be used, but preferably, a lysine residue having a side chain containing a nitrogen atom (amino group), a tyrosine residue having a side chain containing an oxygen atom (hydroxy group), a serine residue, and a threonine residue, and more preferably, a lysine residue.

[0087] In another embodiment, the amino acid residue in the antibody used for the binding of the above sulfur atom (S) and thiol group (SH) may be a cysteine residue. Since the compound used in the present invention is excellent in the efficiency of the reaction, it is considered useful not only for the modification of the thiol group chemically introduced into the antibody but also for the modification of the thiol group in the cysteine residue in the antibody.

[0088] The amino acid residues in the antibody used for the binding of the sulfur atom (S) and the thiol group (SH) can be those present at predetermined positions in the constant region of the antibody heavy chain (for example, CH1, CH2, or CH3, preferably CH2 or CH3).

[0089] In a preferred specific embodiment, the amino acid residues in the antibody used for the binding of the sulfur atom (S) and the thiol group (SH) are lysine residues having a side chain containing a nitrogen atom (amino group). Examples of the lysine residues include lysine residues present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 in the human IgG heavy chain.

[0090] Methods for producing an antibody containing a specific atom or group at a predetermined position by site-selectively modifying a specific amino acid residue at the predetermined position are described, for example, 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. According to the methods described in these documents, a specific amino acid residue at a predetermined position 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.

[0091] Preferably, as the thiol group-introduced antibody, an antibody containing a thiol group that is site-specifically bound via a linker that does not contain a peptide to an atom in the side chain of a specific amino acid residue (preferably, a nitrogen atom in the side chain of a lysine residue) present at a predetermined position in the constant region of the heavy chain can be used. By modifying such a thiol group-introduced antibody with a compound represented by "leaving group-L-R", the sulfur atom (S) adjacent to the antibody (Ab) in formula (A) and the thiol group (SH) adjacent to the antibody (Ab) in formula (B) are site-specifically directly bound to an atom in the side chain of the same amino acid residue present at the same position in the constant region of the antibody heavy chain, or a site-specifically bound via a linker of the antibody composition of the present invention can be obtained. As used herein, "site-specific" or "site-selectivity" means that, although a specific amino acid residue is not unevenly distributed in a specific region in an antibody, a predetermined structural unit capable of binding to a specific amino acid residue in the antibody is unevenly distributed in a specific region in the antibody. Therefore, expressions related to site-selectivity such as "having site-specifically", "site-specific binding", and "binding with site-selectivity" refer to the retention rate or binding rate of a predetermined structural unit in a target region containing one or more specific amino acid residues being 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-specific binding or retention can be achieved by using an antibody obtained by the above method in which a specific amino acid residue at a predetermined position can be site-specifically modified with a thiol group as the thiol group-introduced antibody. 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 100%. Confirmation of site-selectivity can be performed, for example, by peptide mapping [see, for example, International Publication No. 2019 / 240287 (WO2019 / 240287A1)].

[0092] As the linker that does not contain a peptide, the above-described divalent group can be used. Preferably, when the amino acid residue in the antibody used for the bond between the sulfur atom (S) and the thiol group (SH) is a lysine residue, as the linker that does not contain a peptide, a carbonyl-containing linker containing a -C(=O)- moiety capable of forming an amide bond with the amino group in the side chain of the lysine residue can be used.

[0093] As the carbonyl-containing linker, it has a -C(=O)- moiety at the terminal and has a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR4-, -C(=O)-NR4-, -NR4-C(=O)-, -C(=S)-NR4-, -NR4-C(=S)-, -O-, -S-, -(O-R5) m1 -, and -(S-R5) m1 -selected from the group consisting of, or a group having a main chain structure containing two or more (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, particularly preferably 2 or 3) of these groups can be used. R4 represents a hydrogen atom or a substituent described later. R5 represents a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. m1 is an integer from 1 to 10, preferably an integer from 1 to 8, more preferably an integer from 1 to 6, even more preferably an integer from 1 to 5, and particularly preferably an integer from 1 to 3.

[0094] Preferably, the carbonyl-containing linker has a -C(=O)- moiety at the terminal and has a divalent group having a main chain structure containing one group selected from the group consisting of alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR4-, -NR4-C(=O)-, -O-, and -(O-R5) m1 -, or - having -C(=O)- at the terminal and having a divalent group having a main chain structure containing two or more groups selected from the group consisting of alkylene, arylene, -C(=O)-, -NR4-, -C(=O)-NR4-, -NR4-C(=O)-, -O-, and -(O-R5) m -, R2 is a hydrogen atom or an alkyl group, R3 is an alkylene or an arylene group, m may be an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5). Alkylene, arylene, and alkyl are the same as those described above.

[0095] More preferably, the carbonyl-containing linker may be a divalent group having a -C(=O)- moiety at the end and a main chain structure containing an alkylene or an arylene or a combination thereof.

[0096] Even more preferably, the carbonyl-containing linker may be a divalent group having a main chain structure containing a -C(=O)- moiety and an alkylene (e.g., -C(=O)-CH2-CH2-).

[0097] The main chain structure of the carbonyl-containing linker may be substituted by one or more (e.g., 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to 3) of the above-described substituents.

[0098] In the present invention, the molecular weight of the partial structure represented by L-R in formula (A) is 700 or less. When the molecular weight of the partial structure represented by L-R is 700 or less, the antibody intermediate having a bioorthogonal functional group which may be protected or a salt thereof, and the thiol group-introduced antibody or a salt thereof have a very small ratio of the molecular weight of the above partial structure to the molecular weight of the whole antibody, and thus separation based on the difference in molecular weight is difficult. The molecular weight of the partial structure represented by L-R is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, particularly preferably 300 or less, 250 or less, 200 or less, or 150 or less.

[0099] In the present invention, the percentage of the amount of the antibody intermediate or a salt thereof with respect to the total amount of the antibody intermediate or a salt thereof and the thiol group-introduced antibody or a salt thereof [100 (%) × (the amount of the antibody intermediate or a salt thereof) / (the total amount of the antibody intermediate or a salt thereof and the thiol group-introduced antibody or a salt thereof)] is 80% or more. Since the compound or a salt thereof used in the present invention is excellent in the reaction efficiency with respect to the thiol group-introduced antibody or a salt thereof, good reactivity expressed by such a percentage can be achieved. Such a percentage is preferably 82% or more, more preferably 84% or more, still more preferably 86% or more, particularly preferably 88% or more, 90% or more, 92% or more, 94% or more, or 96% or more. Such a percentage can be determined based on the measurement value by reversed-phase HPLC under reducing conditions or mass spectrometry (see Examples).

[0100] In the present invention, the aggregation rate of the antibody intermediate or a salt thereof and the thiol group-introduced antibody or a salt thereof is 5% or less. The modification of the thiol group-introduced antibody with the compound used in the present invention can be carried out under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds), and it is difficult to cause antibody aggregation. The aggregation rate is preferably 4.8% or less, more preferably 4.6% or less, still more preferably 4.4% or less, particularly preferably 4.2% or less, 4.0% or less, 3.8% or less, 3.6% or less, or 3.4% or less. The aggregation rate of the antibody can be measured by size exclusion chromatography (SEC)-HPLC (see Examples and ACS Omega 2020, 5, 7193-7200).

[0101] Details of the antibody intermediate or a salt thereof contained in the antibody composition are as described later for the antibody intermediate or a salt thereof.

[0102] The antibody composition of the present invention may further contain, in addition to (A) an antibody intermediate having a bioorthogonal functional group that may be protected or a salt thereof, and (B) a thiol group-introduced antibody or a salt thereof, an antibody used as a raw material for producing the thiol group-introduced antibody. In this case, the characteristics of the antibody used as a raw material for producing the thiol group-introduced antibody can be inherited by the "antibody intermediate having a bioorthogonal functional group that may be protected" and the "thiol group-introduced antibody". Therefore, the antibody used as a raw material for producing the thiol group-introduced antibody can be defined in the same manner as the "antibody" in the "antibody intermediate having a bioorthogonal functional group that may be protected" and the "thiol group-introduced antibody".

[0103] The antibody composition of the present invention can be obtained by reacting a thiol group-introduced antibody with a compound represented by "leaving group-L-R" (Figure 1). From such a reaction, the thiol group in the thiol group-introduced antibody binds to the leaving group-bonding atom in L in the compound represented by "leaving group-L-R", and the leaving group leaves from the leaving group-bonding atom, to obtain an antibody composition containing an antibody intermediate or a salt thereof (reaction product) and a thiol group-introduced antibody or a salt thereof (unreacted substance). The molar ratio of the compound represented by "leaving group-L-R" or a salt thereof to the thiol group-introduced antibody or a salt thereof in the reaction (compound represented by "leaving group-L-R" or a salt thereof / thiol group-introduced antibody or a salt thereof) is not particularly limited because it varies depending on factors such as the type of the compound represented by "leaving group-L-R" or a salt thereof and the thiol group-introduced antibody, but is, for example, 1 to 100, preferably 2 to 80, and more preferably 3 to 50.

[0104] 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).

[0105] Confirmation of the production of the antibody intermediate or its salt can be carried out, for example, by reversed-phase HPLC under reducing conditions or by mass spectrometry, depending on the specific molecular weights of the raw materials and products.

[0106] The antibody composition of the present invention can contain an antibody intermediate having a bioorthogonal functional group that may be protected or its salt at a high content in a state with a low aggregation rate. The antibody intermediate having a bioorthogonal functional group that may be protected or its salt can be used for the production of a conjugate of an antibody and a functional substance or its salt. Therefore, the antibody composition of the present invention can efficiently produce a conjugate of an antibody and a functional substance or its salt in a state with a low aggregation rate.

[0107] 3. Reagent for derivatizing a thiol group-introduced antibody or its salt The present invention provides a reagent for derivatizing a thiol group-introduced antibody or its salt.

[0108] In one embodiment, the reagent of the present invention contains a compound represented by the following formula (I) or a salt thereof.

Chemical formula

[0109] The halogen atom represented by X in formula (I) is the same as those described above.

[0110] Examples of the arylene represented by Q in formula (I) include phenylene, naphthylene, and anthracenylene, with phenylene and naphthylene being preferred, and phenylene being more preferred. Examples of the alkylene represented by Q in formula (I) include linear or branched alkylene, with linear alkylene being preferred. As the linear alkylene, linear alkylene having 1 to 6 carbon atoms is preferred, and linear alkylene having 1 to 4 carbon atoms is more preferred.

[0111] The divalent group represented by Y in formula (I) is the same as those described above.

[0112] In a specific embodiment, the divalent group represented by Y in formula (I) may be a group having a main chain structure containing -(O-R5) m1 -. R5 and m1 are the same as those described above.

[0113] The bioorthogonal functional group which may be protected represented by R in formula (I) is the same as those described above.

[0114] In another embodiment, the reagent of the present invention contains a compound represented by the following formula (II) or a salt thereof.

Chemical formula

[0115] The alkyl represented by R1 in formula (II) is the same as that described above.

[0116] The divalent group represented by Y in formula (II) is the same as that described above.

[0117] The bioorthogonal functional group which may be protected and represented by R in formula (II) is the same as that described above.

[0118] In yet another embodiment, the reagent of the present invention comprises a compound represented by the following formula (III) or a salt thereof.

Chemical formula

[0119] The divalent group represented by Y in formula (III) is the same as that described above.

[0120] The bioorthogonal functional group which may be protected and represented by R in formula (III) is the same as that described above.

[0121] In yet another embodiment, the reagent of the present invention comprises a compound represented by the following formula (IV) or a salt thereof.

Chemical formula

[0122] The divalent group represented by Y in formula (IV) is the same as those described above.

[0123] The optionally protected bioorthogonal functional group represented by R in formula (IV) is the same as those described above.

[0124] More specifically, the compounds represented by formulas (I) to (IV) may be compounds represented by the following formulas (1) to (12).

Chemical formula

Chemical formula

[0125] The thiol group-introduced antibody or a salt thereof derivatized by the reagent of the present invention is as described above for the thiol group-introduced antibody or the antibody. For example, the thiol group in the thiol group-introduced antibody may be selectively bound to an atom (preferably, a nitrogen atom in the side chain of a lysine residue) in the side chain of an amino acid residue (e.g., an amino acid residue having a side chain that is easily modified as described above) in the constant region of the antibody heavy chain via a linker that does not contain a peptide. Further, the thiol group-introduced antibody may be an IgG antibody. When a human IgG antibody is used as the thiol group-introduced antibody, such lysine residues may be present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain in EU numbering.

[0126] The reagent of the present invention can derivatize the thiol group-introduced antibody or a salt thereof by reacting with the thiol group (preferably, the thiol group introduced via the side chain of a lysine residue) in the thiol group-introduced antibody or a salt thereof.

[0127] 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, citrate buffer solution), and a buffer solution is 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).

[0128] 4. Compound or a salt thereof The present invention also provides a compound or a salt thereof.

[0129] In one embodiment, the compound of the present invention is a compound represented by the following formula (I) or a salt thereof. [Chemical formula] (In the formula, X is a halogen atom, Q is arylene or alkylene, Y is a divalent group, R is an optionally protected bioorthogonal functional group.)

[0130] In formula (I), the halogen atom represented by X, the arylene or alkylene represented by Q, the divalent group represented by Y, and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0131] In another embodiment, the compound of the present invention is a compound represented by the following formula (II) or a salt thereof. [Chemical formula] (In the formula, R1 is alkyl, Y is a divalent group, R is an optionally protected bioorthogonal functional group.)

[0132] In formula (II), the alkyl represented by R1, the divalent group represented by Y, and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0133] In yet another embodiment, the compound of the present invention is a compound represented by the following formula (III) or a salt thereof. [Chemical formula] (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group.)

[0134] In formula (III), the divalent group represented by Y and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0135] In yet another embodiment, the compound of the present invention is a compound represented by the following formula (IV) or a salt thereof. [Chemical formula] (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group.)

[0136] In formula (IV), the divalent group represented by Y and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0137] More specifically, the compounds represented by formulas (I) to (IV) may be the compounds represented by the above formulas (2) to (12).

[0138] The compounds represented by formulas (I) to (IV) can be obtained by a synthetic reaction as described in the examples. For example, such a reaction can be carried out at an appropriate temperature (e.g., about 4 to 90 °C) in a suitable organic solvent system. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours.

[0139] Confirmation of the formation of the series of compounds or salts thereof as described above depends on the specific raw materials and the molecular weights of the products, but can be carried out, for example, by NMR and mass spectrometry. Such compounds or salts thereof can be appropriately purified by any method such as chromatography, solvent extraction, recrystallization, etc.

[0140] The compound of the present invention or a salt thereof can be used for derivatization of a thiol group-introduced antibody or a salt thereof. can be.

[0141] 5. Antibody intermediate or a salt thereof The present invention provides an antibody intermediate having a bioorthogonal functional group that may be protected, or a salt thereof.

[0142] In one embodiment, the antibody intermediate of the present invention is an antibody intermediate having a bioorthogonal functional group that may be protected, represented by the following formula (I’). [Chemical formula] (In the formula, Ab is an antibody, S is a sulfur atom, Q is an arylene or an alkylene, Y is a bond or a divalent group, R is a bioorthogonal functional group that may be protected, n is an integer from 1 to 8.)

[0143] The relationship between the antibody represented by Ab in formula (I’) and the antibody and the sulfur atom (S) adjacent thereto is the same as that described above for the thiol group-introduced antibody or antibody. For example, the sulfur atom (S) adjacent to the antibody may be selectively bonded via a peptide-free linker to an atom (preferably, a nitrogen atom in the side chain of a lysine residue) in the side chain of an amino acid residue (e.g., an amino acid residue having a side chain that is easily modified as described above) in the constant region of the antibody heavy chain. Further, the antibody may be an antibody containing an immunoglobulin unit including two heavy chains and two light chains (preferably, an antibody containing an immunoglobulin unit including two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy chain and the light chain). Furthermore, the antibody may be an IgG antibody. When a human IgG antibody is used as the antibody, such lysine residues may be present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain in EU numbering. The site selectivity and its degree are the same as those described above. The confirmation of site selectivity can be confirmed by the method described above.

[0144] In formula (I’), n is an integer from 1 to 8, preferably an integer from 1 to 6, more preferably an integer from 1 to 4 (i.e., 1, 2, 3, or 4).

[0145] Examples of the arylene represented by Q in formula (I’) include phenylene, naphthylene, and anthracenylene, with phenylene and naphthylene being preferred, and phenylene being more preferred. Examples of the alkylene represented by Q in formula (I’) include linear or branched alkylene, with linear alkylene being preferred. As the linear alkylene, linear alkylene having 1 to 6 carbon atoms is preferred, and linear alkylene having 1 to 4 carbon atoms is more preferred.

[0146] The divalent group represented by Y and the optionally protected bioorthogonal functional group represented by R in formula (I’) are the same as those described above.

[0147] In a specific embodiment, the divalent group represented by Y in formula (I’) may be a group having a main chain structure containing -(O-R5) m1 -. R5 and m1 are the same as those described above.

[0148] In another embodiment, the antibody intermediate of the present invention is an antibody intermediate having an optionally protected bioorthogonal functional group represented by the following formula (II’).

Chemical formula

[0149] In formula (II’), the antibody represented by Ab, the relationship between the antibody and the sulfur atom (S) adjacent thereto, the integer represented by n, the divalent group represented by Y, and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0150] In yet another embodiment, the antibody intermediate of the present invention is an antibody intermediate having an optionally protected bioorthogonal functional group represented by the following formula (III’).

Chemical formula

[0151] In formula (III’), the antibody represented by Ab, the relationship between the antibody and the sulfur atom (S) adjacent thereto, the integer represented by n, the divalent group represented by Y, and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0152] In yet another embodiment, the antibody intermediate of the present invention is an antibody intermediate having an optionally protected bioorthogonal functional group represented by the following formula (IV’).

Chemical formula

[0153] In formula (IV’), the antibody represented by Ab, the relationship between the antibody and the adjacent sulfur atom (S), the integer represented by n, the divalent group represented by Y, and the optionally protected bioorthogonal functional group represented by R are the same as those described above.

[0154] More specifically, the antibody intermediates represented by formulas (I’) to (IV’) may be antibody intermediates represented by the following formulas (1’) to (12’).

Chemical formula

Chemical formula

[0155] The aggregation rate of the antibody intermediate or its salt of the present invention may be 5% or less. The modification of the thiol group-introduced antibody with the compound used in the present invention can be carried out under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds), and it is difficult to cause antibody aggregation. The aggregation rate is preferably 4.8% or less, more preferably 4.6% or less, even more preferably 4.4% or less, particularly preferably 4.2% or less, 4.0% or less, 3.8% or less, 3.6% or less, or 3.4% or less. The aggregation rate of the antibody can be measured by size exclusion chromatography (SEC)-HPLC (see Examples and ACS Omega 2020, 5, 7193-7200).

[0156] The antibody intermediate of the present invention or a salt thereof can also have high blood stability.

[0157] The antibody intermediate of the present invention can be obtained by reacting a thiol group-introduced antibody with a compound represented by formula (I) to (IV) or a salt thereof (Figure 1). The molar ratio of the compound represented by formula (I) to (IV) or a salt thereof to the thiol group-introduced antibody or a salt thereof in the reaction (compound represented by formula (I) to (IV) or a salt thereof / thiol group-introduced antibody or a salt thereof) is not particularly limited because it varies depending on factors such as the type of the compound represented by formula (I) to (IV) or a salt thereof and the thiol group-introduced antibody, but is, for example, 1 to 100, preferably 2 to 80, more preferably 3 to 50.

[0158] Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds). For example, such a reaction can be carried out at room temperature (e.g., about 15 to 30 °C) in an appropriate reaction system, such as a buffer solution. The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such a reaction, 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).

[0159] Confirmation of the production of the antibody intermediate or a salt thereof can be carried out, for example, by reverse-phase HPLC under reducing conditions or by mass spectrometry, although it also depends on the specific raw materials and the molecular weight of the product.

[0160] The antibody intermediate of the present invention can be used for the production of a conjugate of an antibody and a functional substance or a salt thereof.

[0161] 6. Conjugate of an antibody and a functional substance or a salt thereof The present invention provides a conjugate of an antibody and a functional substance or a salt thereof.

[0162] In one embodiment, the conjugate of the present invention is a conjugate of an antibody and a functional substance represented by the following formula (I'').

Chemical formula

[0163] The relationship between the antibody represented by Ab in formula (I'') and the sulfur atom (S) adjacent to the antibody is the same as that described above for the thiol group-introduced antibody or the antibody. For example, the sulfur atom (S) adjacent to the antibody may be position-selectively bonded via a peptide-free linker to an atom (preferably, a nitrogen atom in the side chain of a lysine residue) in the side chain of an amino acid residue (e.g., an amino acid residue having a side chain that is easily modified as described above) in the constant region of the antibody heavy chain. Further, the antibody may be an antibody containing an immunoglobulin unit including two heavy chains and two light chains (preferably, an antibody containing an immunoglobulin unit including two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy chain and the light chain). Furthermore, the antibody may be an IgG antibody. When a human IgG antibody is used as the antibody, such lysine residues may be present at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain in EU numbering. The position selectivity and its degree are the same as those described above. The confirmation of the position selectivity can be confirmed by the method described above.

[0164] In formula (I''), n is an integer from 1 to 8, preferably an integer from 1 to 6, and more preferably an integer from 1 to 4 (i.e., 1, 2, 3, or 4).

[0165] The group generated by the reaction between two bioorthogonal functional groups represented by R' in formula (I'') is a group generated by the reaction between a bioorthogonal functional group carried by an antibody intermediate and a bioorthogonal functional group carried by a functional substance (wherein these bioorthogonal functional groups are selected to be a combination capable of reacting with each other). Since combinations of two bioorthogonal functional groups capable of reacting with each other are well-known, those skilled in the art can appropriately select such combinations and appropriately set a divalent group containing a moiety generated by the reaction between two bioorthogonal functional groups capable of reacting with each other. Examples of combinations of bioorthogonal functional groups capable of reacting with each other include, for example, a combination of a thiol residue and a maleimide residue, a combination of a furan residue and a maleimide residue, a combination of a thiol residue and a halocarbonyl residue (in a substitution reaction, a halogen is substituted by a thiol), a combination of an alkyne residue (preferably, a cyclic group having a triple bond between carbon atoms which may be substituted by a substituent as described above) and an azide residue, a combination of a tetrazine residue and an alkene residue, a combination of a tetrazine residue and an alkyne residue, and a combination of a thiol residue and another thiol residue (a disulfide bond). Therefore, the above moiety may be a group generated by the reaction between a thiol residue and a maleimide residue, a group generated by the reaction between a furan residue and a maleimide residue, a group generated by the reaction between a thiol residue and a halocarbonyl residue, a group generated by the reaction between an alkyne residue and an azide residue, or a group generated by the reaction between a tetrazine residue and an alkene residue, or a disulfide group generated by a combination of a thiol residue and another thiol residue. Examples of combinations of bioorthogonal functional groups capable of reacting with each other include, for example, a combination of an alkyne residue (preferably, a cyclic group having a triple bond between carbon atoms which may be substituted by a substituent as described above) and an azide, a combination of a thiol and a maleimide, a combination of a tetrazine and an azide, and a combination of a furan and a maleimide. Preferably, the combination of bioorthogonal functional groups capable of reacting with each other may be a combination of an alkyne residue (preferably, a cyclic group having a triple bond between carbon atoms which may be substituted by a substituent as described above) and an azide.When a combination of an alkyne residue and an azide is used as two bioorthogonal functional groups capable of reacting with each other, R’ is a divalent triazole ring group. When a combination of a ring group having a triple bond between carbon atoms and an azide is used as two bioorthogonal functional groups capable of reacting with each other, R’ is a divalent ring group in which the ring group and the triazole ring are condensed. Since the ring group having a triple bond between carbon atoms may be substituted with a substituent as described above, the divalent ring group in which the ring group and the triazole ring are condensed may also be substituted with a substituent as described above.

[0166] In certain embodiments, the group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other may be a divalent group represented by any one of the following structural formulas.

Chemical formula

[0167] Examples of the arylene represented by Q in formula (I'') include phenylene, naphthylene, and anthracenylene, with phenylene and naphthylene being preferred, and phenylene being more preferred. Examples of the alkylene represented by Q in formula (I'') include linear or branched alkylene, with linear alkylene being preferred. As the linear alkylene, linear alkylene having 1 to 6 carbon atoms is preferred, and linear alkylene having 1 to 4 carbon atoms is more preferred.

[0168] In formula (I''), the divalent group represented by Y and the functional substance represented by Z are the same as those described above.

[0169] In certain embodiments, the divalent group represented by Y in formula (I'') is -(O-R5) m1It may also be a group having a main chain structure containing -. R5 and m1 are the same as those described above.

[0170] In another embodiment, the conjugate of the present invention is a conjugate of an antibody and a functional substance represented by the following formula (II'').

Chemical formula

[0171] The antibody represented by Ab in formula (II''), the relationship between the antibody and the adjacent sulfur atom (S), the integer represented by n, the divalent group represented by Y, the group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other represented by R', and the functional substance represented by Z are the same as those described above.

[0172] In yet another embodiment, the conjugate of the present invention is a conjugate of an antibody and a functional substance represented by the following formula (III'').

Chemical formula

[0173] The antibody represented by Ab in formula (III''), the relationship between the antibody and the sulfur atom (S) adjacent thereto, the integer represented by n, the divalent group represented by Y, the group generated by the reaction between two bioorthogonal functional groups represented by R' that are reactive with each other, and the functional substance represented by Z are the same as those described above.

[0174] In yet another embodiment, the conjugate of the present invention is a conjugate of an antibody and a functional substance represented by the following formula (IV''). [Chemical formula] (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R' is a group generated by the reaction between two bioorthogonal functional groups that are reactive with each other, Z is a functional substance, n is an integer from 1 to 8.)

[0175] The antibody represented by Ab in formula (IV''), the relationship between the antibody and the sulfur atom (S) adjacent thereto, the integer represented by n, the divalent group represented by Y, the group generated by the reaction between two bioorthogonal functional groups represented by R' that are reactive with each other, and the functional substance represented by Z are the same as those described above.

[0176] More specifically, the conjugates represented by formulas (I'') to (IV'') may be conjugates represented by the following formulas (1'') to (12''). [Chemical formula] [Chemical formula] (In the formula, k is an integer of 0 or 1, m is an integer from 1 to 5 (preferably an integer from 1 to 3), m’ is an integer from 1 to 5 (preferably an integer from 1 to 3), R2 and R3 are each independently a hydrogen atom or methyl, R6’ is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z’ is a functional substance, Ab, S, Q, R’, Z, and n adjacent to Ab are the same as those described above.)

[0177] The aggregation rate of the conjugate of the present invention or a salt thereof may be 10% or less. The antibody intermediate or a salt thereof used in the present invention has an aggregation rate of 5% or less, and the conjugate of the present invention or a salt thereof is obtained by subjecting such an antibody intermediate or a salt thereof to a reaction under conditions (mild conditions) that do not cause protein denaturation and degradation ( Example, cleavage of amide bonds), and thus it is difficult to cause antibody aggregation. The aggregation rate is preferably 9% or less, more preferably 8.5% or less, even more preferably 8.0% or less, particularly preferably 7.5% or less, 7.0% or less, 6.5% or less, 6.0% or less, 5.5% or less, 5% or less, 4.8% or less, 4.6% or less, 4.4% or less, 4.2% or less, 4.0% or less, 3.8% or less, 3.6% or less, or 3.4% or less. The aggregation rate of the antibody can be measured by size exclusion chromatography (SEC)-HPLC (see Examples and ACS Omega 2020, 5, 7193-7200).

[0178] The conjugate of the present invention or a salt thereof can also have high blood stability.

[0179] The conjugate of the present invention or a salt thereof can be obtained by reacting the antibody intermediate 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 or degradation of the protein (immunoglobulin / antibody) (e.g., cleavage of amide bonds), i.e., under mild conditions as described above. In the reaction, the molar ratio of the functional substance to the antibody intermediate or a salt thereof (functional substance / antibody intermediate or a salt thereof) varies depending on factors such as the type of the antibody intermediate or a salt thereof, the type of the functional substance, and the reaction time, and thus is not particularly limited. For example, it is 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 antibody intermediate in a short reaction time, a sufficient amount (e.g., an excessive amount) of the functional substance can be used with respect to the antibody intermediate or a salt thereof.

[0180] Confirmation of the production of the conjugate or a salt thereof can be performed, for example, by reverse-phase HPLC under reducing conditions or by mass spectrometry, although it also depends on the specific raw materials and the molecular weight of the product. The conjugate or a salt thereof can be appropriately purified by any method such as chromatography (e.g., affinity chromatography).

[0181] The conjugate of the present invention or a salt thereof can be used, for example, as a medicine or a reagent (e.g., a diagnostic agent, a research reagent).

[0182] 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, glycyllysine 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 cocoa 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.

[0183] 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, orange juice, capsules containing an effective amount of the ligand as a solid or granule Agents, sachets or tablets, suspension formulations 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, etc.

[0184] 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, isotonic 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.

[0185] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug tolerance of the administration target, body weight, age, etc., but can be set as appropriate.

Example

[0186] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0187] Comparative Example 1: Introduction of an azide group with N-(4-azidophenyl)-2-iodo-acetamide

Chemical formula

[0188] In the following comparative examples and examples, as the thiol group-introduced antibody, an antibody derivative (thiol group-introduced trastuzumab) described in Example 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used. This antibody derivative has the following structure in which a thiol group is regioselectively introduced into trastuzumab (humanized IgG1 antibody) via the amino group of the side chain of the lysine residue at position 246 or 248 of the antibody heavy chain (the position of the lysine residue follows EU numbering).

Chemical formula

[0189] To a solution (50 μM) of the thiol group-introduced antibody in buffer (HEPES buffer at pH 8.0), a DMF solution (12.5 mM) of 20 equivalents of N-(4-azidophenyl)-2-iodo-acetamide and a buffer (HEPES buffer at pH 8.0) solution (100 mM) of 50 equivalents of sodium iodide were added. After standing at 37 °C for 2 hours, purification was carried out using NAP-5 Columns (manufactured by GE Healthcare).

[0190] A solution (0.5 mM) of tris(2-carboxyethyl)phosphine hydrochloride was added to the product, and it was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks were observed at 50831 and 50993 where an azide group was introduced into the heavy chain, as well as at 50683 and 50845 which are the same as the raw material, and a light chain peak was observed at 23439.

[0191] Comparative Example 2: Introduction of an azide group with N-[2-[2-(2-Azidoethoxy)ethoxy]ethyl]-2-iodoaacetamide

Chemical Structure

[0192] To a solution of the thiol group-introduced antibody in buffer (HEPES buffer at pH 8.0, 50 μM) was added 50 equivalents of a DMF solution of N-[2-[2-(2-Azidoethoxy)ethoxy]ethyl]-2-iodoacetamide (31.3 mM) and 50 equivalents of a buffer solution (HEPES buffer at pH 8.0, 100 mM) of sodium iodide. After standing at 37 °C for 2 hours, purification was performed using NAP-5 Columns (manufactured by GE Healthcare).

[0193] To the product was added a solution of tris(2-carboxyethyl)phosphine hydrochloride (0.5 mM), and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the starting thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks were observed at 50896 and 51058 where an azide group was introduced into the heavy chain, as well as heavy chain peaks at 50683 and 50845 which are the same as the starting material, and a light chain peak at 23439.

[0194] Example 1: Introduction of an azide group by azide linker (1)

Chemical formula

[0195] To a solution of the thiol group-introduced antibody in buffer (PBS buffer at pH 7.4, 10 mM EDTA, 20 μM) was added 5 equivalents of a DMF solution of azide linker (1, manufactured by Aldrich). After standing at room temperature for 1 hour, purification was performed using NAP-5 Columns (manufactured by GE Healthcare).

[0196] To the product, a tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the thiol group-introduced antibody as the raw material, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 50816 and 50978, and a light chain peak at 23439, which was the same as that of the raw material, was observed.

[0197] Example 2: Introduction of azide group by azide linker (2)

Chemical formula

[0198] (1) Synthesis of azide linker (2) (1-1) Synthesis of 5-azidopentanehydrazide

Chemical formula

[0199] A THF solution (10 mL) of 5-azidopentanoic acid (200 mg, 1.4 mmol) was cooled to 0 °C, and N-methylmorpholine (0.32 mL, 2.1 mmol) and isobutyl chloroformate (0.22 mL, 1.7 mmol) were added. After stirring at 0 °C for 30 minutes, hydrazine monohydrate (0.087 mL, 2.8 mmol) and DIPEA (0.71 mL, 4.2 mmol) were added. After stirring at room temperature for 16 hours, concentration under reduced pressure was performed, and the obtained crude product was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain 5-(4-azidobutyl)-1,3,4-oxadiazole-2-thiol (111 mg, 0.71 mmol, yield 51%).

[0200] MS(ESI) m / z: 158 [M+H]

[0201] (1-2) Synthesis of 5-(4-azidobutyl)-1,3,4-oxadiazole-2-thiol

Chem.

[0202] Carbon disulfide (0.055 mL, 0.92 mmol) and potassium hydroxide (192 mg, 3.4 mmol) were added to an ethanol solution (7.1 mL) of 5-azidopentanehydrazide (111 mg, 0.71 mmol), and the mixture was stirred at 70 °C for 16 h. Then, 6 M aqueous hydrochloric acid was added to adjust the pH of the system to 3.0. After adjustment, liquid-liquid extraction with ethyl acetate was performed, and the crude product obtained by concentrating the organic phase under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain 5-(4-azidobutyl)-1,3,4-oxadiazole-2-thiol (35 mg, 0.18 mmol, yield 25%).

[0203] MS(ESI) m / z:200[M+H]

[0204] (1-3) Synthesis of 2-(4-azidobutyl)-5-methylsulfanyl-1,3,4-oxadiazole

Chem.

[0205] A solution of 5-(4-azidobutyl)-1,3,4-oxadiazole-2-thiol (35 mg, 0.18 mmol) in THF (1.8 mL) was added with iodomethane (0.033 mL, 0.53 mmol) and triethylamine (0.074 mL, 0.53 mmol), and stirred at room temperature for 16 h. The crude product obtained by concentrating the reaction solution under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain 2-(4-azidobutyl)-5-methylsulfanyl-1,3,4-oxadiazole (33 mg, 0.15 mmol, yield 85%).

[0206] MS(ESI) m / z:214[M+H]

[0207] (1-4) Synthesis of azido linker (2)

Chemical formula

[0208] A dichloromethane solution (1.5 mL) of 2-(4-azidobutyl)-5-methylsulfanyl-1,3,4-oxadiazole (33 mg, 0.15 mmol) was cooled to 0 °C, and mCPBA (0.311 g, 1.3 mmol) was added. After stirring at room temperature for 20 h, saturated aqueous sodium hydrogen carbonate solution was added, and liquid separation extraction with ethyl acetate was performed. The crude product obtained by concentrating the organic layer under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain azido linker (2) (26 mg, 0.11 mmol, yield 71%).

[0209] MS(ESI) m / z:246[M+H]

[0210] (2) Introduction of azido group by azido linker (2) To a solution of the thiol group-introduced antibody in buffer (pH 7.4 PBS buffer, 10 mM EDTA) (20 μM), a 5-fold equivalent amount of a DMF solution of azide linker (2) (1.25 mM) was added. After standing at room temperature for 1 hour, purification was performed using NAP-5 Columns (manufactured by GE Healthcare).

[0211] A solution of tris(2-carboxyethyl)phosphine hydrochloride (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 50846 and 51008, and a light chain peak at 23439, which was the same as that of the raw material, was observed.

[0212] Example 3: Introduction of azide group by azide linker (3)

Chemical formula

[0213] (1) Synthesis of azide linker (3) (1-1) Synthesis of 2-(4-azidophenyl)-5-methylsulfanyl-1,3,4-oxadiazole

Chemical formula

[0214] A dichloromethane solution (3.0 mL) of the trifluoroacetate salt (48 mg, 0.15 mmol) of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)aniline was added with 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (137 mg, 0.48 mmol) and DMAP (86 mg, 0.70 mmol), and the mixture was stirred at 50 °C for 16 h. The crude product obtained by concentrating the reaction solution under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain 2-(4-azidophenyl)-5-methylsulfanyl-1,3,4-oxadiazole (26 mg, 0.11 mmol, yield 73%).

[0215] MS(ESI) m / z:234[M+H]

[0216] (1-2) Synthesis of azide linker (3)

Chemical formula

[0217] MS(ESI) m / z:266[M+H]

[0218] (2) Introduction of azide group by azide linker (3) The azide linker (3) was reacted with the thiol group-introduced antibody in the same manner as the method described in Example 2(2).

[0219] To the product, a tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the thiol group-introduced antibody as the raw material, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks were observed at 50841 and 51002 where an azide group was introduced into the heavy chain, and a light chain peak was observed at 23439, which was the same as that of the raw material.

[0220] Example 4: Introduction of an azide group using an azide linker (4)

Chemical formula

[0221] (1) Synthesis of azide linker (4) (1-1) Synthesis of tert-butyl N-[4-(hydrazinecarbonyl)phenyl]carbamate

Chemical formula

[0222] A THF solution (8.4 mL) of 4-(tert-butoxycarbonylamino)benzoic acid (200 mg, 0.84 mmol) was cooled to 0 °C, and N-methylmorpholine (0.14 mL, 1.3 mmol) and isobutyl chloroformate (0.13 mL, 1.0 mmol) were added. After stirring at 0 °C for 30 minutes, hydrazine monohydrate (0.078 mL, 2.5 mmol) and DIPEA (0.30 mL, 1.7 mmol) were added. After stirring at room temperature for 16 hours, the mixture was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-butyl N-[4-(hydrazinecarbonyl)phenyl]carbamate (187 mg, 0.74 mmol, yield 88%).

[0223] MS(ESI) m / z: 252 [M+H]

[0224] (1-2) Synthesis of tert-butyl N-[4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate

Chemical formula

[0225] To a solution of tert-butyl N-[4-(hydrazinecarbonyl)phenyl]carbamate (187 mg, 0.74 mmol) in ethanol (7.4 mL), carbon disulfide (0.058 mL, 0.97 mmol) and potassium hydroxide (125 mg, 2.2 mmol) were added. After stirring at 75 °C for 16 hours, 6M aqueous hydrochloric acid was added to adjust the pH of the system to 3.0. After adjustment, liquid-liquid extraction with ethyl acetate was performed, and the crude product obtained by concentrating the organic phase under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-butyl N-[4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate (99 mg, 0.34 mmol, yield 45%).

[0226] MS(ESI) m / z: 294 [M+H]

[0227] (1-3) Synthesis of tert-butyl N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate

Chemical formula

[0228] A solution of tert-butyl N-[4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate (99 mg, 0.34 mmol) in THF (3.4 mL) was cooled to 0 °C, and iodomethane (0.063 mL, 1.0 mmol) and triethylamine (0.14 mL, 1.0 mmol) were added. The mixture was stirred at room temperature for 3 hours. The crude product obtained by concentrating the reaction mixture under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-butyl N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate (88 mg, 0.29 mmol, 84% yield).

[0229] MS(ESI) m / z:308[M+H]

[0230] (1-4) Synthesis of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)aniline

Chemical formula

[0231] Trifluoroacetic acid (2.9 mL) was added to a solution of tert-butyl N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate (88 mg, 0.29 mmol) in dichloromethane (2.9 mL), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the trifluoroacetate salt of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)aniline (93 mg, 0.29 mmol, 100% yield).

[0232] (1-5) Synthesis of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]acetamide [ka]

[0233] 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)aniline trifluoroacetate (57 mg, 0.18 mmol) dichloromethane To a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (102 mg, 0.43 mmol), EDCI (115 mg, 0.60 mmol), DMAP (9.4 mg, 0.077 mmol), and triethylamine (0.114 mL, 0.82 mmol) were added. After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to give 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]acetamide (73 mg, 0.17 mmol, 94% yield).

[0234] MS(ESI) m / z:423[M+H]

[0235] (1-6) Synthesis of azide linker (4) [ka]

[0236] A dichloromethane solution (1.7 mL) of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]acetamide (73 mg, 0.17 mmol) was cooled to 0 °C, and mCPBA (185 mg, 0.75 mmol) was added. After stirring at room temperature for 20 hours, saturated aqueous sodium hydrogen carbonate solution was added, and liquid separation extraction with ethyl acetate was performed. The crude product obtained by concentrating the organic layer under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain azide linker (4) (1.2 mg, 0.0026 mmol, yield 1.8%).

[0237] MS(ESI) m / z:455[M+H]

[0238] (2) Introduction of azide group by azide linker (4) The azide linker (4) was reacted with the thiol group-introduced antibody in the same manner as the method described in Example 2(2).

[0239] A solution of tris(2-carboxyethyl)phosphine hydrochloride (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 51057 and 51219, and a light chain peak was observed at 23439, which was the same as that of the raw material.

[0240] Example 5: Introduction of azide group by azide linker (5)

Chemical formula

[0241] (1) Synthesis of azide linker (5) (1-1) Synthesis of 4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)benzoic acid [Chem.]

[0242] To a dichloromethane solution (10 mL) of 4-tert-butoxycarbonylbenzoic acid (307.0 mg, 1.38 mmol) were added hydrazine monohydrate (0.0558 mL, 1.79 mmol), EDCI (411.1 mg, 2.14 mmol), DMAP (18.1 mg, 0.14 mmol), and triethylamine (0.462 mL, 3.34 mmol). After stirring at room temperature for 16 h, the mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography to obtain a mixture of tert-butyl 4-(hydrazinecarbonyl)benzoate.

[0243] To an ethanol solution (10 mL) of the obtained mixture were added carbon disulfide (0.0471 mL, 0.78 mmol) and potassium hydroxide (103.7 mg, 1.80 mmol). After stirring at 70 °C for 16 h, 6 M aqueous hydrochloric acid was added to adjust the pH of the system to 3.0. After the adjustment, liquid-liquid extraction with ethyl acetate was performed, and the crude product obtained by concentrating the organic phase under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain 4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)benzoic acid (72.9 mg, 0.33 mmol, 2-step yield 24%).

[0244] MS (ESI) m / z: 223 [M + H]

[0245] (1 - 2) Synthesis of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)benzoic acid [Chem.]

[0246] A solution of 4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)benzoic acid (72.9 mg, 0.33 mmol) in THF (3.3 mL) was cooled to 0 °C, and iodomethane (0.616 mL, 0.99 mmol) and triethylamine (0.228 mL, 1.60 mmol) were added. The mixture was stirred at room temperature for 3 hours. The crude product obtained by concentrating the reaction mixture under reduced pressure was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to give 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)benzoic acid (66.2 mg, 0.28 mmol, 88% yield).

[0247] MS(ESI) m / z:237[M+H]

[0248] (1-3)N-[2-[2-[2-[2-[(4-azidobenzoyl)amino]ethoxy]ethoxy]ethoxy]ethyl]-4-(5-methy lsulfanyl-1,3,4-oxadiazol-2-yl)benzamide synthesis

Chemical formula

[0249] To a dichloromethane solution (2.8 mL) of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)benzoic acid (66.2 mg, 0.28 mmol) were added N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]-4-azido-benzamide (0.190 mg, 0.42 mmol), EDCI (88.5 mg, 0.46 mmol), DMAP (7.2 mg, 0.059 mmol) and triethylamine (0.116 mL, 0.84 mmol). After stirring at room temperature for 5 hours, the crude product obtained by concentrating the reaction solution under reduced pressure was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to give N-[2-[2-[2-[2-[(4-azidobenzoyl)amino]ethoxy]ethoxy]ethoxy]ethyl]-4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)benzamide (104 mg, 0.19 mmol, yield 67%).

[0250] MS(ESI) m / z:556[M+H]

[0251] (1-4) Synthesis of azido linker (5)

Chemical Structure

[0252] A dichloromethane solution (1.9 mL) of N-[2-[2-[2-[2-[(4-azidobenzoyl)amino]ethoxy]ethoxy]ethoxy]ethyl]-4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)benzamide (104 mg, 0.19 mmol) was cooled to 0 °C, and mCPBA (195.4 mg, 0.79 mmol) was added. After stirring at room temperature for 20 hours, saturated aqueous sodium hydrogen carbonate solution was added, and liquid separation extraction with ethyl acetate was performed. The crude product obtained by concentrating the organic layer under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain azide linker (5) (84.8 mg, 0.14 mmol, yield 76%).

[0253] MS(ESI) m / z:588[M+H]

[0254] (2) Introduction of azide group by azide linker (5) Azide linker (5) was reacted with the thiol group-introduced antibody in the same manner as described in Example 2(2).

[0255] A solution of tris(2-carboxyethyl)phosphine hydrochloride (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the reported method (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 51189 and 51350, and the same light chain peak as the raw material was observed at 23439.

[0256] Example 6: Introduction of azide group by azide linker (6)

Chemical formula

[0257] (1) Synthesis of azide linker (6)

Chemical formula

[0258] N-Succinimidyl 3-(2-Pyridyldithio)propionate (55.2 mg, 0.177 mmol) was dissolved in CH2Cl2 (2.0 mL), and 11-Azido-3,6,9-trioxaundecan-1-amine (35 μL, 0.177 mmol) was added, followed by stirring at room temperature for 18 hours. After confirming the reaction by LC / MS, it was dissolved in an aqueous solution of 0.05% trifluoroacetic acid and subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as the filler. Elution was carried out 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, and after removing acetonitrile by concentration under reduced pressure, lyophilization was performed to obtain azido linker (6) (72.2 mg, 0.173 mmol).

[0259] 1 H NMR (400 MHz, Chloroform-d) δ = 8.55 (s, 1H), 7.78 (s, 1H), 7.22 (s, 1H), 6.73 (s, 1H), 3.76 - 3.63 (m, 10H), 3.60 (dd, J = 5.6, 4.5, 2H), 3.49 (q, J = 5.2, 2H), 3.41 (t, J = 5.0, 2H), 3.13 (tt, J = 7.0, 2.4, 2H), 2.66 (td, J = 6.9, 2.2, 2H). MS (ESI) m / z: z = 1416 [M + H] +

[0260] (2) Introduction of azide groups by azido linker (6) The azido linker (6) reagent was reacted with the thiol group-introduced antibody in the same manner as described in Example 2(2). The reaction solution was replaced with 20 mM ammonium acetate buffer, and when the mass was measured by ESI-TOFMS according to the reported method (Anal. Chem., 2019, 91, 20, 12724 - 12732), a peak of 149009 with two azide groups introduced into the antibody was confirmed.

[0261] Example 7: Introduction of an azide group by azide linker (7)

Chemical formula

[0262] (1) Synthesis of azide linker (7)

Chemical formula

[0263] 4-Methyl-4-(pyridin-2-yldisulfanyl)pentanoic acid (100.0 mg, 0.39 mmol) was dissolved in THF (3.0 mL), and 11-Azido-3,6,9-trioxaundecan-1-amine (77 μL, 0.39 mmol), DCC (97.0 mg, 0.47 mmol), and NHS (54.0 g, 0.47 mmol) were added, followed by stirring at room temperature for 18 hours. After confirming the reaction by LC / MS, it 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 containing 0.05% trifluoroacetic acid and acetonitrile, 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 azide linker (7) (39.9 mg, 0.087 mmol).

[0264] 1 H NMR (400 MHz, Chloroform-d) δ = 8.52 (d, J = 5.0, 1H), 7.81 (d, J = 8.2, 1H), 7.72 (t, J = 7.9, 1H), 7.17 (t, J = 6.2, 1H), 6.15 (s, 1H), 3.76 - 3.62 (m, 10H), 3.57 (dd, J = 5.6, 4.6, 2H), 3.44 (dt, J = 15.2, 5.1, 4H), 2.42 - 2.27 (m, 2H), 2.06 - 1.94 (m, 2H), 1.33 (s, 6H). MS (ESI) m / z: z = 1 458 [M + H] +

[0265] (2) Introduction of azide group by azide linker (7) The azide linker (7) reagent can be reacted with the thiol group-introduced antibody in the same manner as described in Example 2(2).

[0266] Example 8: Introduction of DBCO group by alkyne linker (8)

Chemical formula

[0267] (1) Synthesis of alkyne linker (8)

Chemical formula

[0268] To a dichloromethane solution (1 mL) of 3-Amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone (20 mg, 0.072 mmol) at room temperature, diisopropylcarbodiimide (10.9 mg, 0.087 mmol) and bromoacetylbenzoic acid (21 mg, 0.087 mmol) were added. After stirring at room temperature for 16 hours, concentration under reduced pressure was carried out, and the obtained crude product was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain alkyne linker (8) (12.3 mg, 0.25 mmol, yield 34%).

[0269] MS(ESI) m / z:501[M+H]

[0270] (2) Introduction of DBCO group by alkyne linker (8) The alkyne linker (7) was reacted with the thiol group-introduced antibody in the same manner as the method described in Example 2(2). A tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 51109 and 51270, and a light chain peak identical to that of the raw material was observed at 23439.

[0271] Example 9: Introduction of a DBCO group using an alkyne linker (9)

Chemical formula

[0272] Using the same method as the method described in Example 2(2), 10 equivalents of the above alkyne linker (9, manufactured by Tokyo Chemical Industry Co., Ltd.) were reacted. A tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 51123 and 51291, and a light chain peak identical to that of the raw material was observed at 23439.

[0273] Example 10: Introduction of a DBCO group using an alkyne linker (10)

Chemical formula

[0274] In the same manner as the method described in Example 2(2), 10 equivalents of the above alkyne linker (10) was reacted. A tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the thiol group-introduced antibody as the raw material, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with azide groups introduced were observed at 51371 and 51532, and the same light chain peak as the raw material, 23439, was observed.

[0275] Example 11: Introduction of a DBCO group by an alkyne linker (11)

Chemical formula

[0276] (1) Synthesis of the alkyne linker (11)

Chemical formula

[0277] To a dichloromethane solution (1 mL) of 3-Amino-1-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-1-propanone (14 mg, 0.051 mmol) at room temperature, WSC (15 mg, 0.079 mmol), HOBT (15 mg, 0.111 mmol), triethylamine (12 μL, 0.162 mmol), DMAP (0.5 mg, 0.004 mmol) and 3-pyridyldithiopropionic acid (11 mg, 0.050 mmol) were added. After stirring at room temperature for 3 hours, concentration under reduced pressure was performed, and the obtained crude product was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain the alkyne linker (10) (4.2 mg, 0.0096 mmol, yield 19%).

[0278] MS(ESI) m / z:501[M+H]

[0279] (2) Introduction of DBCO group by alkyne linker (11) The above alkyne linker (11) was reacted with the thiol group-introduced antibody in the same manner as the method described in Example 2(2). The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724-12732). As a result, a peak of 149151 in which two azide groups were introduced into the antibody was confirmed.

[0280] Example 12: Measurement of aggregation rate of bioorthogonal group-introduced antibody by SEC HPLC For the bioorthogonal group-introduced antibodies of the antibodies synthesized in Comparative Examples 1 and 2 and Examples 1 to 6, 8 to 11, the respective aggregation rates were measured under the following conditions according to the previously reported method (ACS Omega 2020, 5, 7193-7200).

[0281] Measurement system: 1260 HPLC system (manufactured by Agilent) Column: AdvanceBio SEC 300Å 2.7μm, 4.6mm×150mm manufactured by Agilent Flow rate: 0.25 mL / min Eluent: Aqueous solution of 100 mM sodium dihydrogen phosphate / sodium hydrogen phosphate, 250 mM sodium chloride (pH 6.8), 10% v / v isopropanol Detector: UV (280 nm)

[0282] [Table 1]

[0283] As a result, the aggregation rate of the bioorthogonal group-introduced antibody obtained using the compound of the example was less than 3.5%, which was lower than that of the bioorthogonal group-introduced antibody obtained using the compound of the comparative example (Table 1).

[0284] Example 13: Measurement of the ratio of unreacted heavy chain by reverse-phase HPLC under reducing conditions (1) Preparation of measurement sample An aqueous solution of DL-dithiothreitol (prepared by adding an aqueous solution of 1 M DL-dithiothreitol to an aqueous solution of 8 M guanidine hydrochloride) was added to the product, and the mixture was heated at 80 °C for 10 minutes.

[0285] (2) Analysis by reverse-phase HPLC For the bioorthogonal functional group-introduced antibodies synthesized in Comparative Examples 1 and 2 and Examples 1 to 5 and 8 to 10, reverse-phase HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). The measurement was carried out using the following conditions.

[0286] Measurement system: 1260 HPLC system (manufactured by Agilent) Column: AdvanceBio RP-mAb Diphenyl 3.5 μm, 2.1 mm × 100 mm manufactured by Agilent Gradient: Linear gradient of eluent A / B Flow rate: 0.4 mL / min Eluent A: Water, 0.1% v / v trifluoroacetic acid Eluent B: Acetonitrile, 0.1% v / v trifluoroacetic acid Detector: UV (280 nm)

[0287] The ratio of the heavy chain of the reaction was calculated by (peak area of unreacted heavy chain) / (peak area of all heavy chains of the antibody).

[0288]

Table 2

[0289] From the results in Table 2 above, it was revealed that the conversion rates of the bioorthogonal functional group-introduced antibodies synthesized in Examples 1 to 5 and 8 to 10 exceeded 85% from the thiol-introduced antibody as the raw material.

[0290] Note that, as in Examples 6 and 11, compounds having disulfide bonds are converted into thiol group-introduced antibodies, which are raw materials, by cleaving the disulfide bonds with DTT in the pretreatment step of reverse-phase HPLC measurement. Therefore, the unreacted heavy chain ratio (%) cannot be determined by this method. Thus, the determination of the unreacted heavy chain ratio (%) for the compounds of Examples 6 and 11 was carried out in the following example.

[0291] Example 14: Measurement of the Ratio of Unreacted Thiol Group-Introduced Antibody by ESI-TOFMS (1) Glycan Cleavage of Bioorthogonal Functional Group-Introduced Antibody The bioorthogonal functional group-introduced antibodies synthesized in Examples 6 and 11 were subjected to glycan cleavage and post-treatment using PNGase F (New England BioLabs, catalog number P0704) according to the manufacturer's protocol and the method of a previous report (WO2019240288A1). HIC-HPLC analysis was performed.

[0292] (2) ESI-TOFMS Analysis of Glycan-Cleaved Product and Measurement of the Ratio of Unreacted Antibody by DAR Calculator (1) The mass of the glycan-cleaved product obtained in (1) was measured by ESI-TOFMS according to the method of a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). Table 3 shows the ratio of unreacted antibody calculated from the DAR peak and %Area by a DAR calculator (Agilent software) for the obtained MS data.

[0293] The ratio of unreacted antibody was calculated from the following formula. Ratio of unreacted antibody (%) = (A + B / 2) / (A + B + C) A: Peak area of the mass number corresponding to the raw material thiol-introduced antibody (Compound 28) B: Peak area of the mass number corresponding to the raw material thiol-introduced antibody (Compound 28) with one bioorthogonal functional group introduced C: Peak area of the mass number corresponding to the raw material thiol-introduced antibody (Compound 28) with two bioorthogonal functional groups introduced

[0294] [Table 3]

[0295] From the results in Table 3 above, it was revealed that in the synthesis of antibody derivatives using the compounds of Examples 6 and 11, the conversion rate of the antibody derivatives introduced with bioorthogonal functional groups from the thiol-introduced antibodies as raw materials exceeded 90%.

[0296] Example 15: Synthesis of ADC and ADC mimic by click reaction with bioorthogonal functional group-introduced antibody (1) Synthesis of ADC by click reaction According to the previous reports (WO2019 / 240287A1 and WO2019 / 240288A1), a 7-equivalent amount of a DMF solution (5 mM) of DBCO-VC-PAB-MMAE (manufactured by ABZENA) was added to the azide-introduced trastuzumab obtained in Examples 1 to 6, and after stirring at room temperature for 20 hours, purification was performed using NAP-5 Columns (manufactured by GE Healthcare) to obtain ADC.

[0297] (2) Synthesis of ADC mimic by click reaction According to the previous reports (WO2019 / 240287A1 and WO2019 / 240288A1), Carboxyrhodamine110-PEG4-DBCO (manufactured by Broadpharm) was added to the azide-introduced trastuzumab obtained in Examples 1 to 6 to obtain ADC mimic.

[0298] Similarly, Carboxyrhodamine110-PEG3-azide (manufactured by Broadpharm) was added to the DBCO-introduced trastuzumab obtained in Examples 8 to 11 to obtain ADC mimic.

[0299] Example (3) Analysis of ADC and ADC mimic The ESI-TOFMS analysis of the ADC synthesized in Example 13(1) and the ADC mimic synthesized in Example 13(2) was performed according to the previous report (WO2019 / 240287A1), and it was confirmed that the DAR was 2.

[0300] Example 16: Evaluation of ADC mimic by stability test using rat plasma The blood stability of various ADC mimics synthesized in Example 15(2) was evaluated. Specifically, the blood stability of the ADC mimic was evaluated by analyzing the amount of fluorescent molecules that dropped off from the ADC mimic when the ADC mimic was incubated in rat blood as follows.

[0301] (1) Synthesis of control ADC mimic The following azide antibody described in the previous report (WO2019 / 240287A1) was reacted with Carboxyrhodamine110-PEG4-DBCO (manufactured by Broad Pharm) according to Example 15(2) to lead to an ADC mimic.

Chemical formula

[0302] (2) Stability test in plasma To 700 μL of rat plasma (manufactured by Charles River), ADC mimic was added to a concentration of 0.1 mg / mL and then sterile filtered. This solution was dispensed into six Eppendorf tubes at 50 μL each. Three of the six samples were stored in an incubator set at 37 °C for 4 days. The remaining three were stored in a -80 °C freezer for 4 days in the same manner. 100 μL of acetonitrile was added to each sample, stirred by vortexing, and then centrifuged to obtain a precipitate. The resulting supernatant solution was collected and subjected to HPLC analysis.

[0303] (3) Analysis of the amount of fluorescent molecules that dropped off using HPLC analysis The measurement was performed by liquid chromatography / fluorescence detection method to measure the fluorescence molecular weight detached from the ADC mimic. Three samples stored in a freezer (-80 °C) for 96 hours in Example 16(1) were used as those at Day = 0, and three samples stored at 37 °C for 96 hours in Example 14(1) were used as those at Day = 4. The difference in fluorescence intensity between Day = 4 and Day = 0 was analyzed.

[0304] The results were evaluated as shown in the following table. As a control, by comparing the fluorescence intensity of the test compound using the ADC mimic synthesized in Example 16(1), the ratio of the relative reduction rate was calculated using the following formula (A).

[0305] Increase in fluorescence intensity of the fluorescent molecule detached from the Example ADC mimic = [(Fluorescence intensity at Day = 4) - (Fluorescence intensity at Day = 0)]

[0306] Increase in fluorescence intensity of the fluorescent molecule detached from the Control ADC mimic = [(Fluorescence intensity at Day = 4) - (Fluorescence intensity at Day = 0)]

[0307] Ratio of increase = Increase in fluorescence intensity of the Example ADC mimic / Increase in fluorescence intensity of the Control ADC mimic

[0308]

Table 4

[0309] As a result, the ADC mimics synthesized in Example 15(1) and (2) were all more than 10 times more stable than the control synthesized in Example 16(1).

[0310] Example 17: Synthesis of Antibody-Protein Conjugate by Click Reaction with Bioorthogonal Functional Group-Introduced Antibody (1) Introduction of Linker into Protein To lysozyme (in a pH 7.4 PBS buffer solution), 6 equivalents of a DMF solution of N-Hydroxysuccinimidyl-4-azidobenzoate were added, and after stirring at room temperature for 3 hours, purification was carried out using NAP-5 Columns (manufactured by GE Healthcare) to obtain azide-introduced lysozyme. When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724 - 12732), a peak of 14450 with an azide group introduced into lysozyme was confirmed.

[0311] Also, an azide group was introduced into bovine serum albumin by the same method. When the mass was measured by ESI-TOFMS, a peak of 66579 with an azide group introduced into bovine serum albumin was confirmed.

[0312] (2) Synthesis of antibody-protein conjugate by click reaction According to the previously reported (WO2019 / 240287A1 and WO2019 / 240288A1), azide-introduced lysozyme synthesized in Example 17(1) was added to the alkyne-introduced trastuzumab obtained in Example 8 to obtain a trastuzumab-lysozyme conjugate. When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724 - 12732), a peak of 178610 with two lysozymes introduced into trastuzumab was confirmed.

[0313] Also, bovine serum albumin synthesized in Example 17(1) was added to the alkyne-introduced trastuzumab obtained in Example 8 by the same method to obtain a trastuzumab-lysozyme conjugate. When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724 - 12732), a peak of 283466 with two bovine serum albumins introduced into trastuzumab was confirmed.

[0314] Using the same method, the azide-introduced lysozyme synthesized in Example 17(1) was added to the alkyne-introduced trastuzumab obtained in Example 9 to obtain a trastuzumab-lysozyme complex. When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 178669 with two lysozymes introduced into trastuzumab was confirmed. A peak of 178669 with two lysozymes introduced into trastuzumab was confirmed.

[0315] Using the same method, the azide-introduced lysozyme synthesized in Example 17(1) was added to the alkyne-introduced trastuzumab obtained in Example 10 to obtain a trastuzumab-lysozyme complex. When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 179154 with two lysozymes introduced into trastuzumab was confirmed.

[0316] Using the same method, the azide-introduced lysozyme synthesized in Example 17(1) was added to the alkyne-introduced trastuzumab obtained in Example 11 to obtain a trastuzumab-lysozyme complex. When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 178647 with two lysozymes introduced into trastuzumab was confirmed.

[0317] Example 18: Introduction of bioorthogonal functional groups at positions Lys288 / 290 (18-1) 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, modification of the anti-HER2 antibody trastuzumab using the compound, and analysis thereof (18-1-1) Synthesis of an IgG1 Fc-binding peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2, an affinity substance for soluble proteins, was synthesized by the Fmoc solid-phase synthesis method. The peptide synthesizer used was Liberty Blue manufactured by CEM. All reagents were purchased from Watanabe Chemical Industries. Resin was Fmoc-NH-SAL-PEG Resin, HL. Double coupling was performed for arginine (R), cysteine (C), and histidine (H). Cleavage from the resin was carried out by stirring 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 resulting crystals for ether precipitation, and the resulting white crystals were recovered by filtration. This was dissolved in a 0.1% aqueous solution of trifluoroacetic acid and subjected to reverse-phase high-performance liquid chromatography using octadecyl group chemically bonded silica gel as the filler. Elution was performed 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, and acetonitrile was removed only by concentration under reduced pressure, followed by lyophilization.

[0318] (18-1-2) Formation of an intramolecular disulfide bond between Cys at positions 5 and 34 of Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (18-1-1) The synthesized peptide was dissolved in DMSO, and 0.1 M Tris-HCl pH 8.0 was added. Glutathione oxidized form 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, and this was dissolved in 0.05% 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 0.05% 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 target product (20.0 mg, 4.70 μmol).

[0319] MS(ESI) m / z: z = 4 1063.65[M + 4H] 4+ , z = 5 851.15[M + 5H] 5+

[0320] (18-1-3) Synthesis of thioester linker (18-1-3-1)

Chemical formula

[0321] (18-1-3-2)

Chem.

[0322] (1.2 g, 3.04 mmol) of the compound synthesized in (18-1-3-1) was dissolved in a mixed solvent of DMF / H2O = 5 / 1, TCEP·HCl (1.74 g, 6.08 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the mixture was extracted with ethyl acetate and water, and the organic layer was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fraction containing the product was collected, the organic solvent was removed by concentration under reduced pressure, and then vacuum drying was carried out to obtain the above compound (1.18 g, 6.5 mmol).

[0323] (18-1-3-3)

Chem.

[0324] (18-1-3-4)

Chem.

[0325] (18 - 1 - 3 - 3)-synthesized compound (134 mg, 0.45 mmol) was dissolved by adding CH2Cl2 (2.25 mL) and triethylamine (157 μL, 1.13 mmol). Pentafluorophenyl trifluoroacetate (154 μL, 0.90 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 fraction containing the product was collected, the organic solvent was removed by concentration under reduced pressure, and then vacuum drying was carried out to obtain the above compound (96 mg, 0.20 mmol).

[0326] 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).

[0327] (18 - 1 - 4) Peptide and linker binding

Chemical Structure

[0328] (18 - 1 - 2)-synthesized Ac - FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC - NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) was dissolved in N,N - dimethylformamide (1.00 mL), and the thioester linker synthesized in (1 - 3) (96.0 mg, 201 μmol) was added, and the mixture was stirred at room temperature for 24 hours. This was dissolved in 0.05% aqueous trifluoroacetic acid solution, and octadecyl group chemical bond It was subjected to reverse-phase high-performance liquid chromatography using synthetic 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 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).

[0329] MS(ESI)m / z:z = 4 1136.80[M + 4H] 4+

[0330] (18 - 1 - 5) Specific modification of anti-HER2 IgG antibody trastuzumab and analysis by ESI-TOFMS (18 - 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 to the antibody) of 10 mM peptide reagent was added, and it 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.

[0331] (18 - 1 - 6) Confirmation of heavy-chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To the antibody-peptide conjugate produced in (18 - 1 - 5), 2 μL (equivalent to the antibody) of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, and it 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, and peaks were observed at 55033 with one linker introduced into the heavy chain and 23439 same as the raw material for the light chain for the product.

[0332] (18-1-7) Confirmation of the peptide / antibody binding ratio by the DAR calculator for the specific conjugate of trastuzumab (18-1-5) For the MS data analyzed in (18-1-5), Table 5 shows the results of confirming the peptide / antibody binding ratio by the DAR calculator (Agilent software). The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 5 was 2.0. Therefore, the generation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0333] [Chemical formula] 〔Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with two carbonyl groups (C=O) 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 binding ratio r between Ig and two carbonyl groups (C=O) adjacent to Ig is 2.0.〕

[0334] [Table 5] <s>

[0335] (18-1-8) Production of a thiol group-introduced antibody derivative by cleavage of the thioester group To the antibody intermediate obtained in (18-1-7), a hydroxylamine solution was added according to a previously reported method (WO2019240287A1), 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.

[0336] [Chemical formula] 〔Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with two carbonyl groups (C=O) 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. The average binding ratio r between Ig and two carbonyl groups (C=O) adjacent to Ig is 2.0.〕

[0337] (18-1-9) Peptide mapping by trypsin treatment (1-8) For the trastuzumab thiol-introduced product obtained in (1-8), peptide mapping was performed in the following steps.

[0338] (18-1-9-1) Trypsin treatment of the trastuzumab thiol-introduced product 10 μL of the sample solution, 10 μL of a 20 mM aqueous solution of dithiothreitol dissolved in 50 mM ammonium bicarbonate buffer and 40% trifluoroethanol were added to a 1.5 mL low-binding microtest tube, 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.

[0339] (18-1-9-2) LC-MS / MS measurement of trastuzumab (Analytical instrument) Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific) Mass spectrometer: Triple quadrupole mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

[0340] (HPLC analysis conditions) Trap column: Acclaim PepMap (registered trademark) 100, 75 μm x 2 cm (Thermo Fisher Scientific) Analysis column: ESI-column (NTCC-360 / 75-3-125, 75 μm × 12.5 cm, 3 μm (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)

[0341] (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).

[0342] (18-1-9-3) Analysis of the modification sites of trastuzumab Regarding the analysis of the modification sites for the LC-MS / MS measurement results, it was performed using BioPharma Finder 3.0 (Thermo Fisher Scientific). Analysis using BioPharma Finder was performed with the S / N Threshold set to 1 and the MS Noise Level set 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) carbamidomethylated by iodoacetamide) were set. In addition, a filter was set so that only those with a Confidence Score of 80 or higher, 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, as data for the amino acid sequences of the modification site search targets, (1) and (2) shown in Fig. 6 were used.

[0343] (18-1-9-4) Analysis results of modification sites of trastuzumab by LC-MS / MS As a result of the analysis using LC-MS / MS, the MS spectrum of a peptide fragment of 18 amino acids containing a modification site on a lysine residue (a thiol-introduced form (+145.019Da) carbamidomethylated by iodoacetamide) of trastuzumab by trypsin digestion, FNWYVDGVEVHNAKTKPR (SEQ ID NO: 3), was observed (Fig. 7). The measured value was m / z 577.03606, the theoretical value was 577.03557, and it was tetravalent. A product ion with m / z 682.13 (theoretical value: 682.01), corresponding to y16 which is trivalent and 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 (Fig. 8). Also Analysis by BioPharma Finder showed that modification occurred highly selectively at the lysine residue at position 288 or 290 (Figure 9). From this result, it was found that in the trastuzumab thiol derivative obtained in the above (18-1-8), conjugation proceeded selectively at Lys288 and Lys290 in the EU numbering on the heavy chain of the antibody.

[0344] (18-2-1) Synthesis of thioester linker (18-2-1-1)

Chemical formula

[0345] The compound (295 mg, 1.62 mmol) synthesized in (18-1-3-2) was dissolved in CH2Cl2 (13.5 mL), 3-(tert-Butoxycarbonyl)benzoic acid (300 mg, 1.35 mmol), DIPEA (700 μL, 2.03 mmol), and PyBOP (843 mg, 1.62 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated. Elution was performed with a 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 (259 mg, 0.64 mmol).

[0346] (18-2-1-2)

Chemical formula

[0347] The compound (259 mg, 0.64 mmol) synthesized in (18-2-1-1) was dissolved in a mixed solution of CH2Cl2 / TFA = 1 / 1 and stirred at room temperature for 1 hour. After confirming by TLC (hexane / ethyl acetate = 5 / 1) that it dropped to the origin, the reaction solution was concentrated and then dried under vacuum to obtain the above compound (227 mg, 0.66 mmol).

[0348] (18-2-1-3)

Chemical formula

[0349] To the compound (227 mg, 0.66 mmol) synthesized in (18-2-1-2), CH2Cl2 (3.3 mL) and triethylamine (230 μL, 1.65 mmol) were added and dissolved After that, pentafluorophenyl trifluoroacetate (225 μL, 1.32 mmol) was added at 0 °C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. It was eluted 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 dried under vacuum 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).

[0350] (18-2-2) Binding of peptide and linker

Chemical formula

[0351] (18-1-2) The Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) synthesized in (18-1-2) 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 an aqueous solution of 0.05% trifluoroacetic acid and subjected to reverse-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 activator (10.0 mg, 2.19 μmol).

[0352] MS(ESI) m / z: z = 4 1145.6[M + 4H] 4+

[0353] (18-2-3) Specific modification of anti-HER2 antibody trastuzumab and analysis by ESI-TOFMS (18-2-2) The peptide linker conjugate synthesized in (18-2-2) 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. Peaks of 152691 with one conjugated peptide introduced, 157163 with two conjugated peptides introduced, and 161634 with three conjugated peptides introduced were confirmed.

[0354] (18 - 2 - 4) Confirmation of Heavy Chain Selectivity by ESI - TOFMS Analysis of the Specific Modification of Trastuzumab under Reducing Conditions (18 - 2 - 3) 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 (18 - 2 - 3), and the mixture was stirred at room temperature for 15 minutes. When the mass was measured by ESI - TOFMS, the raw trastuzumab showed a heavy chain peak at 50596 and a light chain peak at 23439. For the reaction product, 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.

[0355] (18 - 2 - 5) Confirmation of Peptide / antibody Binding Ratio of the Specific Modification of Trastuzumab by DAR calculator Table 6 shows the results of confirming the peptide / antibody binding ratio for the MS data analyzed in (18 - 2 - 3) using a DAR calculator (Agilent software). The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 6 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.

[0356]

Chemical formula

[0357]

Table 6

[0358] Production of Thiol-Group-Introduced Antibody Derivative by Cleavage of Thioester Group (18-2-6) The thiol-group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (18-2-5) to the cleavage reaction of the thioester group described in (18-1-8).

[0359] Peptide Mapping by Trypsin Treatment (18-2-7) For the trastuzumab-thiol introduced product obtained in (18-2-6), peptide mapping was performed in the following steps. Peptide mapping was carried out.

[0360] Trypsin Treatment of Trastuzumab-Thiol Introduced Product (18-2-7-1) Trypsin treatment of the trastuzumab-thiol introduced product obtained in (18-2-6) was carried out in the same manner as in (18-1-9-1).

[0361] LC-MS / MS Measurement of Trastuzumab (18-2-7-2) LC-MS / MS measurement was carried out under the same conditions as in (18-1-9-2).

[0362] Analysis of Modified Sites of Trastuzumab (18-2-7-3) Analysis was carried out in the same manner as in (18-1-9-3).

[0363] Analysis Results of Modified Sites of Trastuzumab by LC-MS / MS (18-2-7-4) 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-introduced derivative (+145.019 Da) that underwent carbamidomethylation with iodoacetamide), the MS spectrum of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 3) (measured value: m / z 577.03571, theoretical value: 577.03557, tetravalent) was observed (Figure 10), and a product ion of 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 11). Also, the analysis with BioPharma Finder showed that the 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 derivative obtained in the above (18-2-6), site-selective conjugation proceeded at Lys288 and Lys290 in the EU numbering on the heavy chain of the antibody.

[0364] (18-3-1) Synthesis of thioester linker (18-3-1-1) [Chemical formula]

[0365] (18-1-3-2) The synthesized compound (220 mg, 1.21 mmol) was dissolved in CH2Cl2 (12.0 mL), 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 carried out 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, concentrated under reduced pressure to remove the organic solvent, and then dried in vacuo to obtain the above compound (205 mg, 0.63 mmol).

[0366] (18-3-1-2)

Chemical formula

[0367] (18-3-1-1) CH2Cl2 (3.15 mL) and triethylamine (220 μL, 1.58 mmol) were added to the synthesized compound (205 mg, 0.63 mmol) and dissolved. 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 carried out 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, concentrated under reduced pressure to remove the organic solvent, and then dried in vacuo to obtain the above compound (200 mg, 0.41 mmol).

[0368] 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).

[0369] (18 - 3 - 2) Binding of Peptide and Linker

Chemical Structure

[0370] (18 - 1 - 2) Ac - FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC - NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) was dissolved in N,N - dimethylformamide (1.00 mL), linker (69.0 mg, 141 μmol) was added, and the mixture was stirred at room temperature for 24 hours. This was dissolved in 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, eluted with a mixed solution of water containing 0.05% trifluoroacetic acid and acetonitrile, and 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 - thiophenol activator (7.5 mg, 1.65 μmol).

[0371] MS(ESI) m / z: z = 4 1140.50[M + 4H] 4+

[0372] (18 - 3 - 3) Specific Modification of Anti - HER2 Antibody Trastuzumab and Analysis by ESI - TOFMS The peptide linker conjugate synthesized in (18-3-2) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of the anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), 3.38 μL of the 10 mM peptide reagent (10 equivalents relative to the antibody) 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 were confirmed at 152676 for the introduction of 1 conjugated peptide, 157126 for the introduction of 2 conjugated peptides, and 161572 for the introduction of 3 conjugated peptides.

[0373] (18-3-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specific modification of trastuzumab under reducing conditions To the antibody-peptide conjugate generated in (18-3-3), 2 μL of a 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent amount relative to the antibody) 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 55048 for the introduction of 1 linker into the heavy chain and at 23439, the same as the starting material, for the light chain.

[0374] (18-3-5) Confirmation of the peptide / antibody binding ratio of the specific modification of trastuzumab by DAR calculator Table 7 shows the results of confirming the peptide / antibody binding ratio for the MS data analyzed in (18-3-3) using a DAR calculator (software from Agilent). The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 7 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.

[0375]

Chemical formula

[0376]

Table 7

[0377] (18 - 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 (18 - 3 - 5) to the thioester group cleavage reaction described in (18 - 1 - 8).

[0378] (18 - 4 - 1) Synthesis of a thioester linker (18 - 4 - 1 - 1)

Chemical formula

[0379] 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. 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 (104 mg, 0.26 mmol).

[0380] (18-4-1-2)

Chemical formula

[0381] (104 mg, 0.26 mmol) of the compound synthesized in (18-4-1-1) was dissolved in a mixed solution of CH2Cl2 / 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).

[0382] (18-4-1-3)

Chemical formula

[0383] The compound synthesized in (18-4-1-2) (104 mg, 0.37 mmol) was dissolved by adding CH2Cl2 (1.85 mL) and triethylamine (130 μL, 0.93 mmol). N-Succinimidyl Trifluoroacetate (156 mg, 0.74 mmol) was added at 0 °C, and the mixture was 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).

[0384] 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).

[0385] (18-4-2) Binding of the peptide and the linker

Chemical formula

[0386] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (29.7 mg, 7.00 μmol, where the two cysteines at the 5th and 34th positions form intramolecular disulfide bonds respectively) synthesized in (18-1-2) was dissolved in N,N-dimethylformamide (1.00 mL), a linker (66.8 mg, 0.14 mmol) was added, and the mixture was stirred at room temperature for 4 hours. This was dissolved in an aqueous solution of 0.05% trifluoroacetic acid 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 containing 0.05% trifluoroacetic acid and acetonitrile, and 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 peptide thioester linker conjugate-NHS activator (13 mg, 2.82 μmol).

[0387] MS(ESI) m / z: z = 4 1153.10[M+4H] 4+

[0388] (18-4-3) Specific modification of anti-HER2 antibody trastuzumab and analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-4-2) 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 152722 with one conjugated peptide introduced, 157215 with two conjugated peptides introduced, and 161708 with three conjugated peptides introduced were confirmed.

[0389] (18-4-4) 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 (equal volume to the antibody) was added to the antibody-peptide conjugate generated in (18-4-3), 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.

[0390] (18-4-5) Confirmation of the peptide / antibody binding ratio of the specific modified form of trastuzumab by DAR calculator Regarding the MS data analyzed in (18-4-3), Table 8 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 8 was 1.8. Therefore, the generation of the antibody intermediate (average peptide / antibody binding ratio 1.8) represented by the following structural formula was confirmed.

[0391] [Chemical formula] Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with two carbonyl groups (C=O) 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 binding ratio r between Ig and two carbonyl groups (C=O) adjacent to Ig is 1.8.

[0392] [Table 8]

[0393] (18-4-6) 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 (18-4-5) to the cleavage reaction of the thioester group described in (18-1-8), thereby obtaining the thiol group-introduced antibody derivative described in (18-1-8).

[0394] (18-5-1) Binding of Peptide and Linker

Chemical Structure

[0395] The linker was bound to Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC-NH2 (SEQ ID NO: 5) (30.0 mg, 7.06 μmol, where the two cysteines at the 5th and 34th positions each form an intramolecular disulfide bond) synthesized by the method described in (18-1-1) in the same manner as in Example 2 (2-2), and the above peptide thioester linker conjugate-thiophenol activator (10.0 mg, 2.19 μmol) was obtained.

[0396] MS(ESI) m / z: z = 4 1145.6 [M+4H] 4+

[0397] (18-5-2) Specific Modification of Anti-HER2 Antibody Trastuzumab and Analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-5-1) 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.

[0398] (18-5-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specific modified form of trastuzumab under reducing conditions To the antibody-peptide conjugate generated in (18-5-2), 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, peaks were observed at 55077 with one linker introduced into the heavy chain and at 23439, the same as the starting material, for the light chain.

[0399] (18-5-4) Confirmation of the peptide / antibody binding ratio of the specific modified form of trastuzumab by DAR calculator Table 9 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 9 was 2.0. Therefore, the generation of the antibody intermediate represented by the following structural formula (average peptide / antibody binding ratio 2.0) was confirmed.

[0400] [Chemical formula] 〔Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with two carbonyl groups (C=O) 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. The average binding ratio r between Ig and two carbonyl groups (C=O) adjacent to Ig is 2.0.〕

[0401]

Table 9

[0402] (18-5-5) 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 (18-5-4) to the cleavage reaction of the thioester group described in (18-1-8), thereby obtaining the thiol group-introduced antibody derivative described in (18-1-8).

[0403] (18-6-1) Binding of a peptide and a linker

Chemical formula

[0404] A linker was bound to Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 6) (30.0 mg, 7.06 μmol, where the two cysteines at the 5th and 34th positions form an intramolecular disulfide bond) synthesized by the method described in (18-1-1) in the same manner as in (18-2-2), to obtain the above peptide thioester linker conjugate-thiophenol activator (22.2 mg, 4.82 μmol).

[0405] MS(ESI) m / z: z = 4 1152.4 [M + 4H] 4+

[0406] (18 - 6 - 2) Specific Modification of Anti - HER2 Antibody Trastuzumab and Analysis by ESI - TOFMS (18 - 6 - 1) The peptide linker conjugate synthesized in (18 - 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), and 3.38 μL (10 equivalents relative to the antibody) of 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 raw material trastuzumab. Peaks at 152720 with one conjugated peptide, 157216 with two conjugated peptides, and 161716 with three conjugated peptides were confirmed.

[0407] (18 - 6 - 3) Confirmation of Heavy - Chain Selectivity by ESI - TOFMS Analysis of the Specifically Modified Trastuzumab under Reducing Conditions (18 - 6 - 2) 2 μL (equivalent amount relative to the antibody) of 100 mM tris(2 - carboxyethyl)phosphine hydrochloride solution was added to the antibody - peptide complex generated in (18 - 6 - 2), 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. For the reaction product, peaks were observed at 55091 with one linker introduced into the heavy chain and 23439, the same as the raw material, for the light chain.

[0408] (18 - 6 - 4) Confirmation of Peptide / antibody Binding Ratio of the Specifically Modified Trastuzumab by DAR calculator Regarding the MS data analyzed in (18-6-2), the results of confirming the peptide / antibody binding ratio using a 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.

[0409] [Chemical formula] [Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with two carbonyl groups (C=O) 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 binding ratio r between Ig and two carbonyl groups (C=O) adjacent to Ig is 2.0. ]

[0410] [Table 10]

[0411] (18-6-5) 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 (18-6-4) to the thioester group cleavage reaction described in (18-1-8) to obtain the thiol group-introduced antibody derivative described in (18-1-8).

[0412] (18-7-1) Binding of a peptide and a linker [Chemical formula] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO: 7.

[0413] Ac-NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 7) (30.0 mg, 7.06 μ mol, 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 (18-2-2) to obtain the above peptide thioester linker conjugate-thiophenol activator (12.0 mg, 2.69 μmol).

[0414] MS(ESI) m / z: z = 4 1115.8 [M+4H] 4+

[0415] (18-7-2) Specific modification of anti-HER2 antibody trastuzumab and analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-7-1) 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), and 3.38 μL (10 equivalents relative to the antibody) of 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 raw material trastuzumab. Peaks at 152573 with one conjugated peptide and 156927 with two conjugated peptides were confirmed.

[0416] (18-7-3) 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 generated in (18-7-2), 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 starting material trastuzumab was observed at 50596 and the light chain peak was observed at 23439. For the reactant, a peak was observed at 54942 where one linker was introduced into the heavy chain and at 23439 which was the same as that of the starting material for the light chain.

[0417] (18-7-4) Confirmation of the peptide / antibody binding ratio of the specific modified form of trastuzumab by DAR calculator Regarding the MS data analyzed in (18-7-2), Table 11 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 11 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.

[0418] [Chemical formula] [Here, Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms an amide bond with two carbonyl groups (C=O) 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 binding ratio r between Ig and two carbonyl groups (C=O) adjacent to Ig is 2.0. ]

[0419] [Table 11]

[0420] (18-7-5) 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 (18-7-4) to the cleavage reaction of the thioester group described in (18-1-8), thereby obtaining the thiol group-introduced antibody derivative described in (18-1-8).

[0421] (18-7-6) Peptide mapping by trypsin treatment For the trastuzumab thiol-introduced product obtained in (18-7-5), peptide mapping was performed in the following steps.

[0422] (18-7-6-1) Trypsin treatment of the trastuzumab thiol-introduced product Trypsin treatment of the trastuzumab thiol-introduced product obtained in (18-7-5) was performed in the same manner as in (18-1-9-1).

[0423] (18-7-6-2) LC-MS / MS measurement of trastuzumab LC-MS / MS measurement was performed under the same conditions as in (18-1-9-2).

[0424] (18-7-6-3) Analysis of the modification site of trastuzumab Analysis was performed in the same manner as in (18-1-9-3).

[0425] (18-7-6-4) 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-introduced derivative (+145.019 Da) that has undergone carbamidomethylation with iodoacetamide), the MS spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 3) (measured value: m / z 769.04506, theoretical value: 769.04482, trivalent) was observed (Figure 13), and a product ion with m / z 1022.71 (theoretical value: 1022.51) corresponding to bivalent 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 14). Also, analysis with BioPharma Finder showed that modification of the lysine residue at position 288 or 290 occurs highly selectively (Figure 15). From these results, it was found that in the trastuzumab thiol-introduced derivative obtained in (18-7-5) above, site-selective conjugation proceeds at Lys288 and Lys290 in the EU numbering on the heavy chain of the antibody.

[0426] (18-8-1) Binding of the Peptide and the Linker [Chemical Structure] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO: 8.

[0427] The linker was bound to Ac-MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (SEQ ID NO: 8) (30.0 mg, 7.06 μmol, where the two cysteines at the 5th and 34th positions each form an intramolecular disulfide bond) synthesized by the method described in (18-1-1) in the same manner as in (18-2-2), and the above peptide thioester linker conjugate-thiophenol activator (16.8 mg, 3.87 μmol) was obtained.

[0428] MS(ESI) m / z: z = 4 1087.3 [M+4H]4+

[0429] (18 - 8 - 2) Specific Modification of Anti - HER2 Antibody Trastuzumab and Analysis by ESI - TOFMS (18 - 8 - 1) The peptide linker conjugate synthesized in (18 - 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), and 3.38 μL (10 equivalents relative to the antibody) of 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 152457 with one conjugated peptide, 156692 with two conjugated peptides, and 160929 with three conjugated peptides were confirmed.

[0430] (18 - 8 - 3) Confirmation of Heavy - Chain Selectivity by ESI - TOFMS Analysis of the Specific Modification of Trastuzumab under Reducing Conditions (18 - 8 - 2) To the antibody - peptide complex generated in (18 - 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 starting material trastuzumab. For the reaction product, peaks were observed at 54829 with one linker introduced into the heavy chain and 23439 (the same as the starting material) for the light chain.

[0431] (18 - 8 - 4) Confirmation of Peptide / antibody Binding Ratio of the Specific Modification of Trastuzumab by DAR calculator (18 - 8 - 2) For the MS data analyzed in (18 - 8 - 2), the results of confirming the peptide / antibody binding ratio by DAR calculator (Agilent software) are shown in Table 12. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 12 was 2.0. Therefore, the generation of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0432]

Chem.

[0433]

Table 12

[0434] (18 - 8 - 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 (18 - 8 - 4) to the cleavage reaction of the thioester group described in (18 - 1 - 8) to obtain the thiol group - introduced antibody derivative described in (18 - 1 - 8).

[0435] (18 - 9 - 1) Binding of a peptide and a linker

Chem.

[0436] (18 - 9 - 1) Ac - QCQRRFYEALHDPNLNEEQRNARIRSIKEEC - NH2 (SEQ ID NO: 9) (30.0 mg, 7.06 μmo) synthesized by the method described in (18 - 9 - 1) 1. However, the 5th and 34th cysteines each form an intramolecular disulfide bond. A linker was attached in the same manner as (18-2-2) to obtain the above-mentioned peptide thioester linker conjugate-thiophenol activator (14.1 mg, 3.35 μmol).

[0437] MS(ESI) m / z: z = 4 1054.4 [M + 4H] 4+

[0438] (18-9-2) Specific modification of anti-HER2 antibody trastuzumab and analysis by ESI-TOFMS (18-9-1) 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), and 3.38 μL (10 equivalents relative to the antibody) of 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 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.

[0439] (18-9-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of the specifically modified trastuzumab under reducing conditions To the antibody-peptide conjugate generated in (18-9-2), 2 μL (equivalent amount relative to the antibody) of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution was added, followed by stirring 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. For the reaction product, peaks were observed at 54698 with one linker introduced into the heavy chain and 23439, the same as the raw material, for the light chain.

[0440] (18-9-4) Confirmation of the peptide / antibody binding ratio of the specifically modified trastuzumab by DAR calculator Regarding the MS data analyzed in (18-9-2), Table 13 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 13 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.

[0441]

Chemical formula

[0442]

Table 13

[0443] (18-9-5) 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 (18-9-4) to the thioester group cleavage reaction described in (18-1-8) to obtain the thiol group-introduced antibody derivative described in (18-1-8).

[0444] (18-10-1) Introduction of a bioorthogonal functional group to the thiol introduced at the Ly288 / Lys290 position of the antibody Using the azide linker (1) of Example 1, the thiol-introduced antibody synthesized in Example (18-1-8) at positions Ly288 / Lys290 was modified. A tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, and it was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks of 50816 and 50978 with azide groups introduced into the heavy chain and the same light chain peak of 23439 as the raw material were observed.

[0445] Using the same method, the thiol-introduced antibody synthesized in Example (18-1-8) at positions Ly288 / Lys290 was modified using the azide linker (2) of Example 2. A tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, and it was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported (WO2019 / 240287A1). For the raw material thiol group-introduced antibody, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks of 50848 and 51011 with azide groups introduced into the heavy chain and the same light chain peak of 23439 as the raw material were observed.

[0446] Using the same method, the thiol-introduced antibody synthesized in Example (18-1-8) at positions Ly288 / Lys290 was modified using the azide linker (6) synthesized in Example 6. The reaction solution was replaced with 20 mM ammonium acetate buffer, and when the mass was measured by ESI-TOFMS according to the method of the previously reported (Anal.Chem.,2019,91,20,12724-12732), a peak of 149023 with two azide groups introduced into the antibody was confirmed.

[0447] Using the same method, the thiol-introduced antibody synthesized in Example (18-1-8) was modified with the alkyne linker (8) of Example 8. A tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, and the mixture was stirred at room temperature for 10 minutes. The mass was measured by ESI-TOFMS according to the previously reported method (WO2019 / 240287A1). For the thiol group-introduced antibody as the raw material, heavy chain peaks were observed at 50683 and 50845, and a light chain peak was observed at 23439. For the product, heavy chain peaks with an alkyne group introduced were observed at 51137 and 51304, and a light chain peak was observed at 23439, which was the same as that of the raw material.

[0448] Using the same method, the thiol-introduced antibody synthesized in Example (18-1-8) was modified with the alkyne linker (11) synthesized in Example 11. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS according to the method reported previously (Anal.Chem.,2019,91,20,12724-12732). As a result, a peak of 149185 with two alkyne groups introduced into the antibody was confirmed. was.

[0449] Example 19: Synthesis of ADC with DAR = 4 Using a Branched Reagent (19-1) Synthesis of Branched Azide Reagent (19-1-1) Synthesis of tert-Butyl N-[5-(tert-butoxycarbonylamino)-1-(hydrazinecarbonyl)pentyl]carbamate

[0450]

Chemical Structure

[0451] To 9H-Fluoren-9-ylmethyl N-[2,6-bis(tert-butoxycarbonylamino)hexanoylamino]carbamate (191 mg, 0.328 mmol), 3 mL of an acetonitrile solution containing 5% piperidine was added, and the mixture was stirred at room temperature for 3 hours. After confirming the disappearance of the starting material by TLC, the crude product obtained by concentration under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-Butyl N-[5-(tert-butoxycarbonylamino)-1-(hydrazinecarbonyl)pentyl]carbamate (136 mg).

[0452] MS(ESI) m / z: 361[M+H]

[0453] 1 H NMR (400 MHz, Chloroform-d) δ = 7.97 (s, 1H), 5.33 (s, 1H), 4.74 (s, 1H), 4. I2 - 4.04 (m, 1H), 3.95 (s, 2H), 3.11 (q, J = 6.6, 2H), 1.84 - 1.77 (m, 1H), 1.72 - 1.65 (m, 1H), 1.54 - 1.37 (m, 4H), 1.45 (s, 18H).

[0454] (19 - 1 - 2) Synthesis of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-sulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamete

[0455]

Chemical Structure

[0456] To a solution of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(hydrazinecarbonyl)pentyl]carbamate (136.2 mg, 0.328 mmol) in ethanol (8 mL) were added carbon disulfide (0.034 mL, 0.567 mmol) and potassium hydroxide (66.4 mg, 1.18 mmol), and the mixture was stirred at 70 °C for 20 h. 1N hydrochloric acid was added to the reaction solution, and the mixture was concentrated under reduced pressure and then extracted with ethyl acetate. Next, the crude product obtained by concentrating the organic layer under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-sulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamete (94 mg, 0.234 mmol, 71% yield over two steps).

[0457] MS(ESI) m / z:401[M-H]

[0458] 1 H NMR (400 MHz, Chloroform-d) δ = 5.14 (s, 1H), 4.74 (s, 1H), 4.55 (s, 1H), 3.06 (d, J=6.7, 2H), 1.84-1.76 (m, 2H), 1.46-1.41 (m, 2H), 1.38 (s, 18H), 1.22-1.20 (m, 2H), 0.81 (t, J=6.8, 1H).

[0459] (19-1-3) Synthesis of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamate

[0460]

Chemical Structure

[0461] A solution of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-sulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamate (94.1 mg, 0.234 mmol) in THF (3 mL) was cooled to 0 °C, and methyl iodide (0.044 mL, 0.702 mmol) and triethylamine (0.097 mL, 0.702 mmol) were added. The mixture was allowed to return to room temperature and stirred, and the disappearance of the starting material was confirmed by TLC. The crude product obtained by concentrating the reaction mixture under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamate (81 mg, 0.194 mmol, 83% yield).

[0462] MS(ESI) m / z:417[M+H]

[0463] 1 H NMR (400 MHz, Chloroform-d) δ = 5.08 (s, 1H), 4.89 (s, 1H), 4.53 (s, 1H), 3.05 (q, J=6.6, 2H), 2.64 (s, 3H), 1.89-1.74 (m, 2H), 1.48-1.41 (m, 4H), 1.37 (d, J=2.5, 18H).

[0464] (19-1-4)Synthesis of 1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentane-1,5-diamine

[0465]

Chemical Structure

[0466] To a dichloromethane solution (5 mL) of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamate (80.9 mg, 0.194 mmol) was added trifluoroacetic acid (5 mL), and the mixture was stirred at room temperature for 2 hours. Then, the reaction solution was concentrated under reduced pressure. To the resulting residue were added 1N hydrochloric acid and ethyl acetate, and the target product was extracted into the aqueous layer. After freeze-drying, 1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentane-1,5-diamine was obtained (57 mg).

[0467] MS(ESI) m / z:217[M+H]

[0468] 1 H NMR (400 MHz, Methanol-d) δ = 4.81 (dd, J=8.3, 5.8, 1H), 2.97 (t, J=7.7, 2H), 2.79 (s, 3H), 2.22-2.09 (m, 2H), 1.82-1.73 (m, 2H), 1.61-1.50 (m, 2H).

[0469] (19-1-5) Synthesis of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[5-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-5-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]acetamide

[0470]

Chemical Structure

[0471] To a DMF solution (3 mL) of 1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentane-1,5-diamine (57.3 mg, 0.200 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (134.2 mg, 0.700 mmol), triethylamine (0.139 mL, 1.00 mmol), N,N-dimethylaminopyridine (4.9 mg, 0.040 mmol), and 11-Azido―3,6,9-trioxaundecanoic acid (102.0 mg, 0.44 mmol) were added. After stirring at room temperature for 3 hours, since starting materials still remained, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (40.0 mg, 0.209 mmol), triethylamine (0.070 mL, 0.505 mmol), and 11-Azido―3,6,9-trioxaundecanoic acid (37.0 mg, 0.160 mmol) were added and stirring was continued for an additional 1 hour. The reaction solution was diluted with acetonitrile water and purified by preparative HPLC, and the fraction containing the product was collected. After removing acetonitrile by concentration under reduced pressure, the aqueous solution was lyophilized to obtain (1-4) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[5-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-5-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]acetamide (48 mg, 0.0734 mmol, 38% yield over two steps).

[0472] MS(ESI) m / z:647[M+H]

[0473] 11H NMR (400 MHz, Chloroform-d) δ = 7.46 (d, J=9.0, 1H), 7.01 (t, J=6.1, 1H), 5.35 (td, J=8.5, 6.2, 1H), 4.07 (d, J=3.2, 2H), 4.00 (s, 2H), 3.79 - 3.58 (m, 20H), 3.40 (dt, J=5.7, 4.3, 4H), 3.30 (q, J=6.9, 2H), 2.72 (s, 3H), 2.15 - 2.01 (m, 1H), 2.10 - 1.84 (m, 1H), 1.68 - 1.53 (m, 2H), 1.53 - 1.35 (m, 2H).

[0474] (19 - 1 - 6) Synthesis of 2 - [2 - [2 - (2 - azidoethoxy)ethoxy]ethoxy] - N - [5 - [[2 - [2 - [2 - (2 - azidoethoxy)ethoxy]ethoxy]acetyl]amino] - 5 - (5 - methylsulfonyl - 1,3,4 - oxadiazol - 2 - yl)pentyl]acetamide

[0475]

Chem.

[0476] To a dichloromethane solution (2 mL) of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[5-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-5-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]acetamide (47.5 mg, 0.0734 mmol) was added 3-chloroperbenzoic acid (containing about 30% water) (112.4 mg, 0.456 mmol). After stirring at room temperature for 5 hours, the reaction solution was concentrated under reduced pressure, and the residue obtained was purified by preparative HPLC. The fraction containing the product was collected, concentrated under reduced pressure to remove acetonitrile, and then the aqueous solution was lyophilized to obtain the target product (92 mg). Since residual impurities were still observed, it was repurified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[5-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-5-(5-methylsulfonyl-1,3,4-oxadiazol-2-yl)pentyl]acetamide (31 mg, 0.0457 mmol, yield 62%).

[0477] MS(ESI) m / z:679[M+H]

[0478] 1 H NMR (400 MHz, Chloroform-d) δ = 7.67 (d, J=8.6, 1H), 7.04 (t, J=5.6, 1H), 5.46 (td, J=8.5, 6.2, 1H), 4.10 (d, J=5.4, 2H), 4.00 (s, 2H), 3.76-3.64 (m, 20H), 3.49 (s, 3H), 3.44-3.37 (m, 4H), 3.32 (q, J=6.8, 2H), 2.23-2.08 (m, 1H), 2.08-1.94 (m, 1H), 1.56-1.40 (m, 2H), 1.38-1.21 (m, 2H).

[0479] (19-2) Conjugation of Branched Azide Reagent and Thiol-Group Introduced Antibody The azide linker synthesized in Example 19-1-6 was reacted with the thiol-group introduced antibody by the same method as described in Example 2(2).

[0480] When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 149620 with four azide groups introduced into the antibody was confirmed.

[0481] (19-3) Synthesis of ADC by Click Reaction According to the reports (WO2019 / 240287A1 and WO2019 / 240288A1) and Example 15-1, to the branched azide-introduced trastuzumab obtained in Example 19-2, a DMF solution ([[]] 5 mM) of DBCO-VC-PAB-MMAE (manufactured by ABZENA) was added in an amount of 20 equivalents, and after stirring at room temperature for 20 hours, purification was carried out using NAP-5 Columns (manufactured by GE Healthcare) to obtain ADC.

[0482] When the mass was measured by ESI-TOFMS according to the method reported previously (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 155284 with four DBCO-VC-PAB-MMAE introduced into the antibody was confirmed.

[0483] (19-4) Synthesis of Branched Protected Thiol Reagent (19-4-1) Synthesis of tert-Butyl(1,3-dihydroxypropan-2-yl)carbamate

[0484]

Chemical Structure

[0485] To a methanol solution (1.8 mL) of 2 - Amino - 1,3 - propanediol (186.2 mg, 2.00 mmol), tert - butyl alcohol (1.8 mL) was added, and a tert - butyl alcohol solution (1.4 mL) of Di - tert - butyl dicarbonate (597.3 mg, 2.60 mmol) was added dropwise. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure. When hexane (4 mL) was added to the obtained crude product, a white solid precipitated. The solid was filtered, washed with hexane (3 mL), and dried on a vacuum line to obtain tert - Butyl(1,3 - dihydroxypropan - 2 - yl)carbamate (352.9 mg, 1.845 mmol, yield 92%).

[0486] MS(ESI) m / z:213[M+Na]

[0487] 1 H NMR (400 MHz, Chloroform - d) δ = 5.22 (s, 1H), 3.84 (qd, J = 11.0, 4.3, 4H), 3.71 (s, 1H), 1.48 (s, 9H)

[0488] (19 - 4 - 2)Synthesis of tert - Butyl N - [2 - allyoxy - 1 - (allyloxymethyl)ethyl]carbamete

[0489]

Chemical Structure

[0490] A solution of tert-Butyl(1,3-dihydroxypropan-2-yl)carbamate (324.9 mg, 1.70 mmol) in DMF (4 mL) was added with Allyl bromide (0.547 mL, 6.34 mmol), and potassium hydroxide (359.0 mg, 6.40 mmol) was added little by little. After stirring at room temperature for 4 hours, dichloromethane and water were added to the residue obtained by concentrating the reaction solution under reduced pressure for extraction. After washing the organic layer with saturated brine, the crude product obtained by concentrating under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain tert-Butyl N-[2-allyoxy-1-(allyloxymethyl)ethyl]carbamete (326.6 mg, 1.20 mmol, yield 71%).

[0491] MS(ESI) m / z:294[M+Na]

[0492] 1 H NMR (400 MHz, Chloroform-d) δ = 5.95-5.86 (m, 2H), 5.29(dq, J=17.2, 1.7, 2H), 5.20 (dq, J=10.4, 1.4, 2H), 4.94 (s, 1H) 4.01 (dt, J=5.5, 1.5, 4H), 3.91 (s, 1H), 3.58 (dd, J=9.4, 4.3, 2H), 3.51 (dd, J=9.4, 6.0, 2H), 1.47 (s, 9H).

[0493] (19-4-3)Synthesis of S-[3-[3-acetylsulfanylpropoxy]-2-(tert-butoxycarbonylamino)propoxy]propyl]ethanethioate

[0494]

Chemical Structure

[0495] To a toluene solution (7.4 mL) of tert-Butyl N-[2-allyoxy-1-(allyloxymethyl)ethyl]carbamete (323.8 mg, 1.19 mmol) was added 2,2′-azobisisobutyronitrile (1.993 g, 25.66 mmol). Thioacetic acid (0.890 mL, 11.88 mmol) was added to the suspension reaction solution in 5 portions, and the mixture was stirred at 65 °C for 21 hours. The crude product obtained by returning the reaction solution to room temperature and concentrating it under reduced pressure was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain S-[3-[3-acetylsulfanylpropoxy]-2-(tert-butoxycarbonylamino)propoxy]propyl]ethanethioate (362.0 mg, 0.854 mmol, yield 72%).

[0496] MS(ESI) m / z:446[M+Na]

[0497] 1 H NMR (400 MHz, Chloroform-d) δ = 4.95 (s, 1H), 3.87 (s, 1H), 3.55 - 3.44 (m, 8H), 3.03 - 2.89 (m, 4H), 2.35 (s, 6H), 1.93 - 1.76 (m, 4H), 1.47 (s, 9H).

[0498] (19 - 4 - 4) Synthesis of S-[3-[3-(3-acetylsulfanylpropoxy)-2-amino―propoxy]propyl]ethanethioate

[0499]

Chemical formula

[0500] To a dichloromethane solution (2 mL) of S-[3-[3-acetylsulfanylpropoxy]-2-(tert-butoxycarbonylamino)propoxy]propyl]ethanethioate (360.1 mg, 0.850 mmol) was added trifluoroacetic acid (2 mL), and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, 0.1 N hydrochloric acid (10 mL) was added to the residue obtained, and the aqueous solution obtained by concentrating again under reduced pressure was freeze-dried to obtain the hydrochloride of S-[3-[3-(3-acetylsulfanylpropoxy)-2-amino-propoxy]propyl]ethanethioate (384.4 mg).

[0501] MS(ESI) m / z:324[M+H]

[0502] 1 H NMR (400 MHz, Chloroform-d) δ = 4.51 (s, 2H), 3.82-3.64 (m, 5H), 3.64-3.45 (m, 4H), 3.10-2.92 (m, 4H), 2.37 (s, 6H), 1.95-1.81 (m, 4H).

[0503] (19-4-5)Synthesis of S-[3-[3-(3-acetylsulfanylpropoxy)-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]propoxy]propyl]ethanethioate

[0504]

Chemical formula

[0505] To a solution of 3-maleimidopropionic acid (17.6 mg, 0.10 mmol) in THF (1.5 mL) was added a solution of o-Benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (56.9 mg, 0.15 mmol), triethylamine (0.021 mL, 0.15 mmol), and S-[3-[3-(3-acetylsulfanylpropoxy)-2-amino-propoxy]propyl]ethanethioate (53.2 mg, 0.11 mmol) in THF (0.5 mL). After stirring at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain S-[3-[3-(3-acetylsulfanylpropoxy)-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]propoxy]propyl]ethanethioate (18.7 mg, 0.039 mmol, 41% yield).

[0506] MS(ESI) m / z:475[M+H]

[0507] 1 H NMR (400 MHz, Chloroform-d) δ = 6.72 (s, 2H), 6.43 (s, 1H), 4.18 (s, 1H), 3.88 (t, J=7.0, 2H), 3.59-3.38 (m, 8H), 3.07-2.88 (m, 4H), 2.61 (t, J=7.1, 2H) 2.36 (s, 6H), 1.88-1.84 (m, 4H).

[0508] (19-5) Conjugation of Branched Protecting Thiol Reagent and Thiol Group-Introduced Antibody The branched linker synthesized in Example 19-4-5 was reacted with the thiol group-introduced antibody in the same manner as described in Example 2(2).

[0509] When the mass was measured by ESI-TOFMS according to the method of the previous report (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 149392 with four protected thiol groups introduced into the antibody was confirmed.

[0510] (19-6) Production of a branched thiol group-introduced antibody derivative by cleavage of a thioester group To the antibody intermediate obtained in (19-5), a hydroxylamine solution was added according to the previous report (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 149231 where the deprotection reaction had proceeded.

[0511] (19-7) Synthesis of ADC mimic (19-7-1) Synthesis of Maleimide-VC-Pyrene Maleimide-VC-Pyrene was synthesized as follows. It was synthesized in one step from commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) and known Sarcosine-pyrene (WO2018 / 218004A1).

[0512]

Chemical formula

[0513] Commercially available MC-VC-PAB-PNP (CAS No: 159857-81-5) (15.5 mg, 0.021 mmol) was dissolved in dichloromethane (1 mL), and a dimethylformamide solution (0.5 mL) of N,N-diisopropylethylamine (0.025 mL, 0.142 mmol) and known Sarcosine-pyrene (WO2018 / 218004A1) (7.6 mg, 0.025 mmol) was added, followed by stirring for 17 hours. After purification by reverse-phase preparative chromatography, the fraction containing the product was collected, acetonitrile was removed by concentration under reduced pressure, and freeze-drying was performed to obtain Maleimide-VC-Pyrene (7.3 mg, 0.008 mmol).

[0514] 1 H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.34 (d, J = 9.2 Hz, 2H), 8.32 - 8.23 (m, 4H), 8.16 (s, 2H), 8.10 - 8.00 (m, 4H), 7.80 (d, J = 8.8 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 6.99 (s, 2H), 5.96 (m, 1H), 5.40 (s, 2H), 5.01 (s, 2H), 4.95 (d, J = 6.0 Hz, 2H), 4.38 (m, 1H), 4.19 (m, 1H), 3.03 - 2.92 (m, 3H), 2.67 (m, 1H), 2.33 (m, 1H), 2.20 - 2.07 (m, 2H), 1.97 (m, 1H), 1.67 (m, 1H), 1.59 (m, 1H), 1.51 - 1.45 (m, 6H), 1.26 - 1.15 (m, 3H), 0.83 (dd, J = 12.8, 6.8 Hz, 6H)

[0515] MS (ESI) m / z: 901.45 [M + H] +

[0516] (19 - 7 - 2) Synthesis of ADC mimic To a buffer (pH 7.4 PBS buffer) solution (20 μM) of the thiol group-introduced antibody obtained in Example 19-6, 10 equivalents of a DMF solution (1.25 mM) of Maleimide-VC-Pyrene synthesized in Example 19-7-1 was added. After allowing to stand at room temperature for 2 hours, purification was performed using NAP-5 Columns (manufactured by GE Healthcare) to obtain an ADC mimic.

[0517] When the mass was measured by ESI-TOFMS according to the method of a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), a peak of 152843 in which four Maleimide-VC-Pyrenes were introduced into the antibody was confirmed.

Claims

1. Formula (II), (III), or (IV): 【Chemical 1】 (In the formula, R １ is alkyl, Y is a divalent group, R is an optionally protected bioorthogonal functional group. [Chemical Formula 2] (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group; or [Chemical Formula 3] (In the formula, Y is a divalent group, and R is an optionally protected bioorthogonal functional group.

2. The reagent according to claim 1, wherein the reagent is a reagent for derivatizing a thiol group-introduced antibody or its salt by reacting with a thiol group introduced via a side chain of an amino acid residue other than a cysteine residue in the thiol group-introduced antibody or its salt.

3. The reagent according to claim 1 or 2, wherein the thiol group in the thiol group-introduced antibody is regioselectively bound to a nitrogen atom in the side chain of a lysine residue in the constant region of the antibody heavy chain via a linker that does not contain a peptide.

4. The reagent according to any one of claims 1 to 3, wherein the thiol group-introduced antibody is an IgG antibody.

5. 5. The reagent according to claim 3, wherein the lysine residue is located at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering.

6. The divalent group is one group selected from the group consisting of alkylene, arylene, -C(=O)-, -NR ２ -, -C(=O)-NR ２ -, -NR ２ -C(=O)-, -O-, and -(O-R ３ ) ｍ -, or The divalent group is a divalent group in which two or more groups selected from the group consisting of alkylene, arylene, -C(=O)-, -NR ２ -, -C(=O)-NR ２ -, -NR ２ -C(=O)-, -O-, and -(O-R ３ ) ｍ - are linked R ２ is a hydrogen atom or an alkyl group, R ３ is alkylene or arylene, The reagent according to any one of claims 1 to 5, wherein m is an integer of 1 to 5.

7. The compound is represented by the following formulas (3) to (12): 【Chemical Formula 4】 【Chemical Formula 5】 (In the formula, k is an integer of 0 or 1; m is an integer from 1 to 5, m' is an integer from 1 to 5, R ２ and R ３ are each independently a hydrogen atom or a methyl group, R ６ is a bioorthogonal functional group that may be protected, R １ 、 Q, and R are the same as those defined in claim 1.) The reagent according to any one of claims 1 to 6, which is selected from the group consisting of compounds represented by

8. Formula (II), (III), or (IV): ​ (In the formula, R １ is alkyl, Y is a divalent group, R is an optionally protected bioorthogonal functional group. 【Chemical Formula 7】 (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group; or [Chemical Formula 8] (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group, or a salt thereof.

9. The compound is represented by the following formulas (3) to (12): 【Chemical Formula 9】 【Chemical 10】 (In the formula, k is an integer of 0 or 1; m is an integer from 1 to 5, m' is an integer from 1 to 5, R ２ and R ３ are each independently a hydrogen atom or a methyl group, R ６ is a bio-orthogonal functional group that may be protected, R １ , Q and R are the same as those defined in claim 8.) The compound according to claim 8 or a salt thereof, which is selected from the group consisting of compounds represented by

10. The following formula (II'), (III'), or (IV'): 【Chemical 11】 (In the formula, Ab is an antibody; S is a sulfur atom, Y is a bond or a divalent group; R is an optionally protected bioorthogonal functional group; n is an integer from 1 to 8. 【Chemical 12】 (In the formula, Ab is an antibody; S is a sulfur atom, Y is a bond or a divalent group; R is an optionally protected bioorthogonal functional group; n is an integer from 1 to 8; or 【Chemical 13】 (In the formula, Ab is an antibody; S is a sulfur atom, Y is a bond or a divalent group; R is an optionally protected bioorthogonal functional group; and n is an integer of 1 to 8. An antibody intermediate having an optionally protected bioorthogonal functional group represented by the formula:

11. The antibody intermediate or salt thereof according to claim 10, wherein the sulfur atom (S) adjacent to the antibody (Ab) in formula (II'), (III'), or (IV') is bonded directly or via a linker to an atom in the side chain of an amino acid residue other than a cysteine residue in the constant region of the heavy chain of the antibody (Ab).

12. adjacent to the antibody (Ab) in formula (II'), (III'), or (IV') 12. The antibody intermediate or salt thereof according to claim 11, wherein the sulfur atom (S) present in the antibody intermediate or salt thereof is regioselectively bound to a nitrogen atom in the side chain of a lysine residue in the constant region of the antibody (Ab) heavy chain via a linker that does not contain a peptide.

13. The antibody intermediate or salt thereof according to any one of claims 10 to 12, wherein the antibody intermediate is an IgG antibody.

14. 14. The antibody intermediate or salt thereof according to claim 12 or 13, wherein the lysine residue is located at one or more positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of the human IgG heavy chain according to EU numbering.

15. The antibody intermediate has the following formulas (3') to (12'): 【Chemical Formula 14】 【Chemical Formula 15】 (In the formula, k is an integer of 0 or 1; m is an integer from 1 to 5, m' is an integer from 1 to 5, R ２ and R ３ are each independently a hydrogen atom or a methyl group, R ６ is a bioorthogonal functional group that may be protected, and n, S, Q, R, and n adjacent to Ab are the same as defined in claim 10.

16. Formula (II), (III), or (IV): 【Chemical 16】 (In the formula, R １ is alkyl and Y is a divalent group, R is an optionally protected bioorthogonal functional group. 【Chemical 17】 (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group; or 【Chemical 18】 (In the formula, Y is a divalent group, R is a bioorthogonal functional group which may be protected. A compound represented by the formula (II) or a salt thereof is reacted with a thiol group-introducing antibody or a salt thereof, The following formula (II'), (III'), or (IV'): 【Chemical Formula 19】 (In the formula, Ab is an antibody, S is a sulfur atom, n is an integer of 1 to 8, Y and R are the same as those in formula (II).), 【Chemical 20】 (In the formula, Ab is an antibody, S is a sulfur atom, n is an integer of 1 to 8, Y and R are the same as those in formula (III).), or 【Chemical 21】 (In the formula, Ab is an antibody, S is a sulfur atom, n is an integer of 1 to 8, Y and R are the same as those in formula (IV).) A method for producing an antibody intermediate having a bioorthogonal functional group which may be protected or a salt thereof, comprising producing an antibody intermediate having a bioorthogonal functional group which may be protected or a salt thereof represented by the formula (II'), (III'), or (IV').

17. The following formula (II''), (III''), or (IV''): 【Chemical 22】 (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z is a functional substance, n is an integer of 1 to 8.) 【Chemical 23】 (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z is a functional substance, n is an integer of 1 to 8.) or 【Chemical 24】 (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z is a functional substance, n is an integer of 1 to 8.) A conjugate of an antibody and a functional substance or a salt thereof represented by the formula (II''), (III''), or (IV'').

18. The conjugate is selected from the group consisting of conjugates represented by the following formula (3'') to (12''): 【Chemical 25】 【Chemical 26】 (In the formula, k is an integer of 0 or 1, m is an integer of 1 to 5, m' is an integer of 1 to 5, R ２ and R ３ are each independently a hydrogen atom or a methyl group, R ６ ' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z' is a functional substance, Ab, S adjacent to Ab, Q, R', Z, and n are the same as those defined in claim 17.) The conjugate or a salt thereof according to claim 17, selected from the group consisting of conjugates represented by the formula (3'') to (12'').

19. The following formula (II'), (III'), or (IV'): 【Chemical 27】 (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R is an optionally protected bioorthogonal functional group, n is an integer from 1 to 8.) 【Chemical Formula 28】 (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R is an optionally protected bioorthogonal functional group, n is an integer from 1 to 8.) or 【Chemical 29】 (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group, R is an optionally protected bioorthogonal functional group, n is an integer from 1 to 8.) represented by an antibody intermediate having an optionally protected bioorthogonal functional group or a salt thereof is reacted with a functional substance having a bioorthogonal functional group capable of reacting with the bioorthogonal functional group carried by the antibody intermediate, The following formula (II''), (III''), or (IV''): 【Chemical 30】 (In the formula, Ab, S, Y, and n adjacent to Ab are the same as those in formula (II'), R' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z is a functional substance.) 【Chemical 31】 (In the formula, Ab, S, Y, and n adjacent to Ab are the same as those in formula (III'), R' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z is a functional substance.) or 【Chemical 32】 (In the formula, Ab, S, Y, and n adjacent to Ab are the same as those in formula (IV'), R' is a group generated by the reaction between two bioorthogonal functional groups capable of reacting with each other, Z is a functional substance.) to produce a conjugate of an antibody and a functional substance or a salt thereof, When the antibody intermediate or a salt thereof has an alkyne residue, the antibody intermediate or a salt thereof is reacted with a functional substance having an azide, When the antibody intermediate or a salt thereof has an azide, the antibody intermediate or a salt thereof is reacted with a functional substance having an alkyne residue, a method for producing a conjugate of an antibody and a functional substance or a salt thereof.

20. The following formula (II), (III), or (IV): 【Chemical 33】 (In the formula, R １ is alkyl, Y and R are the same as those in formula (II').) 【Chemical 34】 (In the formula, Y and R are the same as those in formula (III').) or 【Chemical 35】 (In the formula, Y and R are the same as those in formula (III').) represented by a compound or a salt thereof is reacted with a thiol group-introduced antibody or a salt thereof, The method according to claim 19, further comprising producing an antibody intermediate having an optionally protected bioorthogonal functional group represented by the formula (II'), (III'), or (IV') or a salt thereof.

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