Compound or its salt, and antibody obtained therefrom

Abstract

The present invention provides a desired antibody or antibody composition by modifying a thiol group chemically introduced into an antibody. More specifically, the present invention provides an antibody composition, etc., having desired properties, the antibody composition comprising (A) an antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (A) (in the formula, Ab is a prescribed antibody, S is a sulfur atom, L is a divalent group, R is an optionally protected bioorthogonal functional group, and n is an integer of 1-8) or a salt thereof, and (B) a thiol-group-introduced antibody represented by formula (B) (in the formula, Ab and n are the same as in formula (A), and SH is a thiol group) or a salt thereof.

Description

[Technical field]

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

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

[0003] ADCs are produced by binding functional groups in the side chains of specific amino acid residues present in antibodies to drugs. An example of such a functional group used in producing ADCs is a thiol group in the side chain of a cysteine ​​residue present in an antibody. Known techniques for modifying thiol groups in antibodies include a technique using 3-arylpropionitrile compounds (Patent Document 1) and a technique using aryl / heteroaryl sulfone / sulfoxide compounds (Patent Document 2). The above prior art techniques for modifying thiol groups in antibodies modify the thiol groups in the side chains of cysteine ​​residues in antibodies (e.g., thiol groups in the side chains of cysteine ​​residues naturally present in antibodies and cysteine ​​residues genetically engineered into antibodies).

[0004] However, to the best of the inventors' knowledge, the prior art does not teach or suggest modifying a thiol group that has been chemically introduced into an antibody (e.g., a thiol group that has been introduced into an 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] International Publication No. 2015 / 001117 [Patent Document 2] International Publication No. 2014 / 144878 Summary of the Invention [Problem to be solved by the invention]

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

[0007] As a result of intensive research, the present inventors have found that a specific compound can highly efficiently modify thiol groups chemically introduced into antibodies. Such specific compounds have excellent reaction efficiency, and are therefore considered to be useful not only for modifying thiol groups chemically introduced into antibodies, but also for modifying thiol groups in cysteine ​​residues in antibodies. In addition, such specific compounds can be used to produce desired antibodies in which thiol groups in the antibodies are highly efficiently modified.

[0008] The present inventors have also found that antibodies modified with the specific compounds are superior in desirable properties such as low antibody aggregation.

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

[0010] That is, the present invention is as follows. [1] A compound comprising: (A) an antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (A) or a salt thereof; and (B) an antibody having a thiol group introduced therein represented by formula (B) or a salt thereof, 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 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; 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 to the total amount of the antibody intermediate or its salt and the thiol group-introduced antibody or its salt [100(%) × (the amount of the antibody intermediate or its salt) / (the total amount of the antibody intermediate or its salt and the thiol group-introduced antibody or its salt)] is 80% or more, An antibody composition, wherein the aggregation rate of the antibody intermediate or its salt, and the thiol group-introduced antibody or its salt 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 of [1] to [3], wherein 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 regioselectively bound to a 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 via a linker that does not contain a peptide. [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 according to 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 formulas (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 azide. [9] The antibody composition according to [8], wherein the alkyne residue is an optionally substituted cyclic group having a triple bond between carbon atoms.

[10] A derivatization reagent for a thiol group-introduced antibody or its salt, comprising a compound represented by formula (I), (II), (III), or (IV) or a salt thereof.

[11] The reagent according to

[10] , wherein the reagent is a reagent for derivatizing a thiol group-introduced antibody or a salt thereof 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 a salt thereof.

[12] The reagent according to

[10] or

[11] , wherein the thiol group in the thiol group-introduced antibody is site-selectively bonded 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.

[13] Any of the reagents according to

[10] to

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

[14] The reagent according to

[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 according to EU numbering.

[15] The divalent group is an alkylene, an arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, or -(O-R3) m -, or The divalent group is an alkylene, an arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, or -(O-R3) m - is a divalent group formed by linking two or more groups selected from the group consisting of R2 is a hydrogen atom or an alkyl group; R3 is alkylene or arylene; Any of the reagents

[10] to

[14] , wherein m is an integer of 1 to 5.

[16] The reagent according to 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 formulas (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 antibody intermediate of

[19] or a salt thereof, wherein the sulfur atom (S) adjacent to the antibody (Ab) in formula (I'), (II'), (III'), or (IV') is directly bonded or bonded 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 antibody (Ab) heavy chain.

[21] The antibody intermediate of

[20] or a salt thereof, wherein a sulfur atom (S) adjacent to the antibody (Ab) in formula (I'), (II'), (III'), or (IV') is regioselectively bonded 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.

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

[19] to

[21] , wherein the antibody intermediate is an IgG antibody.

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

[21] or

[22] , 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 according to EU numbering.

[24] The antibody intermediate according to any one of

[19] to

[23] above, or a salt thereof, wherein the antibody intermediate is selected from the group consisting of antibody intermediates represented by formulas (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 formulae (1″) to (12″).

[28] A method comprising 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 its salt has an alkyne residue, the antibody intermediate or its salt is reacted with an azide-containing functional material; 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] The method of

[28] , further 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. Effect of the Invention

[0011] The antibody composition of the present invention enables efficient production of a conjugate of an antibody and a functional substance, or a salt thereof, with a low aggregation rate. The reagent of the present invention is capable of derivatizing 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 producing 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 (eg, a diagnostic agent, a research reagent). [Brief description of the drawings]

[0012] [Figure 1] 1 is a diagram showing an outline of an antibody composition obtained by the reaction of a thiol group-introduced antibody with a compound represented by "leaving group-LR" or a salt thereof (L is a divalent group, and R is an optionally protected bioorthogonal functional group). The antibody composition obtained by such a reaction contains an antibody intermediate having an optionally protected bioorthogonal functional group represented by formula (A) or a salt thereof, and a thiol group-introduced antibody represented by formula (B) or a salt thereof. [Diagram 2] FIG. 2 is a diagram showing an outline of modification of a thiol group-introduced antibody with the compound of the present invention represented by formula (I) or a salt thereof. [Diagram 3] FIG. 3 is a diagram showing an outline of modification of a thiol group-introduced antibody with the compound of the present invention represented by formula (II) or a salt thereof. [Figure 4] FIG. 4 is a diagram showing an outline of modification of a thiol group-introduced antibody with the compound of the present invention represented by formula (III) or a salt thereof. [Diagram 5] FIG. 5 is a diagram showing an outline of modification of a thiol group-introduced antibody with the compound of the present invention represented by formula (IV) or a salt thereof. [Figure 6] FIG. 6 contains (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO:1), and (2) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO:2). [Figure 7]FIG. 7 shows the MS spectrum (measured value: m / z 577.03606, theoretical value: 577.03557, 4-valent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 3), a peptide consisting of 18 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab. [Figure 8] FIG. 8 shows the CID spectrum of the product ion at m / z 682.13 (theoretical value: 682.01), which corresponds to triply charged y16, indicating modification of lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering. [Figure 9] FIG. 9 shows the results of searching for peptide fragments containing lysine residue modifications (thiol-introduced derivatives (+145.019 Da) that have been carbamidomethylated with iodoacetamide) in tryptic digests of trastuzumab using BioPharma Finder. The horizontal axis shows the identified lysine residues, and the vertical axis shows the intensity. [Figure 10] FIG. 10 shows the MS spectrum (measured value: m / z 577.03571, theoretical value: 577.03557, 4-valent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 3), a peptide consisting of 18 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab. [Figure 11] FIG. 11 shows the CID spectrum of the product ion at m / z 682.41 (theoretical value: 682.01), which corresponds to triply charged y16, indicating modification of lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering. [Figure 12]FIG. 12 shows the results of searching for peptide fragments containing lysine residue modifications (thiol-introduced derivatives (+145.019 Da) that have been carbamidomethylated with iodoacetamide) in tryptic digests of trastuzumab using BioPharma Finder. The horizontal axis shows the identified lysine residues, and the vertical axis shows the intensity. [Figure 13] FIG. 13 shows the MS spectrum (measured value: m / z 769.04506, theoretical value: 769.04482, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 3), a peptide consisting of 18 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab. [Figure 14] FIG. 14 shows the CID spectrum of the product ion at m / z 1022.71 (theoretical value: 1022.51), which corresponds to divalent y16 and indicates modification of lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering. [Figure 15] FIG. 15 shows the results of searching for peptide fragments containing lysine residue modifications (thiol-introduced derivatives (+145.019 Da) that have been carbamidomethylated with iodoacetamide) in tryptic digests of trastuzumab using BioPharma Finder. The horizontal axis shows the identified lysine residues, and the vertical axis shows the intensity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

[0015] The type of antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a specific glycosylation has been added (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylation consensus sequence), bispecific antibodies, Fc region proteins, and Fc fusion proteins. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, full-length antibodies, or variable regions and antibody fragments (e.g., antibody fragments containing CH1 and CH2 domains, and antibody fragments not containing constant regions) can be used as monoclonal antibodies, with full-length antibodies being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

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

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

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

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

[0020] (3) Neurological disorders CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

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

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

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

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

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

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

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

[0028] The positions of amino acid residues in an antibody and the positions of the constant region of the heavy chain (e.g., CH2 domain) are based on EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, in the case of human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the CH2 region of human IgG, the lysine residue at position 248 corresponds to the 18th amino acid residue in the CH2 region of human IgG, the lysine residue at position 288 corresponds to the 58th amino acid residue in the CH2 region of human IgG, the lysine residue at position 290 corresponds to the 60th amino acid residue in the CH2 region of human IgG, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the CH2 region of human IgG. The designation 246 / 248 indicates that the lysine residue at position 246 or 248 is the target. The designation 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 Radicals 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-like hydrocarbon group means a hydrocarbon group that is 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-like hydrocarbon group include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

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

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

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

[0035] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0036] The monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it is not necessary to be composed only of alicyclic hydrocarbons, and it may contain a chain structure as a part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

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

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

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

[0040] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0041] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it is not necessary to be composed of only an aromatic ring, and it may contain a chain structure or an alicyclic hydrocarbon as a part thereof, and the aromatic ring may be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is even more preferable. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. As the aryl having 6 to 12 carbon atoms, for example, phenyl and naphthyl can be mentioned.

[0042] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0043] Among these, the monovalent hydrocarbon group is preferably an alkyl group, a cycloalkyl group, or an aryl group.

[0044] (Monovalent heterocyclic groups and related terms) The monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocyclic group, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom.

[0045] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the 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] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

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

[0048] (Divalent group) The divalent group is 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 - is one group selected from the group consisting of, or a group having a main chain structure containing two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2 or 3). 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 represents 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 straight chain hydrocarbon group is a straight chain alkylene, a straight chain alkenylene, or a straight chain alkynylene. The straight-chain alkylene is a straight-chain alkylene having 1 to 6 carbon atoms, and preferably a straight-chain alkylene having 1 to 4 carbon atoms. Examples of the straight-chain alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, and preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethylenylene, n-propenylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, and preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of the linear alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. The divalent linear hydrocarbon group is preferably a linear alkylene group.

[0050] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of the arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene group.

[0051] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent aromatic heterocyclic group include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferable. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. The divalent heterocyclic group is preferably a divalent aromatic heterocyclic group.

[0052] Preferably, the divalent group is alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR2-, -NR2-C(=O)-, -O-, and -(O-R3) m -, or 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 - is 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 an alkyl group; R3 is alkylene or arylene; m may be an integer from 1 to 5 (ie, 1, 2, 3, 4, or 5). The alkylene, arylene and alkyl are the same as those defined above.

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

[0054] (substituent) Substituents include: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) aralkyl; (v)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or a monovalent hydrocarbon group; or (vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or a monovalent hydrocarbon group; (vii) Nitro, sulfate, sulfonate, cyano, and carboxyl groups.

[0055] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, and monovalent heterocyclic group in the above-mentioned substituents are the same as those described above.

[0056] The aralkyl refers to an arylalkyl. The definitions, examples and preferred examples of aryl and alkyl in the arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such an aralkyl include benzoyl, phenethyl, naphthylmethyl and naphthylethyl.

[0057] Preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; or (vii) The same groups as those enumerated in (vii) above.

[0058] More preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 12 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0059] Even more preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 6 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R care the same or different and each represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0060] Particularly preferably, the substituents may be: (i) a halogen atom; (ii) alkyl having 1 to 4 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; or (v) The same groups as those listed in (vii) above.

[0061] (Bio-orthogonal functional groups) A bioorthogonal functional group refers to a group that does not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates) or reacts slowly with biological components, but selectively reacts with components other than biological components. Bioorthogonal functional groups are well known in the art (e.g., see Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR 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-introduced antibody to be derivatized by 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 chain of the 20 natural amino acid residues that constitute the protein, or reacts slowly with the side chain, but reacts with the functional group of interest. The 20 natural amino acids that constitute 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 (L). Among these 20 naturally occurring amino acids, glycine, which has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, which have a hydrocarbon side chain (i.e., do not contain a heteroatom selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inactive to normal reactions. Therefore, the bioorthogonal functional group for proteins is a group that does not react or reacts slowly with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, in addition to the side chains of these amino acids that have side chains that are inactive to normal reactions, but reacts with the functional group of interest.

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

[0064] More specifically, the bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [ka] [Where: R 1a , single or multiple R 1b , and one or more R 1c are the same or different and are the above-mentioned substituents or electron-withdrawing groups, · is 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), and preferred are a halogen atom, a boronic acid residue, mesyl, tosyl, and triflate.

[0066] Preferably, the bioorthogonal functional group may be an alkyne residue or an azide. More preferably, the alkyne residue may be a ring group having a triple bond between carbon atoms, which may be substituted with the above-mentioned 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 bioorthogonal functional group described above. The protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of the protecting group. The cleavage of the protecting group can be performed by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of an amide bond). Examples of such a specific treatment include (a) treatment with one or more substances selected from the group consisting of an acidic substance, a basic substance, a reducing agent, an oxidizing agent, and an enzyme, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) leaving the product when a cleavable linker containing a self-degrading cleavable portion is used. Such protecting groups and their cleavage conditions are common 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, JM, Journal of American Chemical Society. 132, 17928 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)).

[0068] Protected bioorthogonal functional groups include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

[0069] More specifically, the protected bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Single or multiple R 2a are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents, · is 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 any function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked together.

[0072] The drug may be a drug for any disease. Examples of such diseases include cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), genetic and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0073] More specifically, the drug may be an anticancer drug. Examples of the anticancer drug include chemotherapeutic agents, toxins, radioisotopes, or substances containing the same. Examples of the chemotherapeutic agents include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of the toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of the 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 More specifically, the drugs include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBDs (pyrrolobenzodiazepines), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0074] A labeling substance is a substance that allows detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2′-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), or substances containing the same.

[0075] Stabilizers are substances that allow for the stabilization of antibodies, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0076] The functional substance may also be a peptide, a protein, a nucleic acid, a low molecular weight organic compound, a sugar chain, a lipid, a high molecular weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Examples of nucleic acids also include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of low molecular weight organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.

[0077] When the functional substance does not have a bioorthogonal functional group that is easily reactive 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, taking into consideration 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 ring 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 azide. In addition, when the bioorthogonal functional group carried by the antibody intermediate is azide, the bioorthogonal functional group carried by the functional substance or the derivatized functional substance may be an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted by a substituent as described above). Derivatization is common knowledge in the art (e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be performed using any crosslinking agent. Alternatively, derivatization may be performed using a specific linker having a desired functional group. For example, such a linker may be one that allows the functional substance and the antibody to be separated by cleavage of the linker in an appropriate environment (e.g., inside or outside a cell). Examples of such linkers include peptidyl linkers that are degraded by specific proteases (e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secretory proteases)) (e.g., U.S. Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), and linkers that can be cleaved at local acidic sites present in the body (e.g., U.S. Pat. Nos. 5,622,929, 5,122,368, and 5,824,805).The linker may be self-immolative (e.g., WO 02 / 083180, WO 04 / 043493, WO 05 / 112919). In the present invention, the derivatized functional substance is also simply referred to as a "functional substance".

[0078] (salt) In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0079] 2. Antibody composition The present invention relates to a compound represented by the following formula (A): [ka] (In the formula, Ab is an antibody that contains two heavy chains and two light chains and contains immunoglobulin units with 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: (B) The following formula (B): [ka] (In the formula, Ab is the same antibody as 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 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; 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 to the total amount of the antibody intermediate or its salt and the thiol group-introduced antibody or its salt [100(%) × (the amount of the antibody intermediate or its salt) / (the total amount of the antibody intermediate or its salt and the thiol group-introduced 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-LR" (Figure 1). Therefore, the common symbols Ab and n in formulae (A) and (B) are the same between formulae (A) and (B). Although the present invention makes it possible to obtain such an antibody composition, the prior art provides no motivation to obtain such an antibody composition.

[0081] Ab in formulas (A) and (B) is an antibody that contains two heavy chains and two light chains and contains an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy and light chains. Examples of such antibodies include IgG antibodies, IgD antibodies, and IgE antibodies that contain two heavy chains and two light chains and contain an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy and light chains, IgA antibodies that contain four heavy chains and four light chains and contain an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy and light chains, and IgM antibodies that contain eight heavy chains and eight light chains and contain an immunoglobulin unit having disulfide bonds between the heavy chains and between the heavy and light chains, with IgG antibodies (e.g., IgG1, IgG2, IgG3, IgG4) being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

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

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

[0084] In formulae (A) and (B), n is an integer of 1 to 8. n is preferably an integer of 1 to 6, and more preferably an integer of 1 to 4 (that is, 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 or via a linker to an atom in the side chain of the same amino acid residue at the same position in the constant region of the antibody heavy chain. The antibody composition of the present invention can be obtained by modifying a thiol group-introduced antibody with a compound represented by "leaving group-LR" (FIG. 1), so that the binding mode of the sulfur atom (S) and the thiol group (SH) to the antibody is the same. That is, when the sulfur atom (S) is directly bound to the same atom in the side chain, the thiol group (SH) is also directly bound to the same atom in the same side chain. In addition, when the sulfur atom (S) is bound to an atom in the side chain via a linker, the thiol group (SH) is also bound 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 binding the sulfur atom (S) and the thiol group (SH) may be an amino acid residue other than a cysteine ​​residue. As the amino acid residue other than a cysteine ​​residue, an amino acid residue having a side chain that is easily modified (e.g., an amino group, a carboxy group, an amide group, a hydroxy group) (e.g., a lysine residue, an aspartic acid residue, a glutamic acid residue, an asparagine residue, a glutamine residue, a threonine residue, a serine residue, a tyrosine residue) can be used, and 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, or a threonine residue, and more preferably, a lysine residue.

[0087] In another embodiment, the amino acid residue in the antibody used for binding the sulfur atom (S) and the thiol group (SH) may be a cysteine ​​residue, because the compound used in the present invention has excellent reaction efficiency and is therefore considered to be 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.

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

[0089] In a preferred embodiment, the amino acid residue in the antibody used for binding the sulfur atom (S) and thiol group (SH) is a lysine residue having a side chain containing a nitrogen atom (amino group), for example, a lysine residue 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 regioselectively modifying specific amino acid residues at predetermined positions to generate antibodies containing specific atoms or groups at predetermined positions are described, for example, in WO / 2018 / 199337, WO / 2019 / 240288, WO / 2019 / 240287, and WO / 2020 / 090979. In addition, according to the methods described in these documents, specific amino acid residues at predetermined positions can be regioselectively modified without using a linker containing a peptide. The peptide portion has potential immunogenicity and is easily hydrolyzed in blood. Therefore, avoiding the use of linkers containing peptide portions is desirable in clinical applications.

[0091] Preferably, the thiol group-introduced antibody can be an antibody containing a thiol group that is regioselectively bound 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) at a predetermined position in the constant region of the heavy chain via a linker that does not contain a peptide. By modifying such an antibody with a compound represented by "leaving group-LR", it is possible to obtain an antibody composition of the present invention in which 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 regioselectively bound directly or via a linker to an atom in the side chain of the same amino acid residue at the same position in the constant region of the antibody heavy chain. In this specification, "regioselective" or "regioselectivity" refers to a situation in which a specific structural unit capable of binding to a specific amino acid residue in an antibody is unevenly distributed in a specific region in the antibody, even though a specific amino acid residue is not unevenly distributed in a specific region in the antibody. Therefore, expressions related to position selectivity, such as "positionally selectively possessed", "positionally selectively bound", and "positionally selectively bound" mean that the retention or binding rate of a specific structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing a plurality of amino acid residues that are the same type as the specific amino acid residues in the target region. Such position-selective binding or retention can be achieved by using, as a thiol group-introduced antibody, an antibody obtained by the above-mentioned method that can positionally selectively modify a specific amino acid residue at a specific position with a thiol group. Such position selectivity is 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%. The position selectivity can be confirmed, for example, by peptide mapping (see, for example, International Publication No. WO2019 / 240287A1).

[0092] The above-mentioned divalent groups can be used as the peptide-free linker. Preferably, when the amino acid residue in the antibody used for binding the sulfur atom (S) and thiol group (SH) is a lysine residue, the peptide-free linker can be a carbonyl-containing linker that contains a -C(=O)- moiety that can form an amide bond with the amino group in the side chain of the lysine residue.

[0093] The carbonyl-containing linker has a -C(=O)- moiety at its terminal and is a divalent linear 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 -, or a group having a main chain structure containing two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2 or 3). R4 represents a hydrogen atom or a substituent described later. R5 represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. m1 represents 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.

[0094] Preferably, the carbonyl-containing linker has a -C(=O)- moiety at its terminus and is selected from the group consisting of alkylene, arylene, -C(=O)-, -NR2-, -C(=O)-NR4-, -NR4-C(=O)-, -O-, and -(O-R5) m1 -, or a divalent group having a main chain structure containing one group selected from the group consisting of It has -C(=O)- at its terminal and is selected from the group consisting of alkylene, arylene, -C(=O)-, -NR4-, -C(=O)-NR4-, -NR4-C(=O)-, -O-, and -(O-R5) m - is 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 an alkyl group; R3 is alkylene or arylene; m may be an integer from 1 to 5 (ie, 1, 2, 3, 4, or 5). The alkylene, arylene and alkyl are the same as those defined above.

[0095] More preferably, the carbonyl-containing linker may be a divalent group having a terminal -C(=O)- moiety and a backbone structure that includes alkylene or arylene, or a combination thereof.

[0096] Even more preferably, the carbonyl-containing linker can be a divalent group having a -C(=O)- moiety and a backbone structure that includes alkylene (eg, -C(=O)-CH2-CH2-).

[0097] The main chain structure of the carbonyl-containing linker may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) of the above-mentioned substituents.

[0098] In the present invention, the molecular weight of the partial structure represented by LR in formula (A) is 700 or less. When the molecular weight of the partial structure represented by LR is 700 or less, the antibody intermediate or its salt having an optionally protected bioorthogonal functional group, and the thiol group-introduced antibody or its salt are difficult to separate based on the difference in molecular weight because the ratio of the molecular weight of the partial structure to the molecular weight of the entire antibody is very small. The molecular weight of the partial structure represented by LR is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and 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 its salt relative to the total amount of the antibody intermediate or its salt and the thiol group-introduced 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-introduced antibody or its salt)] is 80% or more. The compound or its salt used in the present invention has excellent reaction efficiency with the thiol group-introduced antibody or its salt, and therefore can achieve good reactivity expressed as such a percentage. Such a percentage is preferably 82% or more, more preferably 84% or more, even 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 measurements 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 its salt and the thiol group-introduced antibody or its salt is 5% or less. The modification of the thiol group-introduced antibody with the compound used in the present invention can be performed under conditions (mild conditions) that do not cause protein denaturation or decomposition (e.g., cleavage of amide bonds), and is therefore unlikely 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).

[0101] Details of the antibody intermediate or its salt contained in the antibody composition are as described below for the antibody intermediate or its salt.

[0102] The antibody composition of the present invention may further contain an antibody used as a raw material for producing the thiol group-introduced antibody, 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. In this case, the properties 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 way 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-LR" (FIG. 1). By such a reaction, the thiol group in the thiol group-introduced antibody and the leaving group-binding atom in L in the compound represented by "leaving group-LR" are bound, and the leaving group is eliminated from the leaving group-binding atom, thereby obtaining 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). In the reaction, the molar ratio of the compound represented by "leaving group-LR" or a salt thereof to the thiol group-introduced antibody or a salt thereof (compound represented by "leaving group-LR" 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-LR" 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 a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in an appropriate reaction system, for example, a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0105] The production of the antibody intermediate or its salt can be confirmed by, for example, reverse phase HPLC under reducing conditions or mass spectrometry, although the confirmation depends on the specific raw materials and the molecular weight of the product.

[0106] The antibody composition of the present invention can contain a high content of an antibody intermediate or a salt thereof having an optionally protected bioorthogonal functional group in a state where the aggregation rate is low. The antibody intermediate or a salt thereof having an optionally protected bioorthogonal functional group can be used to produce a conjugate of an antibody and a functional substance or a salt thereof. Thus, 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 where the aggregation rate is low.

[0107] 3. Derivatization reagent for thiol group-introduced antibodies or their salts The present invention provides a derivatization reagent for introducing a thiol group into an antibody or a salt thereof.

[0108] In one embodiment, the reagent of the present invention comprises a compound represented by the following formula (I) or a salt thereof: [ka] (In the formula, X is a halogen atom; Q is arylene or alkylene; Y is a bond or a divalent group; R is an optionally protected bioorthogonal functional group.

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

[0110] The arylene represented by Q in formula (I) may be, for example, phenylene, naphthylene, or anthracenylene, with phenylene or naphthylene being preferred, and phenylene being preferred. The alkylene represented by Q in formula (I) may be, for example, linear or branched alkylene, with linear alkylene being preferred. The linear alkylene is preferably a linear alkylene having 1 to 6 carbon atoms, and more preferably a linear alkylene having 1 to 4 carbon atoms.

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

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

[0113] The optionally protected bioorthogonal functional group represented by R in formula (I) is the same as that described above.

[0114] In another embodiment, the reagent of the present invention comprises a compound represented by the following formula (II) or a salt thereof: [ka] (In the formula, R1 is alkyl; Y is a divalent group, R is an optionally protected bioorthogonal functional group.

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

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

[0117] The optionally protected bioorthogonal functional group 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: [ka] (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group.

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

[0120] The optionally protected bioorthogonal functional group represented by R in formula (III) is the same as 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: [ka] (In the formula, Y is a divalent group, R is an optionally protected bioorthogonal functional group.

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

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

[0124] More specifically, the compounds represented by formulas (I) to (IV) may be compounds represented by the following formulas (1) to (12). [ka] [ka] (In the formula, k is an integer of 0 or 1, m is an integer of 1 to 5 (preferably an integer of 1 to 3), m' is an integer of 1 to 5 (preferably an integer of 1 to 3), R2 and R3 each independently represent a hydrogen atom or methyl; R6 is an optionally protected bioorthogonal functional group; X, R1, Q and R are the same as above.

[0125] The thiol group-introduced antibody or its salt to be derivatized by the reagent of the present invention is as described above for the thiol group-introduced antibody or antibody. For example, the thiol group in the thiol group-introduced antibody may be regioselectively bound to an atom in the side chain of an amino acid residue (e.g., an amino acid residue having a side chain easily modified as described above) in the constant region of the antibody heavy chain (preferably, a nitrogen atom in the side chain of a lysine residue) via a linker that does not contain a peptide. The thiol group-introduced antibody may also be an IgG antibody. When a human IgG antibody is used as the thiol group-introduced antibody, such a lysine residue 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 according to EU numbering.

[0126] The reagent of the present invention can derivatize a thiol group-introduced antibody or a salt thereof by reacting with a thiol group (preferably a 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. Such other components include, for example, a solution and a stabilizer (e.g., an antioxidant, a preservative). The solution is preferably an aqueous solution. Examples of the aqueous solution include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), and buffer solutions (e.g., aqueous phosphate solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), with buffer solutions being preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[0128] 4. Compound or 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 formula (I) or a salt thereof: [ka] (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 formula (II) or a salt thereof: [ka] (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 formula (III) or a salt thereof: [ka] (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 formula (IV) or a salt thereof: [ka] (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 compounds represented by the above formulas (2) to (12).

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

[0139] The production of the above-mentioned series of compounds or their salts can be confirmed by, for example, NMR or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Such compounds or their salts 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.

[0141] 5. Antibody intermediate or its salt The present invention provides an antibody intermediate or a salt thereof having an optionally protected bioorthogonal functional group.

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

[0143] The antibody represented by Ab in formula (I') and the relationship between the antibody and the sulfur atom (S) adjacent thereto are the same as those described above for the thiol group-introduced antibody or antibody. For example, the sulfur atom (S) adjacent to the antibody may be regioselectively bound to an atom in the side chain (preferably, a nitrogen atom in the side chain of a lysine residue) 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. The antibody may also be an antibody comprising an immunoglobulin unit comprising two heavy chains and two light chains (preferably, an antibody comprising an immunoglobulin unit comprising two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy chains and the light chains). Furthermore, the antibody may be an IgG antibody. When a human IgG antibody is used as the antibody, such a lysine residue 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 according to EU numbering. The regioselectivity and its degree are the same as those described above. The regioselectivity can be confirmed by the method described above.

[0144] In formula (I'), n is an integer of 1 to 8, preferably an integer of 1 to 6, and more preferably an integer of 1 to 4 (ie, 1, 2, 3, or 4).

[0145] The arylene represented by Q in formula (I') may be, for example, phenylene, naphthylene, or anthracenylene, with phenylene or naphthylene being preferred, and phenylene being preferred. The alkylene represented by Q in formula (I') may be, for example, linear or branched alkylene, with linear alkylene being preferred. The linear alkylene is preferably a linear alkylene having 1 to 6 carbon atoms, and more preferably a linear alkylene having 1 to 4 carbon atoms.

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

[0147] In certain embodiments, the divalent group represented by Y in formula (I') is -(O-R5) m1 It may be a group having a main chain structure containing -. 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'): [ka] (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.

[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'): [ka] (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.

[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 formula (IV') below. [ka] (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.

[0153] In formula (IV'), 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.

[0154] More specifically, the antibody intermediates represented by formulas (I') to (IV') may be antibody intermediates represented by the following formulas (1') to (12'). [ka] [ka] (In the formula, k is an integer of 0 or 1, m is an integer of 1 to 5 (preferably an integer of 1 to 3), m' is an integer of 1 to 5 (preferably an integer of 1 to 3), R2 and R3 each independently represent a hydrogen atom or methyl; R6 is an optionally protected bioorthogonal functional group; Ab, S, Q, R and n adjacent to Ab are the same as those described above.

[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 performed under conditions (mild conditions) that do not cause protein denaturation or decomposition (e.g., cleavage of amide bonds), and is therefore unlikely 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 or a salt thereof of the present invention can also have high stability in blood.

[0157] The antibody intermediate of the present invention can be obtained by reacting a thiol group-introduced antibody with a compound represented by any one of formulas (I) to (IV) or a salt thereof (FIG. 1). The molar ratio of the compound represented by any one of formulas (I) to (IV) or a salt thereof to the thiol group-introduced antibody or a salt thereof in the reaction (compound represented by any one of formulas (I) to (IV) or a salt thereof / thiol group-introduced antibody or a salt thereof) is not particularly limited as it varies depending on factors such as the type of the compound represented by any one of formulas (I) to (IV) 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.

[0158] Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in an appropriate reaction system, for example, a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0159] The production of the antibody intermediate or its salt can be confirmed by, for example, reverse phase HPLC under reducing conditions or mass spectrometry, although the confirmation depends on the specific raw materials and the molecular weight of the product.

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

[0161] 6. Conjugate of antibody and functional substance or its salt 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″): [ka] (In the formula, Ab is an antibody, S is a sulfur atom, Q is arylene or alkylene; Y is a bond or a divalent group; R' is a group resulting from the reaction between two bioorthogonal functional groups capable of reacting with each other; Z is a functional substance; n is an integer from 1 to 8.

[0163] The antibody represented by Ab in formula (I'') and the relationship between the antibody and the sulfur atom (S) adjacent thereto are the same as those described above for the thiol group-introduced antibody or antibody. For example, the sulfur atom (S) adjacent to the antibody may be regioselectively bound to an atom in the side chain (preferably, a nitrogen atom in the side chain of a lysine residue) of an amino acid residue in the constant region of the antibody heavy chain (e.g., an amino acid residue having a side chain susceptible to modification as described above) via a linker that does not contain a peptide. The antibody may also be an antibody comprising an immunoglobulin unit comprising two heavy chains and two light chains (preferably, an antibody comprising an immunoglobulin unit comprising two heavy chains and two light chains and having disulfide bonds between the heavy chains and between the heavy chains and the light chains). Furthermore, the antibody may be an IgG antibody. When a human IgG antibody is used as the antibody, such a lysine residue 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 according to EU numbering. The regioselectivity and its degree are the same as those described above. The regioselectivity can be confirmed by the method described above.

[0164] In formula (I″), n is an integer of 1 to 8, preferably an integer of 1 to 6, and more preferably an integer of 1 to 4 (ie, 1, 2, 3, or 4).

[0165] The group generated by the reaction between two mutually reactive 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 so as to be a combination capable of reacting with each other). Combinations of two mutually reactive bioorthogonal functional groups are well known, and therefore a person skilled in the art can appropriately select such a combination and appropriately design a divalent group containing a moiety generated by the reaction of two mutually reactive bioorthogonal functional groups. Examples of combinations of bioorthogonal functional groups that can react with each other include 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 (a halogen is replaced by a thiol by a substitution reaction), a combination of an alkyne residue (preferably a ring 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 (disulfide bond). Thus, the above moiety may be a group generated by the reaction of a thiol residue and a maleimide residue, a group generated by the reaction of a furan residue and a maleimide residue, a group generated by the reaction of a thiol residue and a halocarbonyl residue, a group generated by the reaction of an alkyne residue and an azide residue, or a group generated by the reaction of a tetrazine residue and an alkene residue, or a disulfide group generated by the combination of a thiol residue and another thiol residue. Examples of the combination of bioorthogonal functional groups that can react with each other include a combination of an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with the above-mentioned substituents) 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 that can react with each other may be a combination of an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with the above-mentioned substituents) 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 a 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 a triazole ring are condensed may also be substituted with a substituent as described above.

[0166] In certain embodiments, the group produced 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: [ka] (Here, the white circle and black circle represent bonds. When the bond of the white circle is bonded to an atom present on the Ab binding side, the bond of the black circle may be bonded to Z, and when the bond of the white circle is bonded to Z, the bond of the black circle may be bonded to an atom present on the Ab binding side.)

[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 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 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″): [ka] (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group; R' is a group resulting from the reaction between two bioorthogonal functional groups capable of reacting with each other; Z is a functional substance; n is an integer from 1 to 8.

[0171] 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, the group generated by the reaction between two mutually reactive bioorthogonal functional groups 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″): [ka] (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group; R' is a group resulting from the reaction between two bioorthogonal functional groups capable of reacting with each other; Z is a functional substance; n is an integer from 1 to 8.

[0173] 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, the group generated by the reaction between two mutually reactive bioorthogonal functional groups represented by R', 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″): [ka] (In the formula, Ab is an antibody, S is a sulfur atom, Y is a bond or a divalent group; R' is a group resulting from the reaction between two bioorthogonal functional groups capable of reacting with each other; Z is a functional substance; n is an integer from 1 to 8.

[0175] In formula (IV''), 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, the group formed by the reaction between two mutually reactive bioorthogonal functional groups represented by R', and the functional substance represented by Z are the same as those described above.

[0176] More specifically, the conjugates represented by formulae (I″) to (IV″) may be conjugates represented by the following formulae (1″) to (12″). [ka] [ka] (In the formula, k is an integer of 0 or 1, m is an integer of 1 to 5 (preferably an integer of 1 to 3), m' is an integer of 1 to 5 (preferably an integer of 1 to 3), R2 and R3 each independently represent a hydrogen atom or methyl; R6' is a group resulting from 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 or its salt of the present invention may be 10% or less. The antibody intermediate or its salt used in the present invention has an aggregation rate of 5% or less, and the conjugate or its salt of the present invention can be obtained by subjecting such an antibody intermediate or its salt to a reaction under conditions (mild conditions) that do not cause denaturation or decomposition of proteins (e.g., cleavage of amide bonds), and is therefore unlikely 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 stability in blood.

[0179] The conjugate or salt thereof of the present invention can be obtained by reacting the antibody intermediate or salt thereof of the present invention with a functional substance. Such a reaction can be carried out under conditions (mild conditions as described above) that do not cause denaturation or decomposition (e.g., cleavage of amide bonds) of proteins (immunoglobulin / antibody). In the reaction, the molar ratio of the functional substance to the antibody intermediate or salt thereof (functional substance / antibody intermediate or salt thereof) is not particularly limited because it varies depending on factors such as the type of the antibody intermediate or salt thereof and the functional substance, and the reaction time, but is, for example, 2 or more, preferably 3 or more, and 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 excess amount) of the functional substance can be used relative to the antibody intermediate or salt thereof.

[0180] The production of the conjugate or its salt can be confirmed by, for example, reverse phase HPLC under reducing conditions or mass spectrometry, depending on the specific raw material and the molecular weight of the product. The conjugate or its salt 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 (eg, diagnostic agent, 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 pharma- ceutically acceptable carrier. Examples of medicamentously acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cacao butter, polyethylene glycol, and white kerosene. However, these are not limited to these. The conjugate of the present invention or a salt thereof may also have an optional modification that provides stability (eg, PEGylation).

[0183] Formulations suitable for oral administration include liquid preparations in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granules; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0184] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[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 which the composition is administered, the drug tolerance, body weight, age, etc. of the subject, but can be determined appropriately. EXAMPLES

[0186] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0187] Comparative Example 1: Introduction of azide groups using N-(4-azidophenyl)-2-iodo-acetamide [ka]

[0188] In the following Comparative Examples and Examples, the antibody derivative (thiol group-introduced trastuzumab) described in Examples 81-7 of International Publication No. 2019 / 240287 (WO2019 / 240287A1) was used as the thiol group-introduced antibody. 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 is according to EU numbering). [ka] (In the above structure, NH-CH2-CH2-CH2-CH2- extending from the antibody heavy chain corresponds to the side chain of a lysine residue, and a thiol-containing group, HS-CH2-CH2-C(=O), is added to the amino group in the side chain of this lysine residue.)

[0189] To a solution (50 μM) of thiol-group-introduced antibody in buffer (HEPES buffer, pH 8.0), 20 equivalents of a DMF solution (12.5 mM) of N-(4-azidophenyl)-2-iodo-acetamide and 50 equivalents of a buffer solution (HEPES buffer, pH 8.0) of sodium iodide (100 mM) were added, and the mixture was left to stand at 37°C for 2 hours, after which it was purified using NAP-5 Columns (GE Healthcare).

[0190] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845 and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain at 50831 and 50993, and the same heavy chain peaks as the raw material at 50683 and 50845 and a light chain peak at 23439.

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

[0192] To a solution (50 μM) of thiol group-introduced antibody in buffer (HEPES buffer, pH 8.0), 50 equivalents of a DMF solution (31.3 mM) of N-[2-[2-(2-Azidoethoxy)ethoxy]ethyl]-2-iodoacetamide and 50 equivalents of a buffer solution (HEPES buffer, pH 8.0) of sodium iodide (100 mM) were added, and the mixture was left to stand at 37°C for 2 hours, after which it was purified using NAP-5 Columns (GE Healthcare).

[0193] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845 and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain at 50896 and 51058, and the same heavy chain peaks as the raw material at 50683 and 50845 and a light chain peak at 23439.

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

[0195] Five equivalents of a DMF solution (1.25 mM) of azide linker (1, manufactured by Aldrich) was added to a solution (20 μM) of thiol group-introduced antibody in buffer (pH 7.4 PBS buffer, 10 mM EDTA), and the mixture was left to stand at room temperature for 1 hour, after which it was purified using NAP-5 Columns (manufactured by GE Healthcare).

[0196] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439. The product had azide groups introduced into the heavy chain at 50816 and 50978, and a light chain peak at 23439, the same as the raw material.

[0197] Example 2: Introduction of an azide group using azide linker (2) [ka]

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

[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, the mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography. The fractions containing the product were 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 [ka]

[0202] Carbon disulfide (0.055mL, 0.92mmol) and potassium hydroxide (192mg, 3.4mmol) were added to an ethanol solution (7.1mL) of 5-azidopentanehydrazide (111mg, 0.71mmol), and the mixture was stirred at 70°C for 16 hours. After that, 6M aqueous hydrochloric acid was added to adjust the pH of the system to 3.0. After the adjustment, separation and extraction with ethyl acetate were 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 (35mg, 0.18mmol, yield 25%).

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

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

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

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

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

[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 hours, a saturated aqueous sodium bicarbonate solution was added, and separation and extraction with ethyl acetate were performed. The organic layer was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography. The fractions containing the product were 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 an azide group using the azide linker (2) Five equivalents of a DMF solution (1.25 mM) of the azide linker (2) was added to a thiol group-introduced antibody in a buffer (pH 7.4 PBS buffer, 10 mM EDTA) (20 μM), and the mixture was left to stand at room temperature for 1 hour, after which it was purified using NAP-5 Columns (GE Healthcare).

[0211] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439. The product had azide groups introduced into the heavy chain at 50846 and 51008, and a light chain peak at 23439, the same as the raw material.

[0212] Example 3: Introduction of an azide group using azide linker (3) [ka]

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

[0214] 2-Azido-1,3-dimethylimidazolinium hexafluorophosphate (137 mg, 0.48 mmol) and DMAP (86 mg, 0.70 mmol) were added to a dichloromethane solution (3.0 mL) of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)aniline trifluoroacetate (48 mg, 0.15 mmol), and the mixture was stirred at 50° C. for 16 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography. The fractions containing the product were 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) [ka] A dichloromethane solution (1.1 mL) of 2-(4-azidophenyl)-5-methylsulfanyl-1,3,4-oxadiazole (26 mg, 0.11 mmol) was cooled to 0°C, and mCPBA (113 mg, 0.45 mmol) was added. After stirring at room temperature for 20 hours, a saturated aqueous solution of sodium bicarbonate was added, and separation and extraction with ethyl acetate were performed. The organic layer was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to obtain azido linker (3) (23 mg, 0.086 mmol, yield 78%).

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

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

[0219] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439. The product had azide groups introduced into the heavy chain at 50841 and 51002, and a light chain peak at 23439, the same as the raw material.

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

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

[0222] A solution of 4-(tert-butoxycarbonylamino)benzoic acid (200 mg, 0.84 mmol) in THF (8.4 mL) 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 resulting crude product was purified by column chromatography. The fractions containing the product were 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] Synthesis of (1-2)tert-butyl N-[4-(5-sulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate [ka]

[0225] Carbon disulfide (0.058mL, 0.97mmol) and potassium hydroxide (125mg, 2.2mmol) were added to an ethanol solution (7.4mL) of tert-butyl N-[4-(hydrazinecarbonyl)phenyl]carbamate (187mg, 0.74mmol), and the mixture was stirred at 75℃ for 16 hours. After that, 6M aqueous hydrochloric acid was added to adjust the pH of the system to 3.0. After the adjustment, separation and extraction with ethyl acetate were 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 (99mg, 0.34mmol, yield 45%).

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

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

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

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

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

[0231] Trifluoroacetic acid (2.9 mL) was added to a dichloromethane solution (2.9 mL) of tert-butyl N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]carbamate (88 mg, 0.29 mmol), and the mixture was stirred at room temperature for 1 hour. The reaction solution 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] Synthesis of (1-5)2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]acetamide [ka]

[0233] 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (102mg, 0.43mmol), EDCI (115mg, 0.60mmol), DMAP (9.4mg, 0.077mmol) and triethylamine (0.114mL, 0.82mmol) were added to a dichloromethane solution (1.8mL) of 4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)aniline trifluoroacetate (57mg, 0.18mmol). After stirring at room temperature for 2 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 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-N-[4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)phenyl]acetamide (73 mg, 0.17 mmol, yield 94%).

[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, a saturated aqueous sodium bicarbonate solution was added, and separation and extraction with ethyl acetate were performed. The organic layer was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain azido linker (4) (1.2 mg, 0.0026 mmol, yield 1.8%).

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

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

[0239] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439. The product had azide groups introduced into the heavy chain and light chain peaks at 51057 and 51219, as well as at 23439, the same as the raw material.

[0240] Example 5: Introduction of an azide group using azide linker (5) [ka]

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

[0242] Hydrazine monohydrate (0.0558mL, 1.79mmol), EDCI (411.1mg, 2.14mmol), DMAP (18.1mg, 0.14mmol) and triethylamine (0.462mL, 3.34mmol) were added to a dichloromethane solution (10mL) of 4-tert-butoxycarbonylbenzoic acid (307.0mg, 1.38mmol). After stirring at room temperature for 16 hours, 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] Carbon disulfide (0.0471 mL, 0.78 mmol) and potassium hydroxide (103.7 mg, 1.80 mmol) were added to the ethanol solution (10 mL) of the obtained mixture, and the mixture was stirred at 70°C for 16 hours. After that, 6 M aqueous hydrochloric acid was added to adjust the pH of the system to 3.0. After the adjustment, separation and 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, 24% yield in two steps).

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

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

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

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

[0248] Synthesis of (1-3)N-[2-[2-[2-[2-[(4-azidobenzoyl)amino]ethoxy]ethoxy]ethoxy]ethyl]-4-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)benzamide [ka]

[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), 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) were added. After stirring at room temperature for 5 hours, the reaction solution was concentrated under reduced pressure, and the crude product obtained 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 azide linker (5) [ka]

[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, a saturated aqueous sodium bicarbonate solution was added, and separation and extraction with ethyl acetate were performed. The organic layer was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography. The fraction containing the product was collected and concentrated under reduced pressure to obtain azido linker (5) (84.8 mg, 0.14 mmol, yield 76%).

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

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

[0255] Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain and light chain peaks at 51189, 51350, and 23439, the same as the raw material.

[0256] Example 6: Introduction of an azide group using azide linker (6) [ka]

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

[0258] N-Succinimidyl 3-(2-Pyridyldithio)propionate (55.2 mg, 0.177 mmol) was dissolved in CHCl (2.0 mL), 11-Azido-3,6,9-trioxaundecan-1-amine (35 μL, 0.177 mmol) was added, and the mixture was stirred at room temperature for 18 hours. After confirming the reaction by LC / MS, the mixture was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid. Each fraction was confirmed by LC-MS. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain azido linker (6) (72.2 mg, 0.173 mmol).

[0259] 1 H NMR (400MHz, 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=1 416[M+H] +

[0260] (2) Introduction of an azide group using the azide linker (6) The thiol group-introduced antibody was reacted with the azide linker (6) reagent in the same manner as 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 previously reported method (Anal. Chem., 2019, 91, 20, 12724-12732). A peak of 149009 was confirmed, in which two azide groups were introduced into the antibody.

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

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

[0263] 4-Methyl-4-(pyridin-2-yldisulfanyl)pentanoic acid (100.0 mg, 0.39 mmol) was dissolved in THF (3.0 mL), 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 and stirred at room temperature for 18 hours. After confirming the reaction by LC / MS, the mixture was dissolved in 0.05% trifluoroacetic acid aqueous solution and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as a packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the azido linker (7) (39.9 mg, 0.087 mmol).

[0264] 1 H NMR(400MHz,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 an azide group using the azide linker (7) The thiol group-introduced antibody can be reacted with the azidolinker (7) reagent in the same manner as described in Example 2(2).

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

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

[0268] Diisopropylcarbodiimide (10.9 mg, 0.087 mmol) and bromoacetylbenzoic acid (21 mg, 0.087 mmol) were added 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. After stirring at room temperature for 16 hours, the mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to obtain the 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 via alkyne linker (8) The thiol group-introduced antibody was reacted with an alkyne linker (7) in the same manner as described in Example 2(2). Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain at 51109 and 51270, and a light chain peak at 23439, the same as the raw material.

[0271] Example 9: Introduction of DBCO group via alkyne linker (9) [ka]

[0272] In the same manner as described in Example 2(2), 10 equivalents of the above alkyne linker (9, manufactured by Tokyo Chemical Industry Co., Ltd.) were reacted. Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain at 51123 and 51291, and a light chain peak at 23439, the same as the raw material.

[0273] Example 10: Introduction of DBCO group via alkyne linker (10) [ka]

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

[0275] Example 11: Introduction of DBCO group via alkyne linker (11) [ka]

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

[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), 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 at room temperature. After stirring at room temperature for 3 hours, the mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography. The fractions containing the product were 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 via alkyne linker (11) The above alkyne linker (11) 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 the mass was measured by ESI-TOFMS according to the method previously reported (Anal.Chem., 2019, 91, 20, 12724-12732). A peak of 149151 was confirmed, in which two azide groups were introduced into the antibody.

[0280] Example 12: Measurement of aggregation rate of bioorthogonally functionalized antibodies by SEC HPLC The aggregation rates of the antibodies synthesized in Comparative Examples 1 and 2 and Examples 1 to 6 and 8 to 11 to which bioorthogonal functional groups had been introduced were measured under the following conditions according to a previous report (ACS Omega 2020, 5, 7193-7200).

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

[0282] [Table 1]

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

[0284] Example 13: Determination of the percentage of unreacted heavy chain by reversed-phase HPLC under reducing conditions (1) Preparation of measurement samples An aqueous solution of DL-dithiothreol (a 1 M aqueous solution of DL-dithiothreol added to an 8 M aqueous solution of 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 The bioorthogonal functionalized antibodies synthesized in Comparative Examples 1 and 2 and Examples 1 to 5 and 8, 9, and 10 were subjected to reverse phase HPLC analysis according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732). The measurement was performed under the following conditions.

[0286] Measurement system: 1260 HPLC system (Agilent) Column: Agilent AdvanceBio RP-mAb Diphenyl 3.5μm, 2.1mm×100mm Gradient: Linear gradient of eluent A / B Flow rate: 0.4mL / 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 proportion of reactive heavy chains was calculated by (peak area of ​​unreacted heavy chains) / (peak area of ​​total antibody heavy chains).

[0288] [Table 2]

[0289] The results in Table 2 above reveal that the bioorthogonal functional group-introduced antibodies synthesized in Examples 1 to 5 and 8, 9, and 10 had a conversion rate of more than 85% from the starting thiol-introduced antibody.

[0290] In addition, in the case of compounds having disulfide bonds such as those in Examples 6 and 11, the disulfide bonds are cleaved by DTT in the pretreatment step of reversed-phase HPLC measurement, and the compounds are converted into the starting thiol group-introduced antibody, so the percentage (%) of unreacted heavy chains cannot be determined by this method. Therefore, the percentage (%) of unreacted heavy chains for the compounds of Examples 6 and 11 was determined in the next Example.

[0291] Example 14: Measurement of the proportion of unreacted thiol group-introduced antibody by ESI-TOFMS (1) Glycosylation of antibodies with bioorthogonal functional groups The bioorthogonal functionalized antibodies synthesized in Examples 6 and 11 were subjected to glycosylation and post-treatment using PNGase F (New England BioLabs, catalog number P0704) according to the manufacturer's protocol and the previously reported method (WO2019240288A1). HIC-HPLC analysis was performed.

[0292] (2) ESI-TOFMS analysis of glycan cleavage products and measurement of the proportion of unreacted antibodies using the DAR calculator The mass of the glycan cleavage product obtained in (1) was measured by ESI-TOFMS according to the method previously reported (Anal. Chem., 2019, 91, 20, 12724-12732). The percentage of unreacted antibody calculated from the DAR peak and %Area using the DAR calculator (Agilent software) for the obtained MS data is shown in Table 3.

[0293] The proportion of unreacted antibody was calculated according to the following formula. Percentage of unreacted antibodies (%) = (A+B / 2) / (A+B+C) A: Peak area of ​​mass number corresponding to the raw material thiol-introduced antibody (compound 28) B: Peak area corresponding to the mass number of the starting thiol-introduced antibody (compound 28) to which one bioorthogonal functional group was introduced. C: Peak area corresponding to the mass number of the starting thiol-introduced antibody (compound 28) to which two bioorthogonal functional groups have been introduced.

[0294] [Table 3]

[0295] The results in Table 3 above reveal that in the synthesis of antibody derivatives using the compounds of Examples 6 and 11, the antibody derivatives having bioorthogonal functional groups had a conversion rate of more than 90% from the starting thiol-introduced antibody.

[0296] Example 15: Synthesis of ADCs and ADC mimics by click reaction with bioorthogonally functionalized antibodies (1) Synthesis of ADCs via Click Reaction According to previous reports (WO2019 / 240287A1 and WO2019 / 240288A1), 7 equivalents of a DMF solution (5 mM) of DBCO-VC-PAB-MMAE (ABZENA) was added to the azide-introduced trastuzumab obtained in Examples 1 to 6, and the mixture was stirred at room temperature for 20 hours. The mixture was then purified using NAP-5 Columns (GE Healthcare) to obtain an ADC.

[0297] (2) Synthesis of ADC mimics by click reaction According to 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 an 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 an ADC mimic.

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

[0300] Example 16: Evaluation of ADC mimics by stability test using rat plasma The blood stability of each of the 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 detached from the ADC mimic when the ADC mimic was incubated in rat blood, as described below.

[0301] (1) Synthesis of control ADC mimic The following azide antibody described previously (WO2019 / 240287A1) was reacted with Carboxyrhodamine110-PEG4-DBCO (Broadpharm) in accordance with Example 15(2) to produce an ADC mimic. [ka]

[0302] (2) Plasma stability test ADC mimic was added to 700μL of rat plasma (Charles River) to a concentration of 0.1mg / mL, and then sterilized and filtered. This solution was dispensed into six Eppendorf tubes at 50μL each. Three of the six samples were stored in an incubator set at 37℃ for four days. The remaining three were similarly stored in a freezer at -80℃ for four days. 100uL of acetonitrile was added to each sample, stirred with a vortex mixer, and centrifuged to obtain precipitates. The resulting supernatant solution was collected and subjected to HPLC analysis.

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

[0304] The results were evaluated as shown in the table below. The ADC mimic synthesized in Example 16(1) was used as a control to compare the fluorescence intensity of the Example compounds, and the ratio of the relative decrease rates was calculated using the following formula (A).

[0305] Increase in fluorescence intensity of fluorescent molecules shed from the ADC mimic of the embodiment = [(fluorescence intensity on Day = 4) - (fluorescence intensity on Day = 0)]

[0306] Increase in fluorescence intensity of fluorescent molecules shed from control ADC mimic = [(fluorescence intensity on Day 4) - (fluorescence intensity on Day 0)]

[0307] Ratio of increase amount = increase amount of fluorescence intensity of the example ADC mimic / increase amount of fluorescence intensity of the control ADC mimic

[0308] [Table 4]

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

[0310] Example 17: Synthesis of antibody-protein conjugates by click reaction with bioorthogonally functionalized antibodies (1) Introduction of a linker into a protein Six equivalents of N-Hydroxysuccinimidyl-4-azidobenzoate in DMF was added to lysozyme (pH 7.4 PBS buffer solution), stirred at room temperature for 3 hours, and purified using NAP-5 Columns (GE Healthcare) to obtain azide-introduced lysozyme. When the mass was measured by ESI-TOFMS according to the method previously reported (Anal.Chem.,2019,91,20,12724-12732), a peak of 14450 was confirmed, where the azide group was introduced into lysozyme.

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

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

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

[0314] In a similar manner, 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 previously reported (Anal.Chem.,2019,91,20,12724-12732), a peak of 178669 was confirmed, where two lysozymes were introduced into trastuzumab.

[0315] In a similar manner, 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 previously reported (Anal.Chem., 2019, 91, 20, 12724-12732), a peak of 179154 was confirmed, where two lysozymes were introduced into trastuzumab.

[0316] In a similar manner, 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 previously reported (Anal.Chem., 2019, 91, 20, 12724-12732), a peak of 178647 was confirmed, where two lysozymes were introduced into trastuzumab.

[0317] Example 18: Introduction of bioorthogonal functional groups at Lys288 / 290 positions (18-1) Synthesis of compounds (peptide thioester linker-thiophenol activated compounds) having affinity for soluble proteins, cleavable moieties and reactive groups, and modification and analysis of the anti-HER2 antibody trastuzumab using the compounds (18-1-1) Synthesis of IgG1 Fc-binding peptide The peptide Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4), which is an affinity substance for soluble proteins, was synthesized by Fmoc solid-phase synthesis. The peptide synthesizer used was Liberty Blue manufactured by CEM. All reagents used were manufactured by Watanabe Chemical Industry Co., Ltd. The resin used was Fmoc-NH-SAL-PEG Resin, HL, arginine (R), cysteine ​​(C), and histidine (H) were double-coupled. Cleavage from the resin was carried out under the condition of 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 to perform ether precipitation, and the resulting white crystals were collected by filtration. This was dissolved in a 0.1% trifluoroacetic acid aqueous solution, subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing, and eluted with a mixed solution of water and acetonitrile containing 0.1% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected, concentrated under reduced pressure to remove only acetonitrile, and then freeze-dried.

[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) The peptide synthesized in (18-1-1) was dissolved in DMSO, and 0.1M Tris-HCl pH 8.0 was added. Oxidized glutathione was added to this solution and stirred at room temperature for 20 hours. The reaction solution was stopped by adding 2M aqueous trifluoroacetic acid solution, and this was dissolved in 0.05% aqueous trifluoroacetic acid solution, subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as a packing material, 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) [ka] 3,3'-Dithiodipropionic Acid (1g, 5.0mmol) was dissolved in THF (10mL), and DMF (100μL) and Oxalyl Chloride (1.5mL, 15.0mmol) were added at 0℃, and the mixture was stirred at 0℃ for 15 minutes and at room temperature for 1 hour. Then, Benzenethiol (1.53mL, 15.0mmol), Pyridine (4mL, 50mmol), and CHCl (10mL) were added at 0℃, and the mixture was stirred at room temperature for 3 hours. After confirming the reaction with TLC (hexane / ethyl acetate = 5 / 1), the crystals were removed, and the organic layer was extracted with ethyl acetate and 1M aqueous hydrochloric acid, and then concentrated. The mixture 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, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the above compound (1.2g, 3.04mmol).

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

[0322] The compound (1.2g, 3.04mmol) synthesized in (18-1-3-1) was dissolved in a mixed solvent of DMF / H2O = 5 / 1, and TCEP·HCl (1.74g, 6.08mmol) was added and stirred at room temperature for 1 hour. After confirming the reaction with TLC (hexane / ethyl acetate = 5 / 1), the organic layer was extracted with ethyl acetate and water, and then 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, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the above compound (1.18g, 6.5mmol).

[0323] (18-1-3-3) [ka] The compound (200mg, 1.10mmol) synthesized in (18-1-3-2) was dissolved in acetonitrile (2.0mL), and glutaric anhydride (125.5mg, 1.10mmol), DMAP (6.72mg, 0.06mmol), and pyridine (0.22mL) were added and stirred at room temperature for 4 hours. After confirming the reaction by TLC (dichloromethane / methanol = 10 / 1), it was extracted with ethyl acetate and 0.5M HCl, and the organic layer was concentrated. It was eluted with a mixed solution of dichloromethane and methanol, and each fraction was confirmed by TLC (dichloromethane / methanol = 10 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the above compound (135mg, 0.45mmol).

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

[0325] The compound (134 mg, 0.45 mmol) synthesized in (18-1-3-3) was dissolved in CH2Cl2 (2.25 mL) and triethylamine (157 μL, 1.13 mmol). Pentafluorophenyl trifluoroacetic acid (154 μL, 0.90 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (96 mg, 0.20 mmol).

[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) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:4.

[0328] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the 5th and 34th cysteines each form a disulfide bond within the molecule) synthesized in (18-1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and the thioester linker (96.0 mg, 201 μmol) synthesized in (1-3) was added and stirred at room temperature for 24 hours. This was dissolved in 0.05% trifluoroacetic acid aqueous solution, and subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain the above peptide thioester linker conjugate-thiophenol activated product (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 its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-1-4) 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 HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was replaced with 20 mM ammonium acetate buffer, and the mass was measured by ESI-TOFMS, and a peak was observed at 148223 for the raw material trastuzumab. The product was confirmed to have peaks of 152660 with one binding peptide introduced, 157093 with two binding peptides introduced, and 161528 with three binding peptides introduced.

[0331] (18-1-6) Confirmation of heavy chain selectivity of specific modified forms of trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-1-5) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50596 and a light chain peak at 23439, while the product had a heavy chain with one linker introduced at 55033 and a light chain peak at 23439, the same as the raw material.

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

[0333] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0334] [Table 5]

[0335] (18-1-8) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups Hydroxylamine solution was added to the antibody intermediate obtained in (18-1-7) according to the previous report (WO2019240287A1) and left at room temperature for 1 hour. After 2 hours, the buffer was replaced with 20 mM PBS buffer and 10 mM EDTA (pH 7.4) to obtain a thiol group-introduced antibody derivative. When the mass was measured by ESI-TOFMS, a peak was confirmed at 148409, where the cleavage reaction had progressed.

[0336] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues present at positions 288 / 290 in the two heavy chains according to Eu numbering, The average bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0337] (18-1-9) Peptide mapping by trypsin treatment Peptide mapping was performed on the trastuzumab thiol derivative obtained in (1-8) using the following steps.

[0338] (18-1-9-1) Trypsin treatment of trastuzumab thiol derivative 10μL of sample solution, 50mM ammonium bicarbonate buffer, and 10μL of 20mM dithiothreitol solution dissolved in 40% trifluoroethanol were added to a 1.5mL low-adsorption micro test tube, and the mixture was heated at 65℃ for 1 hour, after which 10μL of 50mM iodoacetamide solution was added and reacted at room temperature for 30 minutes under light shielding. After the reaction, 40μL of 50mM ammonium bicarbonate buffer was added and stirred, and 10μL of 20ng / μL trypsin solution was added and enzyme digestion was performed at 37℃ for 16 hours. After digestion, 2μL of 20% trifluoroacetic acid solution was added to stop the reaction, and LC-MS / MS measurement was performed.

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

[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, 75μm×12.5cm, 3μm (Nikkyo Technos)) Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid in water Flow rate: 300nL / min Sample injection volume: 1μL Gradient conditions (B%): 2% (0.0-0.5 min), 2% → 30% (0.5-23.5 min), 30% → 75% (23.5-25.5 min), 75% (25.5-35.0 min)

[0341] (Mass spectrometer analysis conditions) Ionization method: ESI, positive mode Scan Type: Data Dependent Acquisition Activation Type: Collision Induced Dissociation(CID) Data acquisition was performed using the accompanying software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).

[0342] (18-1-9-3) Analysis of modification sites of trastuzumab Modification site analysis for the LC-MS / MS measurement results was performed using BioPharma Finder 3.0 (Thermo Fisher Scientific). Analysis with 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. The digestive enzyme was set to trypsin and the specificity was set to High. Static modification was set to carbamidomethyl (+57.021 Da) as a modification of cysteine ​​residues by iodoacetamide. For dynamic modifications, oxidation of methionine residues (+15.995 Da) and modification of lysine residues (thiol-introduced product after carbamidomethylation by iodoacetamide (+145.019 Da)) were set. In addition, a filter was set to include only those with a Confidence Score of 80 or more, a Mass Accuracy of 5 ppm or less at the time of peptide identification, and MS / MS observation. Regarding the residue numbers of lysine residues, those on the heavy chain VH domain and light chain are indicated by the numbers in the sequence (i.e., the N-terminal amino acid is numbered 1; the same applies below), while those on the heavy chain CH1, CH2, and CH3 domains are indicated using EU numbering. In addition, (1) and (2) shown in Figure 6 were used as amino acid sequence data to search for modification sites.

[0343] (18-1-9-4) Analysis of modification sites of trastuzumab by LC-MS / MS As a result of analysis using LC-MS / MS, the MS spectrum (measured value: m / z 577.03606, theoretical value: 577.03557, quadrivalent) of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 3), a peptide consisting of 18 amino acid residues, including the modification site of the lysine residue by trypsin digestion of trastuzumab (thiol-introduced product (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) was observed (Figure 7), and the product ion of m / z 682.13 (theoretical value: 682.01), which corresponds to the trivalent y16, indicating the modification of the lysine residue at the 288th or 290th position in the EU numbering of the heavy chain, was confirmed from the CID spectrum (Figure 8). In addition, analysis with BioPharma Finder showed that the modification of the lysine residue at the 288th or 290th position occurred highly selectively (Figure 9). These results demonstrated that in the trastuzumab thiol-introduced antibody obtained in (18-1-8) above, conjugation proceeded site-selectively to Lys288 and Lys290 (EU numbering) on ​​the heavy chain of the antibody.

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

[0345] The compound (295 mg, 1.62 mmol) synthesized in (18-1-3-2) was dissolved in CHCl (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. The mixture was eluted with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 5 / 1). The fractions containing the product were collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the above compound (259 mg, 0.64 mmol).

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

[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 that the solution had fallen to the origin by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated and then vacuum dried to obtain the above compound (227 mg, 0.66 mmol).

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

[0349] The compound (227 mg, 0.66 mmol) synthesized in (18-2-1-2) was dissolved in CHCl (3.3 mL) and triethylamine (230 μL, 1.65 mmol). Pentafluorophenyl trifluoroacetic acid (225 μL, 1.32 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (175.3 mg, 0.34 mmol). 1 H NMR (400 MHz, Chloroform-d) δ = 8.79 (t, J=1.8, 1H), 8.43 (dt, J=7.8, 1.5, 1H), 8.30 (dt, J=7.9, 1.5, 1H), 7.70 (t, J=7.8, 1H), 7.45 (s, 4H), 3.45 (t, J=6.9, 2H), 3.14 (t, J=6.9, 2H).

[0350] (18-2-2) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:4.

[0351] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the 5th and 34th cysteines each form a disulfide bond within the molecule) synthesized in (18-1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and a linker (72.0 mg, 141 μmol) was added and stirred at room temperature for 24 hours. This was dissolved in 0.05% trifluoroacetic acid aqueous solution, subjected to reversed-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as the packing material, eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above peptide thioester linker-thiophenol activated product (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 its analysis by ESI-TOFMS 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), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was 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 were confirmed at 152691, where one binding peptide was introduced, 157163, where two binding peptides were introduced, and 161634, where three binding peptides were introduced.

[0354] (18-2-4) Confirmation of heavy chain selectivity of specific modified forms of trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-2-3) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 55,067, and the light chain had a peak at 23,439, the same as the raw material.

[0355] (18-2-5) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-2-3), and the results are shown in Table 6. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 6 was 2.0. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio 2.0) represented by the following structural formula was confirmed.

[0356] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0357] [Table 6]

[0358] (18-2-6) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (18-2-5) was subjected 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).

[0359] (18-2-7) Peptide mapping by trypsin treatment Peptide mapping was performed on the trastuzumab thiol derivative obtained in (18-2-6) by the following steps.

[0360] (18-2-7-1) Trypsin treatment of trastuzumab thiol derivative The trastuzumab thiol-introduced complex obtained in (18-2-6) was treated with trypsin in the same manner as in (18-1-9-1).

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

[0362] (18-2-7-3) Analysis of modification sites of trastuzumab The analysis was carried out in the same manner as (18-1-9-3).

[0363] (18-2-7-4) Analysis of modification sites of trastuzumab by LC-MS / MS As a result of analysis using LC-MS / MS, the MS spectrum (measured value: m / z 577.03571, theoretical value: 577.03557, quadrivalent) of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 3), a peptide consisting of 18 amino acid residues, including the modification site of the lysine residue by trypsin digestion of trastuzumab (thiol-introduced product (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) was observed (Figure 10), and the product ion of m / z 682.41 (theoretical value: 682.01), which corresponds to the trivalent y16, indicating the modification of the lysine residue at the 288th or 290th position in the EU numbering of the heavy chain, was confirmed from the CID spectrum (Figure 11). In addition, analysis with BioPharma Finder showed that the modification of the lysine residue at the 288th or 290th position occurred highly selectively (Figure 12). These results demonstrated that in the trastuzumab thiol-introduced antibody obtained in (18-2-6) above, conjugation proceeded site-selectively to Lys288 and Lys290 (EU numbering) on ​​the heavy chain of the antibody.

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

[0365] The compound (220 mg, 1.21 mmol) synthesized in (18-1-3-2) was dissolved in CHCl (12.0 mL), and AdipicAcid (530 mg, 3.63 mmol), DIPEA (314 μL, 1.82 mmol), and PyBOP (755 mg, 1.45 mmol) were added and stirred at room temperature for 1 hour. After confirming the reaction by TLC (dichloromethane / methanol = 10 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of dichloromethane and methanol, and each fraction was confirmed by TLC (dichloromethane / methanol = 10 / 1). The fraction containing the product was collected, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the above compound (205 mg, 0.63 mmol).

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

[0367] The compound (205 mg, 0.63 mmol) synthesized in (18-3-1-1) was dissolved in CH2Cl2 (3.15 mL) and triethylamine (220 μL, 1.58 mmol). Pentafluorophenyl trifluoroacetic acid (215 μL, 1.26 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 3 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 3 / 1). The fraction containing the product was collected and concentrated under reduced pressure to remove the organic solvent, followed by vacuum drying to obtain the above compound (200 mg, 0.41 mmol).

[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) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:4.

[0370] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (30.0 mg, 7.06 μmol, where the 5th and 34th cysteines each form a disulfide bond within the molecule) synthesized in (18-1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and a linker (69.0 mg, 141 μmol) was added and stirred at room temperature for 24 hours. This was dissolved in 0.05% trifluoroacetic acid aqueous solution, subjected to reversed-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as the packing material, eluted with a mixed solution of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above peptide thioester linker-thiophenol activated product (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 its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-3-2) was dissolved in dimethyl sulfoxide to 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 and 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 were confirmed at 152676 with one binding peptide introduced, 157126 with two binding peptides introduced, and 161572 with three binding peptides introduced.

[0373] (18-3-4) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of specific modified forms of trastuzumab under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-3-3) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 55,048, and the light chain had a peak at 23,439, the same as the raw material.

[0374] (18-3-5) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-3-3), and the results are shown in Table 7. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 7 was 1.9. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio 1.9) represented by the following structural formula was confirmed.

[0375] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 1.9.

[0376] [Table 7]

[0377] (18-3-6) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (18-3-5) 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).

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

[0379] tBu-3-Sulfanylpropanoate (500 mg, 3.08 mmol) was dissolved in tetrahydrofuran (7 mL), triethylamine (0.64 mL, 4.62 mmol) was added, and malonyl chloride (0.15 mg, 1.54 mmol) was added at 0°C and stirred for 3 hours. After confirming the reaction with 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, concentrated under reduced pressure to remove the organic solvent, and then vacuum dried to obtain the above compound (104 mg, 0.26 mmol).

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

[0381] The compound (104 mg, 0.26 mmol) 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 the solution had fallen to the origin by TLC (hexane / ethyl acetate = 5 / 1), the reaction solution was concentrated and then vacuum dried to obtain the above compound (104 mg, 0.37 mmol).

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

[0383] The compound (104 mg, 0.37 mmol) synthesized in (18-4-1-2) was dissolved in CH2Cl2 (1.85 mL) and triethylamine (130 μL, 0.93 mmol). N-SuccinimidylTrifluoroacetate (156 mg, 0.74 mmol) was added at 0°C and stirred for 1 hour. After confirming the reaction by TLC (hexane / ethyl acetate = 1 / 1), the reaction solution was concentrated. Elution was performed with a mixed solution of hexane and ethyl acetate, and each fraction was confirmed by TLC (hexane / ethyl acetate = 1 / 1). The fraction containing the product was collected and concentrated under reduced pressure to remove the organic solvent, and then vacuum dried 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) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:4.

[0386] Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH2 (SEQ ID NO: 4) (29.7 mg, 7.00 μmol, where the 5th and 34th cysteines each form a disulfide bond within the molecule) synthesized in (18-1-2) was dissolved in N,N-dimethylformamide (1.00 mL), and a linker (66.8 mg, 0.14 mmol) was added and stirred at room temperature for 4 hours. This was dissolved in 0.05% aqueous trifluoroacetic acid solution, subjected to reversed-phase high-performance liquid chromatography using octadodecyl-group chemically bonded silica gel as the packing material, and eluted with a mixture of water and acetonitrile containing 0.05% trifluoroacetic acid, and each fraction was confirmed by LC-MS. The fractions containing the product were collected and concentrated under reduced pressure to remove acetonitrile, and then lyophilized to obtain the above peptide thioester linker-NHS activated product (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 its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-4-2) was dissolved in dimethyl sulfoxide to 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 and 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 were confirmed at 152722 with one binding peptide introduced, 157215 with two binding peptides introduced, and 161708 with three binding peptides introduced.

[0389] (18-4-4) Confirmation of heavy chain selectivity of specific modified forms of trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-4-3) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,594 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 55,091, and the light chain had a peak at 23,439, the same as the raw material.

[0390] (18-4-5) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-4-3), and the results are shown in Table 8. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 8 was 1.8. Therefore, the production of the antibody intermediate (average peptide / antibody binding ratio 1.8) represented by the following structural formula was confirmed.

[0391] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 1.8.

[0392] [Table 8]

[0393] (18-4-6) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (18-4-5) was subjected 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).

[0394] (18-5-1) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:5.

[0395] A linker was bound to Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC-NH2 (sequence number 5) (30.0 mg, 7.06 μmol, with the two cysteines at positions 5 and 34 each forming a disulfide bond within the molecule) synthesized by the method described in (18-1-1) in Example 2 (2-2) in the same manner as in Example 2 (2-2) to obtain the above peptide thioester linker conjugate-thiophenol activated product (10.0 mg, 2.19 μmol).

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

[0397] (18-5-2) Specific modification of anti-HER2 antibody trastuzumab and its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-5-1) was dissolved in dimethyl sulfoxide to 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 and 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 were confirmed at 152707, where one binding peptide was introduced, 157188, where two binding peptides were introduced, and 161676, where three binding peptides were introduced.

[0398] (18-5-3) Confirmation of heavy chain selectivity of specific modified forms of trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-5-2) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 55,077, and the light chain had a peak at 23,439, the same as the raw material.

[0399] (18-5-4) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (5-2), and the results are shown in Table 9. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 9 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0400] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0401] [Table 9]

[0402] (18-5-5) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (18-5-4) was subjected 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).

[0403] (18-6-1) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:6.

[0404] A linker was bound to Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (sequence number 6) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (18-1-1) in the same manner as in (18-2-2) to obtain the above peptide thioester linker conjugate-thiophenol activated product (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 its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-6-1) was dissolved in dimethyl sulfoxide to 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 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 were confirmed at 152720 with one binding peptide introduced, 157216 with two binding peptides introduced, and 161716 with three binding peptides introduced.

[0407] (18-6-3) Confirmation of heavy chain selectivity of specific modified forms of trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-6-2) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 55,091, and the light chain had a peak at 23,439, the same as the raw material.

[0408] (18-6-4) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-6-2), and the results are shown in Table 10. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 10 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0409] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0410] [Table 10]

[0411] (18-6-5) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (18-6-4) was subjected to the cleavage reaction of the thioester group described in (18-1-8) to obtain the antibody derivative having a thiol group introduced therein.

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

[0413] A linker was bound to Ac-NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (sequence number 7) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (18-1-1) in (18-2-2) in the same manner as in (18-2-2) to obtain the above peptide thioester linker conjugate-thiophenol activated product (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 its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-7-1) was dissolved in dimethyl sulfoxide to 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 and 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. A peak of 152573 with one binding peptide introduced and 156927 with two binding peptides introduced were confirmed.

[0416] (18-7-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of specific modified forms of trastuzumab under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-7-2) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 54,942, and the light chain had a peak at 23,439, the same as the raw material.

[0417] (18-7-4) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-7-2), and the results are shown in Table 11. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 11 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0418] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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 bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0419] [Table 11]

[0420] (18-7-5) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The antibody intermediate obtained in (18-7-4) was subjected 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).

[0421] (18-7-6) Peptide mapping by trypsin treatment Peptide mapping was performed on the trastuzumab thiol derivative obtained in (18-7-5) using the following steps.

[0422] (18-7-6-1) Trypsin treatment of trastuzumab thiol derivative The trastuzumab thiol-introduced complex obtained in (18-7-5) was treated with trypsin 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 carried out under the same conditions as for (18-1-9-2).

[0424] (18-7-6-3) Analysis of modification sites of trastuzumab The analysis was carried out in the same manner as (18-1-9-3).

[0425] (18-7-6-4) Analysis of modification sites of trastuzumab by LC-MS / MS As a result of analysis using LC-MS / MS, the MS spectrum (measured value: m / z 769.04506, theoretical value: 769.04482, trivalent) of the peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 3), a peptide consisting of 18 amino acid residues including a modification site of the lysine residue by trypsin digestion of trastuzumab (thiol-introduced product (+145.019 Da) subjected to carbamidomethylation with iodoacetamide), was observed (FIG. 13), and the product ion of m / z 1022.71 (theoretical value: 1022.51), which corresponds to bivalent y16, indicating modification of the lysine residue at the 288th or 290th position in EU numbering of the heavy chain, was confirmed from the CID spectrum (FIG. 14). In addition, analysis with BioPharma Finder showed that modification of the lysine residue at the 288th or 290th position occurred highly selectively (FIG. 15). These results demonstrated that in the trastuzumab thiol-introduced antibody obtained in (18-7-5) above, conjugation had proceeded site-selectively to Lys288 and Lys290 (EU numbering) on ​​the heavy chain of the antibody.

[0426] (18-8-1) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:8.

[0427] A linker was bound to Ac-MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (sequence number 8) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (18-1-1) in (18-2-2) in the same manner as in (18-2-2) to obtain the above peptide thioester linker conjugate-thiophenol activated product (16.8 mg, 3.87 μmol).

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

[0429] (18-8-2) Specific modification of anti-HER2 antibody trastuzumab and its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-8-1) was dissolved in dimethyl sulfoxide to 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 and 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 were confirmed at 152457 with one binding peptide introduced, 156692 with two binding peptides introduced, and 160929 with three binding peptides introduced.

[0430] (18-8-3) Confirmation of heavy chain selectivity by ESI-TOFMS analysis of specific modified forms of trastuzumab under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-8-2) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 54,829, and the light chain had a peak at 23,439, the same as the raw material.

[0431] (18-8-4) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-8-2), and the results 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 production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0432] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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: 8, The average bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0433] [Table 12]

[0434] (18-8-5) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups 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) Bonding of peptide and linker [ka] Both of the above two amino acid sequences are the amino acid sequence of SEQ ID NO:9.

[0436] A linker was attached to Ac-QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH2 (sequence number 9) (30.0 mg, 7.06 μmol, where the two cysteines at positions 5 and 34 each form a disulfide bond within the molecule) synthesized by the method described in (18-9-1) in a manner similar to that described in (18-2-2) to obtain the above peptide thioester linker conjugate-thiophenol activated product (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 its analysis by ESI-TOFMS The peptide linker conjugate synthesized in (18-9-1) was dissolved in dimethyl sulfoxide to a concentration of 10 mM. 500 μg of anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 200 μL (20 μM) of 50 mM HEPES buffer (pH 8.2), and 3.38 μL (10 equivalents relative to the antibody) of 10 mM peptide reagent was added and stirred at room temperature for 1 hour. The reaction solution was 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 were confirmed at 152236, where one binding peptide was introduced, 156430, where two binding peptides were introduced, and 160541, where three binding peptides were introduced.

[0439] (18-9-3) Confirmation of heavy chain selectivity of specific modified forms of trastuzumab by ESI-TOFMS analysis under reducing conditions 2 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride solution (equivalent to the antibody) was added to the antibody-peptide complex produced in (18-9-2) and stirred at room temperature for 15 minutes. When the mass was measured by ESI-TOFMS, the raw material trastuzumab had a heavy chain peak at 50,596 and a light chain peak at 23,439. The reaction product had a heavy chain with one linker introduced at 54,698, and the light chain had a peak at 23,439, the same as the raw material.

[0440] (18-9-4) Confirmation of peptide / antibody binding ratio of specific modified forms of trastuzumab using DAR calculator The peptide / antibody binding ratio was confirmed using the DAR calculator (software from Agilent) for the MS data analyzed in (18-9-2), and the results are shown in Table 13. The average peptide / antibody binding ratio calculated from the DAR peak and %Area in Table 13 was 2.0. Therefore, the production of an antibody intermediate (average peptide / antibody binding ratio of 2.0) represented by the following structural formula was confirmed.

[0441] [ka] [Here, Ig refers to an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and forms amide bonds with two carbonyl groups (C=O) adjacent to Ig via amino groups in the side chains of lysine residues 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:9, The average bond ratio r between Ig and the two carbonyl groups (C=O) adjacent to Ig is 2.0.

[0442] [Table 13]

[0443] (18-9-5) Preparation of thiol group-introduced antibody derivatives by cleavage of thioester groups The thiol group-introduced antibody derivative was obtained by subjecting the antibody intermediate obtained in (18-9-4) 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).

[0444] (18-10-1) Introduction of bioorthogonal functional groups to the thiols at Ly288 / Lys290 of antibodies The azido linker (1) of Example 1 was used to modify the Ly288 / Lys290 thiol-introduced antibody synthesized in Example (18-1-8). Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain at 50816, 50978, and the same light chain peak at 23439 as the raw material.

[0445] In a similar manner, the thiol-introduced antibody at Ly288 / Lys290 position synthesized in Example (18-1-8) was modified using the azide linker (2) of Example 2. Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439, while the product had azide groups introduced into the heavy chain at 50848, 51011, and the same light chain peak at 23439 as the raw material.

[0446] In a similar manner, the antibody synthesized in Example (18-1-8) with thiols introduced at 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 the mass was measured by ESI-TOFMS according to the method previously reported (Anal.Chem., 2019, 91, 20, 12724-12732). A peak of 149023 was confirmed, indicating that two azide groups had been introduced into the antibody.

[0447] In a similar manner, the Ly288 / Lys290 thiol-introduced antibody synthesized in Example (18-1-8) was modified using the alkyne linker (8) of Example 8. Tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 mM) was added to the product, stirred at room temperature for 10 minutes, and the mass was measured by ESI-TOFMS according to a previous report (WO2019 / 240287A1). The raw material thiol group-introduced antibody had heavy chain peaks at 50683 and 50845, and a light chain peak at 23439, while the product had alkyne groups introduced into the heavy chain at 51137 and 51304, and a light chain peak at 23439, the same as the raw material.

[0448] In a similar manner, the antibody synthesized in Example (18-1-8) with thiols introduced at Ly288 / Lys290 was modified using 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 previously reported (Anal.Chem., 2019, 91, 20, 12724-12732). A peak of 149185 was confirmed, in which two alkyne groups were introduced into the antibody.

[0449] Example 19: Synthesis of ADCs with DAR=4 using branched reagents (19-1) Synthesis of branched azide reagents (19-1-1)Synthesis of tert-Butyl N-[5-(tert-butoxycarbonylamino)-1-(hydrazinecarbonyl)pentyl]carbamate

[0450] [ka]

[0451] 9H-Fluoren-9-ylmethyl N-[2,6-bis(tert-butoxycarbonylamino)hexanoylamino]carbamate (191 mg, 0.328 mmol) was added with 3 mL of 5% piperidine-containing acetonitrile solution and stirred at room temperature for 3 hours. After confirming the disappearance of the raw material by TLC, 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-[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), 4.12-4.04 (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] Synthesis of (19-1-2)tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-sulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamete

[0455] [ka]

[0456] Carbon disulfide (0.034mL, 0.567mmol) and potassium hydroxide (66.4mg, 1.18mmol) were added to an ethanol solution (8mL) of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(hydrazinecarbonyl)pentyl]carbamate (136.2mg, 0.328mmol), and the mixture was stirred at 70°C for 20 hours. 1N hydrochloric acid was added to the reaction solution, which was then concentrated under reduced pressure, and the mixture was extracted with ethyl acetate. The organic layer was then concentrated under reduced pressure to obtain a crude product, which was then purified by column chromatography. The fractions containing the product were 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 (94mg, 0.234mmol, 2 steps yield 71%).

[0457] MS(ESI) m / z:401[MH]

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

[0460] [ka]

[0461] A THF solution (3 mL) of tert-butyl N-[5-(tert-butoxycarbonylamino)-1-(5-sulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamate (94.1 mg, 0.234 mmol) 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 returned to room temperature, stirred, and the disappearance of the raw material was confirmed by TLC. The reaction solution 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-[5-(tert-butoxycarbonylamino)-1-(5-methylsulfanyl-1,3,4-oxadiazol-2-yl)pentyl]carbamate (81 mg, 0.194 mmol, yield 83%).

[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] [ka]

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

[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] Synthesis of (19-1-5)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] [ka]

[0471] 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 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). After stirring at room temperature for 3 hours, raw materials were still remaining, so 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 stirred for another hour. The reaction solution was diluted with acetonitrile water and purified by preparative HPLC, and the fraction containing the product was collected. After removing the acetonitrile by concentration under reduced pressure, the aqueous solution was freeze-dried 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, two-step yield 38%).

[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]

Chemical Structure

[0476] 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 (47.5mg, 0.0734mmol) in dichloromethane solution (2mL) was added with 3-chloroperbenzoic acid (containing about 30% water) (112.4mg, 0.456mmol). After stirring at room temperature for 5 hours, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified by preparative HPLC. The fractions containing the product were collected, concentrated under reduced pressure to remove acetonitrile, and the aqueous solution was freeze-dried to obtain the target product (92mg). Since impurities were still present, repurification was performed 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-[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 reagents with thiol-introduced antibodies The azido linker synthesized in Example 19-1-6 was reacted with the thiol group-introduced antibody in the same manner as described in Example 2(2).

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

[0481] (19-3) Synthesis of ADCs via Click Reaction According to previous reports (WO2019 / 240287A1 and WO2019 / 240288A1) and Example 15-1, 20 equivalents of a DMF solution (5 mM) of DBCO-VC-PAB-MMAE (ABZENA) was added to the branched azide-introduced trastuzumab obtained in Example 19-2, and the mixture was stirred at room temperature for 20 hours. The mixture was then purified using NAP-5 Columns (GE Healthcare) to obtain an ADC.

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

[0483] (19-4) Synthesis of branched protected thiol reagents Synthesis of (19-4-1) tert-Butyl(1,3-dihydroxypropan-2-yl)carbamate

[0484] [ka]

[0485] t-Butyl alcohol (1.8mL) was added to a methanol solution (1.8mL) of 2-amino-1,3-propanediol (186.2mg, 2.00mmol), and a t-butyl alcohol solution (1.4mL) of di-tert-butyl dicarbonate (597.3mg, 2.60mmol) was added dropwise. After stirring at room temperature for 17 hours, the reaction solution was concentrated under reduced pressure. When hexane (4mL) was added to the obtained crude product, a white solid precipitated. After filtration, the solid was washed with hexane (3mL) and dried on a vacuum line to obtain tert-Butyl(1,3-dihydroxypropan-2-yl)carbamate (352.9mg, 1.845mmol, 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] Synthesis of (19-4-2)tert-Butyl N-[2-allyoxy-1-(allyloxymethyl)ethyl]carbamate

[0489] [ka]

[0490] Allyl bromide (0.547mL, 6.34mmol) was added to a DMF solution (4mL) of tert-Butyl(1,3-dihydroxypropan-2-yl)carbamate (324.9mg, 1.70mmol), and potassium hydroxide (359.0mg, 6.40mmol) was added little by little. After stirring at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure, and the residue obtained was extracted with dichloromethane and water. The organic layer was washed with saturated saline, and 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 (326.6mg, 1.20mmol, 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] Synthesis of (19-4-3)S-[3-[3-acetylsulfanylpropoxy]-2-(tert-butoxycarbonylamino)propoxy]propyl]ethaneethioate

[0494] [ka]

[0495] 2,2'-Azobisisobutyronitrile (1.993g, 25.66mmol) was added to a toluene solution (7.4mL) of tert-Butyl N-[2-allyoxy-1-(allyloxymethyl)ethyl]carbamete (323.8mg, 1.19mmol). Thioacetic acid (0.890mL, 11.88mmol) was added in five portions to the suspension-like reaction solution, and the mixture was stirred at 65°C for 21 hours. The reaction solution was returned to room temperature and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to obtain S-[3-[3-acetylsulfanylpropoxy]-2-(tert-butoxycarbonylamino)propoxy]propyl]ethanethioate (362.0mg, 0.854mmol, 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] Synthesis of (19-4-4)S-[3-[3-(3-acetylsulfanylpropoxy)-2-amino-propoxy]propyl]ethanethioate

[0499] [ka]

[0500] Trifluoroacetic acid (2 mL) was added to a dichloromethane solution (2 mL) of S-[3-[3-acetylsulfanylpropoxy]-2-(tert-butoxycarbonylamino)propoxy]propyl]ethanethioate (360.1 mg, 0.850 mmol), and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and 0.1N hydrochloric acid (10 mL) was added to the residue obtained. The solution was concentrated under reduced pressure again, and the resulting aqueous solution was freeze-dried to obtain the hydrochloride salt 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] Synthesis of (19-4-5)S-[3-[3-(3-acetylsulfanylpropoxy)-2-[3-(2,5-dioxoppyrrol-1-yl)propanoylamino]propoxy]propyl]ethaneethioate

[0504] [ka]

[0505] 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 a THF solution (0.5 mL) were added to 3-maleimidopropionic acid (17.6 mg, 0.10 mmol) in a THF solution (1.5 mL). After stirring at room temperature for 2 hours, the reaction solution was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography. The fractions containing the product were collected and concentrated under reduced pressure to give S-[3-[3-(3-acetylsulfanylpropoxy)-2-[3-(2,5-dioxoppyrrol-1-yl)propanoylamino]propoxy]propyl]ethanethioate (18.7 mg, 0.039 mmol, yield 41%).

[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 protected thiol reagents with thiol-introduced antibodies 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] The mass was measured by ESI-TOFMS according to the method previously reported (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak of 149392 was confirmed, indicating that four protected thiol groups had been introduced into the antibody.

[0510] (19-6) Preparation of branched thiol-group-introduced antibody derivatives by cleavage of thioester groups Hydroxylamine solution was added to the antibody intermediate obtained in (19-5) according to the previous report (WO2019 / 240287A1) and left at room temperature for 1 hour. After 2 hours, the buffer was replaced with 20 mM PBS buffer and 10 mM EDTA (pH 7.4) to obtain a thiol group-introduced antibody derivative. When the mass was measured by ESI-TOFMS, a peak was confirmed at 149231, where the deprotection reaction had progressed.

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

[0512] [ka]

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

[0514] 1 H NMR(400 MHz,DMSO-d6)δ9.98(s,1H),8.34(d,J=9.2Hz,2H),8.32-8.23(m,4H),8.16(s,2H),8.10-8.00(m,4H),7.80(d ,J=8.8Hz,1H),7.59(d,J=8.4Hz,2H),7.31(d,J=8.0Hz,2H),6.99(s,2H),5.96(m,1H),5.40(s,2H),5.01(s,2H) ),4.95(d,J=6.0Hz,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=12.8,6.8Hz,6H)

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

[0516] (19-7-2)Synthesis of ADC mimics 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, and the mixture was left to stand at room temperature for 2 hours. After that, the mixture was purified using NAP-5 Columns (GE Healthcare) to obtain an ADC mimic.

[0517] The mass was measured by ESI-TOFMS according to the method previously reported (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak of 152843 was confirmed, where four maleimide-VC-pyrenes were introduced into the antibody.

Claims

1. The following formula (I): 【Chemistry 1】 (In the formula, X is a halogen atom; Q is arylene; Y is a bond or a divalent group; the divalent group is a divalent group formed by linking 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 -; R 2 is a hydrogen atom; R 3 is alkylene or arylene; m is an integer from 1 to 5; R is a bioorthogonal functional group. or a salt thereof. A derivatization reagent for a thiol group-introduced antibody or its salt, comprising a compound represented by the following formula:

2. The reagent according to claim 1, wherein the reagent is 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 site-selectively 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 or 4, 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 according to EU numbering.

6. The following formula (I): 【Chemistry 2】 (In the formula, X is a halogen atom; Q is arylene; Y is a divalent group, the divalent group is a divalent group formed by linking 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 -; R 2 is a hydrogen atom; R 3 is alkylene or arylene; m is an integer from 1 to 5; R is a DBCO group. A compound represented by the formula:

7. The following formula (I'): 【Chemistry 3】 (In the formula, Ab is an antibody; S is a sulfur atom; Q is arylene; Y is a bond or a divalent group; the divalent group is a divalent group formed by linking 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 -; R 2 is a hydrogen atom; R 3 is alkylene or arylene; m is an integer from 1 to 5; R is a bioorthogonal functional group; n is an integer from 1 to 8. An antibody intermediate having a bioorthogonal functional group, represented by the formula:

8. The sulfur atom (S) adjacent to the antibody (Ab) in the formula (I') is 8. The antibody intermediate or salt thereof according to claim 7, which is regioselectively bonded to a nitrogen atom in the side chain of a lysine residue in the heavy chain constant region via a linker that does not contain a peptide.

9. The antibody intermediate or a salt thereof according to claim 7 or 8, wherein the antibody intermediate is an IgG antibody.

10. 10. The antibody intermediate or salt thereof according to claim 8 or 9, 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 a human IgG heavy chain according to EU numbering.

11. The following formula (I): 【Chemistry 4】 (In the formula, X is a halogen atom; Q is arylene; Y is a bond or a divalent group; the divalent group is a divalent group formed by linking 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 -; R 2 is a hydrogen atom; R 3 is alkylene or arylene; m is an integer from 1 to 5; R is a bioorthogonal functional group. or a salt thereof with a thiol group-introduced antibody or a salt thereof, The following formula (I'): 【Chemistry 5】 (In the formula, Ab is an antibody; S is a sulfur atom; n is an integer from 1 to 8, Q, Y and R are the same as those in formula (I). A method for producing an antibody intermediate or a salt thereof having a bioorthogonal functional group, comprising producing an antibody intermediate or a salt thereof having a bioorthogonal functional group represented by the formula:

12. The following formula (I″): 【Chemistry 6】 (In the formula, Ab is an antibody; S is a sulfur atom; Q is arylene; Y is a bond or a divalent group; the divalent group is a divalent group formed by linking 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 -; R 2 is a hydrogen atom; R 3 is alkylene or arylene; m is an integer from 1 to 5; R' is a group resulting from the reaction between two bioorthogonal functional groups capable of reacting with each other; Z is a functional substance; n is an integer from 1 to 8. A conjugate of an antibody and a functional substance, represented by the formula:

13. The following formula (I'): 【Chemistry 7】 (In the formula, Ab is an antibody; S is a sulfur atom; Q is arylene; Y is a bond or a divalent group; the divalent group is a divalent group formed by linking 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 -; R 2 is a hydrogen atom; R 3 is alkylene or arylene; m is an integer from 1 to 5; R is a bioorthogonal functional group; n is an integer from 1 to 8. 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, The following formula (I″): 【Chemistry 8】 (In the formula, Ab, S adjacent to Ab, Q, Y and n are the same as those in formula (I'); R' is a group resulting from the reaction between two bioorthogonal functional groups capable of reacting with each other; Z is a functional substance. or a salt thereof, When the antibody intermediate or its salt has an alkyne residue, the antibody intermediate or its salt is reacted with an azide-containing functional material; 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.

14. The following formula (I): 【Chemistry 9】 (In the formula, X is a halogen atom; Q, Y and R are the same as those in formula (I'). or a salt thereof with a thiol group-introduced antibody or a salt thereof, The method of claim 13, further comprising producing an antibody intermediate having a bioorthogonal functional group represented by formula (I') or a salt thereof.

15. A compound represented by the following formula (V) or a salt thereof: 【Chemistry 10】

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