A compound having an affinity substance for an antibody and a bioorthogonal functional group or a salt thereof

Abstract

The present invention provides a technique for achieving modification of an antibody, particularly site-selective modification of an antibody. More specifically, the present invention provides a compound or a salt thereof, etc., having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I): A-L-E-B (I) [In the formula, A is an affinity substance for an antibody, L is a divalent group containing a leaving group, E is a divalent group containing an electrophilic group, the electrophilic group being: (i) connected to the above-mentioned leaving group and (ii) having the ability to react with a nucleophilic group in the above-mentioned antibody, B is a bioorthogonal functional group, and the above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

Description

Technical Field

[0001] The present invention relates to a compound having an affinity substance for an antibody and a bioorthogonal functional group, or a salt thereof, etc. Background Art

[0002] In recent years, research and development of antibody-drug conjugates (Antibody-Drug Conjugates: ADCs) have been actively carried out. As the name indicates, an ADC is a medicament obtained by conjugating a drug (e.g., an anticancer agent) to an antibody, and has direct cytotoxic activity against cancer cells and the like. As a representative ADC, there is T-DM1 (trade name: Kadcyla (registered trademark)) (Non-Patent Documents 1 to 3).

[0003] ADCs represented by T-DM1 have had a problem of their non-uniformity since the initial stage of development. That is, since a low-molecular-weight drug is randomly reacted with about 70 to 80 Lys residues present in the antibody, the drug-antibody ratio (Drug Antibody Ratio: DAR) and the conjugation position are uncertain. It is known that generally in such a random conjugation method, the DAR is in the range of 0 to 8, and a variety of medicaments having different numbers of drug bindings are produced. In recent years, it has been reported that if the number of drug bindings and the binding position of an ADC are changed, the in vivo kinetics, the drug release rate, and the effect will change. Based on these circumstances, for the next-generation ADCs, control of the number and position of the conjugated drugs is required. It is generally considered that if the number and position are determined, problems such as the expected efficacy, the diversity of conjugated medicaments, and batch differences, that is, so-called standardization, will be solved (Non-Patent Document 4).

[0004] Methods for site-selective modification of antibodies have been studied globally, but almost all of them use genetic engineering means or enzyme modification methods. Regarding the genetic engineering modification method, although site-selectivity and number-selectivity can be controlled, problems such as a decrease in the expression efficiency of the antibody itself (a decrease in the total yield when preparing an ADC) have been pointed out. In addition, it has also become a problem that construction of an antibody expression system and the like require a long time (Non-Patent Documents 5 to 7).

[0005] In addition, in recent years, a method for chemically modifying proteins in a mixed environment such as inside cells using a small molecule probe has been reported. This method is used for identification of receptors and the like in imaging or repositioning of low-molecular-weight drugs. In addition, in the biochemical field, an organic chemical protein modification method using a synthetic small molecule probe has attracted attention (Non-Patent Documents 8 to 11).

[0006] Recently, the CCAP (Chemical Conjugation by Affinity Peptide) method has been developed. This method involves reacting a peptide reagent obtained by linking an NHS active ester and a drug to an affinity peptide with an antibody (i.e., a method for producing an ADC via a linker containing a peptide moiety), and has successfully achieved site-selective modification of antibodies. This method is the world's first to successfully perform site-selective modification of the antibody Fc region with a drug by chemical synthesis means, and good results have also been confirmed in actual use [reaction time: 30 minutes, yield: 70% (for DAR1), site selectivity: 100%]. It has been confirmed that DAR can be controlled to 2 by adding about 5 equivalents of the peptide reagent, and the modification site can also be controlled, which is of epoch-making significance in this regard (Patent Document 1).

[0007] Prior Art Documents

[0008] Patent Documents

[0009] Patent Document 1: WO 2016 / 186206;

[0010] Non-Patent Documents

[0011] Non-Patent Document 1: Reichert JM et al., Nat Biotechnol 2005; 23: 1073-8;

[0012] Non-Patent Document 2: Kubota T et al., Cancer Sci 2009; 100: 1566-72;

[0013] Non-Patent Document 3: Wu AM et al., Nat Biotechnol 2005; 23: 1137-46;

[0014] Non-Patent Document 4: Junutula JR et al., Nat Biotechnol 2008; 26: 925-32;

[0015] Non-Patent Document 5: Shen BQ et al., Nat Biotechnol 2012; 30: 184-9;

[0016] Non-Patent Document 6: Hofer T et al., Biochemistry 2009; 48: 12047-57;

[0017] Non-Patent Document 7: Liu W et al., Nat Methods 2007; 4: 239-44;

[0018] Non-Patent Document 8: S.T. Laughlin et al., Science 2008; 320, 664;

[0019] Non-Patent Document 9: A.E. Speers et al., ChemBioChem 2004; 5, 41;

[0020] Non-Patent Document 10: Y. Takaoka et al., Angew. Chem. Int. Ed. 2013; 52, 4088; Non-Patent Document 11: S. Fujishima et al., J. Am. Chem. Soc, 2012; 134: 3961-64. Summary of the Invention

[0021] Problems to be Solved by the Invention

[0022] An object of the present invention is to develop a technique for modifying an antibody, particularly site-selective modification of an antibody.

[0023] Means for Solving the Problems

[0024] The present inventors conducted intensive studies and found that: a compound having a structural unit of A-L-E (where A is an affinity substance for an antibody, L is a divalent group containing a predetermined leaving group, and E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above leaving group, and (ii) capable of reacting with a nucleophilic group in the above antibody.) or a salt thereof can be used for site-specific modification of an antibody. For example, it was found that a specific compound having an affinity substance for an antibody and a bioorthogonal functional group represented by formula (I) can be used for site-specific modification of an antibody (for example, Figure 1 , various examples). It was also found that a compound having an affinity substance for an antibody and a functional substance represented by formula (IV) or a salt thereof can be used for site-specific modification of an antibody (for example, Examples 13 and 14). The present inventors further found that: by using such a compound, an antibody (antibody-drug conjugate (ADC)) etc. that does not contain a peptide moiety as a linker and site-selectively has a functional substance (such as a drug) can be prepared. Avoiding the use of a linker containing a peptide moiety that has potential immunogenicity and is easily hydrolyzed in blood is desirable in the clinical application of ADC. That is, according to the method developed by the present inventors, by chemical synthesis means and without using a linker containing a peptide moiety, the Fc region of an antibody can be site-selectively modified with a drug.

[0025] That is, the present invention is as follows.

[0026] [1] A compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I):

[0027] A-L-E-B (I)

[0028] [In the formula,

[0029] A is an affinity substance for an antibody,

[0030] L is a divalent group containing a leaving group,

[0031] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0032] B is a bioorthogonal functional group,

[0033] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0034] [2][1] The compound or its salt, wherein the above-mentioned affinity substance is a peptide.

[0035] [3][2] The compound or its salt, wherein the above-mentioned peptide is a peptide having the ability to bind to the constant region of a monoclonal antibody.

[0036] [4][2] or [3] The compound or its salt, wherein the above-mentioned peptide is a peptide having the ability to bind to the Fc region of a monoclonal antibody.

[0037] [5][4] The compound or its salt, wherein the above-mentioned peptide is a peptide having the ability to bind to the Fc region of IgG.

[0038] [6][2] - [5] The compound or its salt according to any one of the above, wherein the above-mentioned peptide has 10 to 40 amino acid residues.

[0039] [7][2] - [6] The compound or its salt according to any one of the above, wherein the above-mentioned peptide contains the following amino acid sequence:

[0040] (a) In the amino acid sequence of (a-1-1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO:11), or

[0041] (a-1-2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:12) amino acid sequence,

[0042] Any 1 to 3 amino acid residues in the sequence may be the same or different and are each replaced by 1 amino acid residue selected from lysine residue, aspartic acid residue, and glutamic acid residue,

[0043] in the amino acid sequence of (a-2-1) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO:13), or

[0044] (a-2-2) β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:14),

[0045] any 1 to 3 amino acid residues in the sequence may be the same or different and are each replaced by 1 amino acid residue selected from lysine residue, aspartic acid residue, and glutamic acid residue; and

[0046] (b) has an identity of more than 85% with each of the above amino acid sequences of SEQ ID NOs: 11 to 14.

[0047] A compound or a salt thereof according to any one of [8][2] to [6], wherein the peptide comprises any one of the following amino acid sequences:

[0048] Formula 1-1: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-I-W-C-(X 0-3 ) b (SEQ ID NO:15)

[0049] Formula 1-2: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-V-W-C-(X 0-3 ) b (SEQ ID NO:16)

[0050] Formula 1-3: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-V-V-W-C-(X 0-3 ) b (SEQ ID NO:17)

[0051] Formula 1-4: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-A-V-W-C-(X 0-3 ) b (SEQ ID NO:18)

[0052] Formula 1-5: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-L-W-C-(X 0-3 ) b (SEQ ID NO:19)

[0053] Formula 1-6: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-I-W-C-(X 0-3 ) b (SEQ ID NO:20)

[0054] Formula 1-7: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-F-C-(X 0-3 ) b (SEQ ID NO:21)

[0055] Formula 1-8: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Q-V-W-C-(X 0-3 ) b (SEQ ID NO:22)

[0056] Formula 1-9: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-E-V-W-C-(X 0-3 ) b (SEQ ID NO:23)

[0057] [Wherein,

[0058] (X 0-3 ) a is absent, arginine residue - glycine residue - asparagine residue, glycine residue - asparagine residue, aspartic acid residue, or asparagine residue,

[0059] (X 0-3 ) b is absent, threonine residue - tyrosine residue - histidine residue, or threonine residue,

[0060] Xaa1 is alanine residue,

[0061] Xaa2 is a tyrosine residue, a tryptophan residue, or a histidine residue,

[0062] Xaa3 is a histidine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an arginine residue, or a glycine residue,

[0063] Xaa4 is a lysine residue, an aspartic acid residue, or a glutamic acid residue,

[0064] Xaa5 is a glycine residue, a serine residue, an asparagine residue, a glutamine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a histidine residue, a threonine residue, a leucine residue, an alanine residue, a valine residue, an isoleucine residue, or an arginine residue,

[0065] Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue.]; or

[0066] Formula 2-1: (X 0-3 ’) a -C-(Xaa1’)-(Xaa2’)-(Xaa3’)-(Xaa4’)-(Xaa5’)-(Xaa6’)-L-V-W-C-(X 0-3 ’) b (SEQ ID NO:24)

[0067] [wherein,

[0068] (X 0-3 ’) a and (X 0-3 ’) b are respectively the same as the above-mentioned (X 0-3 ) a and (X 0-3 ) b respectively,

[0069] Xaa1’, Xaa2’, Xaa3’, Xaa4’, Xaa5’, Xaa6’ are respectively the same as the above-mentioned Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6.].

[0070] [9][1] to [8] Any one of the compounds or salts thereof, wherein the leaving group is: (1) a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); or (2) a heteroarylene group.

[0071]

[10] The compound or salt thereof according to any one of [1] to [9], wherein the nucleophilic group is selected from the group consisting of NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine ​​residue.

[0072]

[11] The compound or salt thereof according to any one of [1] to

[10] , wherein the electrophilic group is a group selected from -C(=O)-, -SO2-, and -CH2-.

[0073]

[12] The compound or salt thereof according to any one of [1] to

[11] , wherein the bioorthogonal functional group is a group selected from the group consisting of an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an olefin residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, a thioester residue, an α-halogenated carbonyl residue, an isonitrile residue, a Sydney ketone residue, and a selenium residue.

[0074]

[13] The compound or salt thereof according to any one of [1] to

[12] , wherein the bioorthogonal functional group is a group selected from an azide residue, a thiol residue, an alkyne residue, a maleimide residue, and a disulfide residue.

[0075]

[14] The compound or salt thereof according to any one of [1] to

[13] , wherein the compound represented by the above formula (I) is a compound represented by the following formula (I-1):

[0076] A-L1-L2-E1-E2-E3-B (I-1)

[0077] [Wherein,

[0078] A and B have the same meanings as the corresponding symbols in the above formula (I),

[0079] L1 is a bond or a divalent group,

[0080] L2 is a leaving group,

[0081] E1 is an electrophilic group that (i) is linked to the above-mentioned leaving group and (ii) has the ability to react with the nucleophilic group in the above-mentioned antibody,

[0082] E2 is (a) -X-Y- [wherein, X bonded to E1 is C(R1)(R2) (wherein, R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (wherein, R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (wherein, R4 and R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0083] [Chemical formula 1]

[0084]

[0085] (wherein, ring Z is a divalent ring group in which the ring-constituting atom X' bonded to E1 and the two adjacent ring-constituting atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and the two adjacent ring-constituting atoms are carbon atoms. · represents a bonding site.)

[0086] E3 is a divalent group when E2 is -X-Y-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0087] The above leaving group has the ability to be cleaved and detached from E1 through the reaction between the above nucleophilic group and the above electrophilic group.].

[0088] The compound or its salt of

[15]

[14] , wherein the above L2 is:

[0089] (a) Ring P-Q- [wherein, ring P is a group selected from arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine which may be condensed, a 2,6-diketopiperidine which may be condensed, a 2-ketopyrrolidine which may be condensed, a 2-ketopiperidine which may be condensed, 2-pyridone, and Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0090] (b) Heteroarylene; or

[0091] (c) -Q- [wherein, Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).].

[0092] A compound of

[16] ,

[14] or

[15] or a salt thereof, wherein L2 is a group selected from the following structural formulas:

[0093] [Chemical Formula 2]

[0094]

[0095] (Here, EWG is an electron-withdrawing group,

[0096] m is an integer from 0 to 4,

[0097] n is an integer from 0 to 3,

[0098] R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

[0099] ○ (white circle) is a bonding site with L1, and ● (black circle) is a bonding site with E1.).

[0100] A compound of any one of

[17] , [1] to

[16] or a salt thereof, wherein the main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

[0101] A compound of any one of

[18] ,

[14] to

[17] or a salt thereof, wherein the compound represented by the above formula (I-1) is the compound represented by the following formula (I-2):

[0102] A-L1-L2-E1-X-Y-E3-B (I-2)

[0103] [In the formula,

[0104] A, L1, X, Y and B have the same meanings as the corresponding symbols in the above formula (I-1),

[0105] L2 is:

[0106] (a) Ring P-Q-[Here, ring P is selected from the group consisting of an arylene group which may be substituted by an electron-withdrawing group, a heteroarylene group which may be substituted by an electron-withdrawing group, a 2,5-diketopyrrolidine which may be condensed, a 2,6-diketopiperidine which may be condensed, a 2-ketopyrrolidine which may be condensed, a 2-ketopiperidine which may be condensed, and a 2-pyridone group, and Q is selected from the group consisting of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0107] (b) A heteroarylene group; or

[0108] (c)-Q- [where Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0109] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0110] E3 is a divalent group.],

[0111]

[19]

[14] to

[17] The compound or a salt thereof according to any one of, wherein the compound represented by the above formula (I-1) is the compound represented by the following formula (I-3):

[0112] [Chemical formula 3]

[0113]

[0114] [In the formula,

[0115] A, L1, ring Z, and B have the same meanings as the corresponding symbols in the above formula (I-1),

[0116] L2 is:

[0117] (a) Ring P-Q- [where ring P is a group selected from arylene which may be substituted by an electron-withdrawing group, heteroarylene which may be substituted by an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, 2-pyridone, and Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0118] (b) Heteroarylene; or

[0119] (c)-Q- [where Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0120] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0121] E3 is a bond or a divalent group.],

[0122]

[20] A position-selective modification reagent for antibodies based on bioorthogonal functional groups, the modification reagent comprising a compound or a salt thereof having an affinity substance for antibodies and a bioorthogonal functional group represented by the following formula (I):

[0123] A-L-E-B (I)

[0124] [In the formula,

[0125] A is an affinity substance for antibodies,

[0126] L is a divalent group containing a leaving group,

[0127] E is a divalent group containing an electrophilic group, the electrophilic group being: (i) connected to the above-mentioned leaving group and (ii) having the ability to react with a nucleophilic group in the above-mentioned antibody,

[0128] B is a bioorthogonal functional group,

[0129] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0130]

[21] A method for preparing an antibody or a salt thereof having a bioorthogonal functional group, the method comprising:

[0131] Reacting a compound or a salt thereof having an affinity substance for antibodies and a bioorthogonal functional group represented by the following formula (I) with an antibody to generate an antibody or a salt thereof having a bioorthogonal functional group represented by the following formula (II),

[0132] A-L-E-B (I)

[0133] [In the formula,

[0134] A is an affinity substance for antibodies,

[0135] L is a divalent group containing a leaving group,

[0136] E is a divalent group containing an electrophilic group, the electrophilic group being: (i) connected to the above-mentioned leaving group and (ii) having the ability to react with a nucleophilic group in the above-mentioned antibody,

[0137] B is a bioorthogonal functional group,

[0138] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.];

[0139] Ab-E-B (II)

[0140] [In the formula,

[0141] E and B have the same meanings as the corresponding symbols in the above formula (I),

[0142] Ab is an antibody.

[0143]

[22] A method for preparing an antibody or a salt thereof having a functional substance, the method comprising:

[0144] (1) Reacting a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) with an antibody to form an antibody or a salt thereof having a bioorthogonal functional group represented by the following formula (II),

[0145] A-L-E-B (I)

[0146] [In the formula,

[0147] A is an affinity substance for an antibody,

[0148] L is a divalent group containing a leaving group,

[0149] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0150] B is a bioorthogonal functional group,

[0151] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.

[0152] Ab-E-B (II)

[0153] [In the formula,

[0154] E and B have the same meanings as the corresponding symbols in the above formula (I),

[0155] Ab is an antibody.]; and

[0156] (2) Reacting the antibody or a salt thereof having a bioorthogonal functional group represented by the above formula (II) with a functional substance via the bioorthogonal functional group to form an antibody or a salt thereof having a functional substance represented by the following formula (III):

[0157] Ab-E-B’-F (III)

[0158] [In the formula,

[0159] Ab has the same meaning as the corresponding symbol in the above formula (II),

[0160] E has the same meaning as the corresponding symbol in the above formula (I),

[0161] B’ is a divalent group containing a moiety formed by the reaction between a functional substance and a bioorthogonal functional group,

[0162] F is a functional substance.

[0163]

[23] An antibody or a salt thereof having a bioorthogonal functional group selectively at the position represented by the following formula (II-1):

[0164] Ab-E1-E2-E3-B (II-1)

[0165] [In the formula,

[0166] Ab is an antibody,

[0167] E1 is an electrophilic group linked to a nucleophilic group in the antibody,

[0168] E2 is (a) -X-Y- [wherein X linked to E1 is C(R1)(R2) (wherein R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (wherein R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y linked to E3 is C(R4)(R5) (wherein R4 and R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0169] [Chemical formula 4]

[0170]

[0171] (wherein ring Z is a divalent ring group in which the ring-forming atom X' linked to E1 and its two adjacent ring-forming atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' linked to E1 is a nitrogen atom and the two adjacent ring-forming atoms of the nitrogen atom are carbon atoms. · is a bonding site.),

[0172] E3 is a divalent group when E2 is -X-Y-, and is a bond or a divalent group when E2 is a group represented by formula (i),

[0173] B is a bioorthogonal functional group.

[0174]

[24] The antibody or a salt thereof according to

[23] , wherein the above antibody is an antibody having a bioorthogonal functional group only in the constant region of a monoclonal antibody.

[0175]

[25] The antibody or a salt thereof according to

[23] or

[24] , wherein the above antibody is an antibody having a bioorthogonal functional group only in the Fc region of a monoclonal antibody.

[0176] An antibody or a salt thereof according to any one of

[26] to

[25] , wherein the antibody is a human IgG having a bioorthogonal functional group selectively at a region composed of amino acid residues at positions 246 to 248 or 288 to 290 in the human IgG Fc region.

[0177]

[27] An antibody or a salt thereof according to any one of

[23] to

[26] , wherein the antibody represented by the above formula (II-1) is an antibody selectively having a bioorthogonal functional group represented by the following formula (II-2):

[0178] Ab-E1-X-Y-E3-B (II-2)

[0179] [In the formula,

[0180] Ab, X, Y, and B have the same meanings as the corresponding symbols in the above formula (II-1),

[0181] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0182] E3 is a divalent group. ].

[0183]

[28] An antibody or a salt thereof according to any one of

[23] to

[26] , wherein the antibody represented by the above formula (II-1) is an antibody selectively having a bioorthogonal functional group represented by the following formula (II-3):

[0184] [Chemical formula 5]

[0185]

[0186] [In the formula,

[0187] Ab, the ring-constituting atom X', the ring Z, and B have the same meanings as the corresponding symbols in the above formula (II-1),

[0188] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0189] E3 is a bond or a divalent group. ].

[0190]

[29] An antibody or a salt thereof selectively having a functional substance represented by the following formula (III-1):

[0191] Ab-E1-E2-E3-B'-F (III-1)

[0192] [In the formula,

[0193] Ab is an antibody,

[0194] E1 is an electrophilic group that binds to a nucleophilic group in the antibody.

[0195] E2 is (a) -X-Y- [where X that binds to E1 is C(R1)(R2) (where R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y that binds to E3 is C(R4)(R5) (where R4 and R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0196] [Chemical formula 6]

[0197]

[0198] (wherein ring Z is a divalent ring group in which the ring-forming atom X' that binds to E1 and its two adjacent ring-forming atoms are both carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' that binds to E1 is a nitrogen atom and the two adjacent ring-forming atoms of the nitrogen atom are carbon atoms. · represents a bonding site.)

[0199] E3 is a divalent group when E2 is -X-Y-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0200] B' is a divalent group containing a part formed by the reaction between a functional substance and a bioorthogonal functional group.

[0201] F is a functional substance.]

[0202] The antibody of

[30]

[29] or a salt thereof, wherein the above antibody is an antibody having a bioorthogonal functional group only in the constant region of a monoclonal antibody.

[0203] The antibody of

[31]

[29] or

[30] or a salt thereof, wherein the above antibody is an antibody having a bioorthogonal functional group only in the Fc region of a monoclonal antibody.

[0204] The antibody of any one of

[32]

[29] to

[31] or a salt thereof, wherein the above antibody is a human IgG having a functional substance selectively at a position in the region composed of amino acid residues at positions 246 to 248 or 288 to 290 in the human IgG Fc region.

[0205] The antibody of any one of

[33]

[29] to

[32] or a salt thereof, wherein the above antibody represented by formula (III-1) is an antibody having a functional substance selectively at a position represented by the following formula (III-2):

[0206] Ab-E1-X-Y-E3-B’-F (III-2)

[0207] [In the formula,

[0208] Ab, X, Y, B’ and F have the same meanings as the corresponding symbols in the above formula (III-1),

[0209] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0210] E3 is a divalent group].

[0211]

[34]

[29] to

[32] The antibody or its salt according to any one of them, wherein the antibody represented by the above formula (III-1) is an antibody having a functional substance selectively at the position represented by the following formula (III-3):

[0212] [Chemical formula 7]

[0213]

[0214] [In the formula,

[0215] Ab, ring-constituting atom X’, ring Z, B’ and F have the same meanings as the corresponding symbols in the above formula (III-1),

[0216] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0217] E3 is a bond or a divalent group.].

[0218]

[35] A compound or its salt having an affinity substance for an antibody and a functional substance represented by the following formula (IV):

[0219] A-L-E-F (IV)

[0220] [In the formula,

[0221] A is an affinity substance for an antibody,

[0222] L is a divalent group containing a leaving group,

[0223] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group, and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0224] F is a functional substance,

[0225] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0226]

[36] A position-selective modifying reagent for an antibody based on a functional substance, the modifying reagent comprising a compound or a salt thereof represented by the following formula (IV) and having an affinity substance for an antibody and a functional substance:

[0227] A-L-E-F (IV)

[0228] [In the formula,

[0229] A is an affinity substance for an antibody,

[0230] L is a divalent group containing a leaving group,

[0231] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0232] F is a functional substance,

[0233] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0234]

[37] A method for preparing an antibody or a salt thereof having a functional substance, the method comprising:

[0235] Reacting a compound or a salt thereof represented by the following formula (IV) and having an affinity substance for an antibody and a functional substance with an antibody to generate an antibody or a salt thereof represented by the following formula (III) and having a functional substance:

[0236] A-L-E-F (IV)

[0237] [In the formula,

[0238] A is an affinity substance for an antibody,

[0239] L is a divalent group containing a leaving group,

[0240] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0241] F is a functional substance,

[0242] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.];

[0243] Ab-E-F (III)

[0244] [In the formula,

[0245] Ab is an antibody,

[0246] E and F have the same meanings as the corresponding symbols in the above formula (IV).

[0247] Advantages of the Invention

[0248] The compound or its salt of the present invention having an affinity substance for an antibody and a bioorthogonal functional group or a functional substance can be used, for example, for the site-selective modification of an antibody.

[0249] The antibody or its salt of the present invention having a bioorthogonal functional group site-selectively can be used, for example, as an intermediate in the preparation of an antibody or its salt having a functional substance site-selectively.

[0250] The antibody or its salt of the present invention having a functional substance site-selectively can be used, for example, as a drug or a reagent (for example, a diagnostic agent, a research reagent). Brief Description of the Drawings

[0251] Figure 1 Figure 1 is a schematic diagram showing the outline of the present invention.

[0252] Figure 2 Figure 2 is a graph showing the results of SDS-PAGE-based analysis in the synthesis of the IgG antibody trastuzumab-peptide complex. Lanes 1, 3, 6, 8: molecular weight markers; Lane 2: unreacted IgG antibody trastuzumab (control, the band around 50,000 molecular weight shows the heavy chain, and the band around 25,000 molecular weight shows the light chain); Lane 4: the complex formed by IgG antibody trastuzumab and compound 10 (the band above around 50,000 molecular weight shows that compound 10 is conjugated to the heavy chain of trastuzumab. The band below around 50,000 molecular weight shows the unreacted heavy chain, and the band around 25,000 molecular weight shows the unreacted light chain); Lane 5: the complex formed by IgG antibody trastuzumab and compound 11 (the band above around 50,000 molecular weight shows that compound 11 is conjugated to the heavy chain of trastuzumab. The band below around 50,000 molecular weight shows the unreacted heavy chain, and the band around 25,000 molecular weight shows the unreacted light chain); Lane 7: the reaction mixture after conjugation of IgG antibody trastuzumab and compound 12 (the band around 50,000 molecular weight shows the unreacted heavy chain, and the band around 25,000 molecular weight shows the unreacted light chain. No band conjugated with compound 12 was seen).

[0253] Figure 3 Figure 3 ​​​​​​This is a figure showing the SDS-PAGE analysis results of a trastuzumab-peptide complex synthesized by site-specifically introducing maleimide into IgG antibody trastuzumab and then coupling it with a thiol-containing peptide reagent. Lanes 1 and 9: molecular weight markers. Lane 2: a complex obtained by treating IgG antibody trastuzumab with compound 22 (10 molar equivalents relative to the antibody), then site-selectively introducing maleimide, and then coupling compound 25. The bands above around 50,000 molecular weight show that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25. The bands below around 50,000 molecular weight show unreacted heavy chains, and the bands around 25,000 molecular weight show unreacted light chains. Lane 3: a complex obtained by treating IgG antibody trastuzumab with compound 22 (20 molar equivalents relative to the antibody), then site-selectively introducing maleimide, and then coupling compound 25. The bands above around 50,000 molecular weight show that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25. The bands below around 50,000 molecular weight show unreacted heavy chains, and the bands around 25,000 molecular weight show unreacted light chains. Lane 4: a complex obtained by treating IgG antibody trastuzumab with compound 22 (40 molar equivalents relative to the antibody), then site-selectively introducing maleimide, and then coupling compound 25. The bands above around 50,000 molecular weight show that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25. The bands below around 50,000 molecular weight show unreacted heavy chains, and the bands around 25,000 molecular weight show unreacted light chains. Lane 5: a complex obtained by treating IgG antibody trastuzumab with compound 23 (10 molar equivalents relative to the antibody), then site-selectively introducing maleimide, and then coupling compound 25. The bands above around 50,000 molecular weight show that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25. The bands below around 50,000 molecular weight show unreacted heavy chains, and the bands around 25,000 molecular weight show unreacted light chains. Lane 6: a complex obtained by treating IgG antibody trastuzumab with compound 23 (20 molar equivalents relative to the antibody), then site-selectively introducing maleimide, and then coupling compound 25. The bands above around 50,000 molecular weight show that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25. The bands below around 50,000 molecular weight show unreacted heavy chains, and the bands around 25,000 molecular weight show unreacted light chains. Lane 7: a complex obtained by treating IgG antibody trastuzumab with compound 23 (40 molar equivalents relative to the antibody), then site-selectively introducing maleimide, and then coupling compound 25. The bands above around 50,000 molecular weight show that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25.Bands below around 50,000 in molecular weight show unreacted heavy chains, and bands around 25,000 in molecular weight show unreacted light chains. Lanes 8 and 10: Unreacted IgG antibody trastuzumab (control, bands around 50,000 in molecular weight show heavy chains, and bands around 25,000 in molecular weight show light chains). Lane 11: Reaction mixture obtained by treating IgG antibody trastuzumab with Compound 24 (10 molar equivalents relative to the antibody) and then adding Compound 25. Bands around 50,000 in molecular weight show unreacted heavy chains, and bands around 25,000 in molecular weight show unreacted light chains. Lane 12: Reaction mixture obtained by treating IgG antibody trastuzumab with Compound 24 (20 molar equivalents relative to the antibody) and then adding Compound 25. Bands around 50,000 in molecular weight show unreacted heavy chains, and bands around 25,000 in molecular weight show unreacted light chains. Lane 13: Reaction mixture obtained by treating IgG antibody trastuzumab with Compound 24 (40 molar equivalents relative to the antibody) and then adding Compound 25. Bands around 50,000 in molecular weight show unreacted heavy chains, and bands around 25,000 in molecular weight show unreacted light chains.

[0254] Figure 4 Figure 4 It is a graph showing ESI-TOFMS under reduced conditions of the trastuzumab-maleimide modifier synthesized in (4-5-1). The upper part shows the measurement results of unreacted trastuzumab, and the lower part shows the modifier.

[0255] Figure 5 Figure 5 It is a graph showing ESI-TOFMS under reduced conditions of the trastuzumab-azide modifier (azide-modified antibody 1) synthesized in (6-1-1). The lower part shows the measurement results of unreacted trastuzumab, and the upper part shows the modifier.

[0256] Figure 6 Figure 6 It is a graph showing ESI-TOFMS under reduced conditions of the trastuzumab-azide modifier (azide-modified antibody 3) synthesized in (6-1-2). The lower part shows the measurement results of unreacted trastuzumab, and the upper part shows the modifier.

[0257] Figure 7 Figure 7 It is a graph showing ESI-TOFMS under reduced conditions of the trastuzumab-azide modifier (azide-modified antibody 6) synthesized in (6-1-3). The upper part shows the measurement results of unreacted trastuzumab, and the lower part shows the modifier.

[0258] Figure 8 Figure 8 ​​​​​​​​​​It is a graph of ESI-TOFMS under reduced conditions of the trastuzumab-azide modifier (azide-modified antibody 8) synthesized in (6-1-3). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0259] Figure 9 Figure 9 It is a graph of ESI-TOFMS under reduced conditions of the trastuzumab-azide modifier (azide-modified antibody 10) synthesized in (6-1-3). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0260] Figure 10 Figure 10 It is a graph of ESI-TOFMS under reduced conditions of the adalimumab-azide modifier (azide-modified antibody 28) synthesized in (6-1-4). The upper section shows the measurement results of unreacted adalimumab, and the lower section shows the modifier.

[0261] Figure 11 Figure 11 It is a graph of ESI-TOFMS under reduced conditions of the denosumab-azide modified antibody (azide-modified antibody 29) synthesized in (6-1-4). The upper section shows the measurement results of unreacted denosumab, and the lower section shows the modifier.

[0262] Figure 12 Figure 12 It is a graph of ESI-TOFMS under reduced conditions of the dupilumab-azide modified antibody (azide-modified antibody 30) synthesized in (6-1-4). The upper section shows the measurement results of unreacted dupilumab, and the lower section shows the modifier.

[0263] Figure 13 Figure 13 It is a graph of ESI-TOFMS under reduced conditions of the rituximab-azide modified antibody (azide-modified antibody 31) synthesized in (6-1-4). The lower section shows the measurement results of unreacted rituximab, and the upper section shows the modifier.

[0264] Figure 14 Figure 14 It is a graph of ESI-TOFMS under reduced conditions of the trastuzumab-protected thiol modifier synthesized in (8-1-1). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0265] Figure 15 Figure 15 ​​​​​​​​​​​​​​It is a graph of ESI-TOFMS under reduced conditions of the deprotected trastuzumab-thiol modifier in (8-3-1). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0266] Figure 16 Figure 16 It is a graph of ESI-TOFMS under reduced conditions of the synthesized trastuzumab-azide modifier in (9-1-1). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0267] Figure 17 Figure 17 It is a graph of ESI-TOFMS under reduced conditions of the synthesized trastuzumab-azide modifier in (9-1-5). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0268] Figure 18 Figure 18 It is a graph of ESI-TOFMS under reduced conditions of the synthesized trastuzumab-azide modifier in (9-2-2). The upper section shows the measurement results of unreacted trastuzumab, and the lower section shows the modifier.

[0269] Figure 19 Figure 19 It is a graph of ESI-TOFMS of the synthesized trastuzumab-Cy3 complex in (10-1-1). The lower section shows the measurement results of unreacted trastuzumab, the middle section shows the measurement results of the trastuzumab-azide modifier synthesized in (6-1-1), and the upper section shows the trastuzumab-Cy3 complex.

[0270] Figure 20 Figure 20 It is a graph of ESI-TOFMS under reduced conditions of the processed trastuzumab-Cy3 complex in (10-1-2). The lower section shows the measurement results of unreacted trastuzumab, the middle section shows the measurement results of the trastuzumab-azide modifier synthesized in (6-1-1), and the upper section shows the trastuzumab-Cy3 complex.

[0271] Figure 21 Figure 21 It is a graph of ESI-TOFMS of the synthesized trastuzumab-peptide complex in (10-2-2). The lower section shows the measurement results of unreacted trastuzumab, the middle section shows the measurement results of the trastuzumab-azide modifier synthesized in (6-1-1), and the upper section shows the trastuzumab-Cy3 complex.

[0272] Figure 22 Figure 22 ​​​​​​​​​​​​​​It is a diagram of ESI-TOFMS of the trastuzumab-Cy3 complex processed in (10-2-3) under reducing conditions. The lower section shows the measurement results of unreacted trastuzumab, the middle section shows the measurement results of the trastuzumab-azide modifier synthesized in (6-1-1), and the upper section shows the trastuzumab-peptide complex.

[0273] Figure 23 Figure 23 It is a diagram of ESI-TOFMS of the trastuzumab-maleimide compound complex processed in (10-3-1) under reducing conditions. The upper section shows the measurement results of the thiol-introduced body of trastuzumab, and the lower section shows the trastuzumab-maleimide compound complex.

[0274] Figure 24 Figure 24 It is a diagram of ESI-TOFMS of the reaction product processed in (10-3-2) under reducing conditions. The upper section shows the measurement results of the thiol-introduced body of trastuzumab, and the lower section shows the trastuzumab-maleimide compound complex.

[0275] Figure 25 Figure 25 It is a diagram showing (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO:2), (2) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO:4).

[0276] Figure 26 Figure 26 It is a diagram showing (1) the amino acid sequence of the heavy chain of denosumab (SEQ ID NO:104), (2) the amino acid sequence of the light chain of denosumab (SEQ ID NO:105).

[0277] Figure 27 Figure 27 It is a diagram showing (1) the amino acid sequence of the heavy chain of dupilumab (SEQ ID NO:106), (2) the amino acid sequence of the light chain of dupilumab (SEQ ID NO:107).

[0278] Figure 28 Figure 28 It is a diagram of the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue of trastuzumab modified with azidobenzoic acid by trypsin digestion.

[0279] Figure 29 Figure 29 ​​​​​​​​​​​​​​It is a figure showing the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0280] Figure 30 Figure 30 It is a figure showing the MS spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO:12) containing the modification site of lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0281] Figure 31 Figure 31 It is a figure showing the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO:12) containing the modification site of lysine residue (aminobenzoic acid adduct (+119.037Da)) by trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0282] Figure 32 Figure 32 It is a figure showing the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ IDNO:11) containing the modification site of lysine residue by trypsin digestion of maleimide-modified trastuzumab added with mercaptopropionic acid.

[0283] Figure 33 Figure 33 It is a figure showing the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue by trypsin digestion of maleimide-MPA-modified trastuzumab.

[0284] Figure 34 Figure 34 It is a figure showing the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue by trypsin digestion of alkyl azide-modified trastuzumab.

[0285] Figure 35 Figure 35 ​​​​​​​​​​​​It is a figure showing the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site for lysine residues of alkyl azide-modified trastuzumab digested with trypsin.

[0286] Figure 36 Figure 36 It is a figure showing the MS spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (SEQ ID NO:101) containing the modification site for lysine residues of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0287] Figure 37 Figure 37 It is a figure showing the CID spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (SEQ ID NO:101) containing the modification site for lysine residues of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0288] Figure 38 Figure 38 It is a figure showing the analysis result based on BioPharmaFinder that the lysine residue at position 317 of azidobenzoic acid-modified trastuzumab is highly selectively modified.

[0289] Figure 39 Figure 39 It is a figure showing the MS spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO:12) containing the modification site for lysine residues of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0290] Figure 40 Figure 40 It is a figure showing the CID spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO:12) containing the modification site for lysine residues of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0291] Figure 41 Figure 41 It is a figure showing the analysis result based on BioPharma Finder that the lysine residue at position 288 or 290 of azidobenzoic acid-modified trastuzumab is highly selectively modified.

[0292] Figure 42 Figure 42 ​​​​​​​​​​​​​​It is a figure showing the MS spectrum of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing the modification site of lysine residue after trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0293] Figure 43 Figure 43 It is a figure showing the CID spectrum of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing the modification site of lysine residue after trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0294] Figure 44 Figure 44 It is a figure showing the analysis result based on BioPharma Finder that the lysine residues at positions 246 or 248 of azidobenzoic acid-modified trastuzumab are highly selectively modified.

[0295] Figure 45 Figure 45 It is a figure showing the MS spectrum of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing the modification site of lysine residue after trypsin digestion of acetylthiol-modified trastuzumab.

[0296] Figure 46 Figure 46 It is a figure showing the CID spectrum of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing the modification site of lysine residue after trypsin digestion of acetylthiol-modified trastuzumab.

[0297] Figure 47 Figure 47 It is a figure showing the analysis result based on BioPharma Finder that the lysine residues at positions 246 or 248 of acetylthiol-modified trastuzumab are highly selectively modified.

[0298] Figure 48 Figure 48 It is a figure showing the MS spectrum of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing the modification site of lysine residue after trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0299] Figure 49 Figure 49 ​​​​​​​​​​​​​​It is a graph showing the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue after trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0300] Figure 50 Figure 50 It is a graph showing the MS spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO:12) containing the modification site of lysine residue after trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0301] Figure 51 Figure 51 It is a graph showing the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKT KPR (SEQ ID NO:12) containing the modification site of lysine residue after trypsin digestion of azidobenzoic acid-modified trastuzumab.

[0302] Figure 52 Figure 52 It is a graph showing the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue after trypsin digestion of acetylthiol- and azidobenzoic acid-modified trastuzumab.

[0303] Figure 53 Figure 53 It is a graph showing the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO:11) containing the modification site of lysine residue after trypsin digestion of acetylthiol- and azidobenzoic acid-modified trastuzumab.

[0304] Figure 54 Figure 54 It is a graph showing the MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO:12) containing the modification site of lysine residue after trypsin digestion of acetylthiol- and azidobenzoic acid-modified trastuzumab.

[0305] Figure 55 Figure 55 It is a graph showing the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO:12) containing the modification site of lysine residue after trypsin digestion of acetylthiol- and azidobenzoic acid-modified trastuzumab.

[0306] ​​​​​​​​​​​​​Figure 56 Figure 56 It is a figure showing the analysis results based on BioPharmaFinder that the lysine residues at positions 246 or 248 and 288 or 290 of trastuzumab modified with acetylthiol and azidobenzoic acid are highly selectively modified.

[0307] Figure 57 Figure 57 It is a figure showing the MS spectrum of the peptide fragment CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR (SEQ ID NO: 102) containing the modification sites of lysine residues of denosumab modified with benzoic acid and digested with trypsin.

[0308] Figure 58 Figure 58 It is a figure showing the CID spectrum of the peptide fragment CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR (SEQ ID NO: 102) containing the modification sites of lysine residues of denosumab modified with benzoic acid and digested with trypsin.

[0309] Figure 59 Figure 59 It is a figure showing the analysis results based on BioPharma Finder that the lysine residues at positions 247 or 249 of denosumab modified with benzoic acid are highly selectively modified.

[0310] Figure 60 Figure 60 It is a figure showing the MS spectrum of the peptide fragment YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 103) containing the modification sites of lysine residues of dupilumab modified with benzoic acid and digested with trypsin.

[0311] Figure 61 Figure 61 It is a figure showing the CID spectrum of the peptide fragment YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 103) containing the modification sites of lysine residues of dupilumab modified with benzoic acid and digested with trypsin.

[0312] Figure 62 Figure 62 It is a figure showing the analysis results based on BioPharma Finder that the lysine residues at positions 251 or 253 of dupilumab modified with benzoic acid are highly selectively modified.

[0313] Figure 63 Figure 63 ​​​​​​​​​​​​​​​It is a figure showing the analysis results of the trastuzumab-DM1 conjugate synthesized in (12-1-1) based on ESI-TOFMS (under non-reducing conditions).

[0314] Figure 64 Figure 64 It is a figure showing the analysis results of the trastuzumab-DM1 conjugate synthesized in (12-1-1) based on ESI-TOFMS (under reducing conditions).

[0315] Figure 65 Figure 65 It is a figure showing the analysis results of the trastuzumab-MMAE conjugate synthesized in (12-2-1) based on ESI-TOFMS (under non-reducing conditions).

[0316] Figure 66 Figure 66 It is a figure showing the analysis results of the trastuzumab-MMAE conjugate synthesized in (12-2-1) based on ESI-TOFMS (under reducing conditions).

[0317] Figure 67 Figure 67 It is a figure showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-3-1) based on ESI-TOFMS (under non-reducing conditions).

[0318] Figure 68 Figure 68 It is a figure showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-3-1) based on ESI-TOFMS (under reducing conditions).

[0319] Figure 69 Figure 69 It is a figure showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-4-1) based on ESI-TOFMS (under non-reducing conditions).

[0320] Figure 70 Figure 70 It is a figure showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-4-1) based on ESI-TOFMS (under reducing conditions).

[0321] Figure 71 Figure 71 It is a figure showing (1) the common amino acid sequence (SEQ ID NO:1) of the Fc region in the heavy chain of trastuzumab and the IgG1 Fc region, and (2) the amino acid sequence (SEQ ID NO:3) of the IgG1 Fc region. Detailed implementation mode

[0322] ​​​​​​​​​​​​​​​​1. A compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group

[0323] 1-1. Summary

[0324] The present invention provides a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by formula (I):

[0325] A-L-E-B (I)

[0326] [In the formula,

[0327] A is an affinity substance for an antibody,

[0328] L is a divalent group containing a leaving group,

[0329] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group, and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0330] B is a bioorthogonal functional group,

[0331] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0332] In the writing of formula (I) and other formulas shown in connection with the present invention, the dash (-) indicates that two units on both sides thereof are covalently bonded. Therefore, in formula (I), A is covalently bonded to L, L is covalently bonded to A and E, E is covalently bonded to L and B, and B is covalently bonded to E. The compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by formula (I) shows that the affinity substance for an antibody (A) contains a structural unit having the structure of L-E-B through the covalent bonding between A and L. Therefore, in formula (I), the affinity substance for an antibody (A) may have one structural unit having the structure of L-E-B or multiple (for example, 2 to 5, preferably 2 to 4, more preferably 2 or 3) structural units having the structure of L-E-B (the same or different).

[0333] It is also shown in other formulas that the affinity substance for an antibody (A) or the antibody (Ab) contains a specific structural unit (a structural unit other than A or Ab) in the formula through covalent bonding. Therefore, in other formulas, the affinity substance for an antibody (A) or the antibody (Ab) may also have one specific structural unit or multiple (for example, 2 to 5, preferably 2 to 4, more preferably 2 or 3) specific structural units (the same or different).

[0334] 1-2. Affinity substance for an antibody (A)

[0335] In formula (I), A is an affinity substance for an antibody. An affinity substance for an antibody refers to a substance having a binding ability based on non-covalent bonding to an antibody.

[0336] The affinity substance used in the present invention targets an antibody. The antibody can be an antibody modified with a biomolecule (such as a sugar) or an antibody not modified with a biomolecule. As the antibody, any antibody against any component such as a component from a living organism, a component from a virus, and a component visible in the environment can be used, but an antibody against a component from a living organism or a component from a virus is preferred. As a component from a living organism, for example, components (such as proteins) from animals such as mammals, birds (such as chickens), insects, microorganisms, plants, fungi, and fish can be mentioned. A component from a living organism is preferably a component from a mammal. As mammals, for example, primates (such as humans, monkeys, chimpanzees), rodents (such as mice, rats, guinea pigs, hamsters, rabbits), pets (such as dogs, cats), livestock (such as cows, pigs, goats), draft animals (such as horses, sheep) can be mentioned. A component from a living organism is more preferably a component (such as a protein) from a primate or a rodent, and from the viewpoint of the clinical application of the present invention, it is even more preferably a component (such as a protein) from a human. As a component from a virus, for example, components (such as proteins) from influenza viruses (such as avian influenza virus, swine influenza virus), human immunodeficiency virus, Ebola virus, bacteriophage virus can be mentioned.

[0337] In addition, the antibody is a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody is preferred. As monoclonal antibodies, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies attached with a specified sugar chain (such as antibodies modified in a manner having a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), bispecific antibodies, scFv antibodies, Fab antibodies, F(ab’)2 antibodies, VHH antibodies, Fc region proteins, Fc fusion proteins can be mentioned. The antibody can also be a divalent antibody (such as IgG, IgD, IgE) or an antibody with a valence of four or more (such as IgA antibody, IgM antibody).

[0338] The antibody that is the target of the affinity substance may also be composed of any amino acid residue, preferably composed of the 20 natural L-α-amino acid residues that usually constitute proteins. Examples of such amino acid residues include: L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H) or L-lysine (K) and glycine (G) (hereinafter, the writing of L is omitted). The antibody may be composed of, for example, 100 or more, preferably 120 or more, more preferably 150 or more, still more preferably 180 or more, particularly preferably 200 or more amino acid residues. The antibody may also be, for example, 1000 or less, preferably 900 or less, more preferably 800 or less, still more preferably 700 or less, particularly preferably 600 or less. More specifically, the antibody may be composed of, for example, 100 to 1000, preferably 120 to 900, more preferably 150 to 800, still more preferably 180 to 700 or more, particularly preferably 200 to 600 amino acid residues. In the case where the antibody is an antibody (such as the above-mentioned monoclonal antibody), the amino acid residues of the above number may correspond to the amino acid residues of the heavy chain of the antibody.

[0339] The antibody that is the target of the affinity substance may also be a protein that contains, at one or more positions (preferably multiple positions), a specific amino acid residue having a side chain or a terminus (N-terminus and / or C-terminus), preferably a side chain, that can react with a bioorthogonal functional group as described later. Examples of such specific amino acid residues include the amino acid residues described later, and preferably amino acid residues selected from lysine residues, tyrosine residues, serine residues, threonine residues, and cysteine residues. Considering that the compounds of the present invention can modify antibodies in a position-selective manner, antibodies that contain such specific amino acid residues at multiple positions are preferred. As the multiple positions, any two or more positions are acceptable and there is no particular limitation. For example, they can be three or more positions, preferably five or more positions, more preferably ten or more positions, still more preferably twenty or more positions, and particularly preferably thirty or more positions. The multiple positions can also be, for example, 200 or fewer positions, preferably 180 or fewer positions, more preferably 150 or fewer positions, still more preferably 120 or fewer positions, and particularly preferably 100 or fewer positions. More specifically, the multiple positions can be, for example, 3 to 200 positions, preferably 5 to 180 positions, more preferably 10 to 150 positions, still more preferably 20 to 120 positions, and particularly preferably 30 to 100 positions. Even for antibodies that contain such specific amino acid residues at multiple positions, the compounds of the present invention can modify one or two specific amino acid residues present in a specific region in a position-selective manner. For example, the number of lysine residues in human IgG1, although also depending on the amino acid composition in the variable region, is generally considered to be around 70 to 90. In the present invention, it has been successfully achieved to modify one or two lysine residues present in a specific region of human IgG1 in a position-selective manner.

[0340] More specifically, in the present invention, from the viewpoint of modifying the amino acid residues at specific target sites present in an antibody while maintaining the antibody function (i.e., while maintaining the native folding without denaturing the antibody), position-selective modification of the amino acid residues exposed on the surface of the antibody is preferred. For example, in human IgG such as human IgG1, exposed lysine residues and exposed tyrosine residues are present at the following positions (based on EU numbering, refer to http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0341] (1) Exposed lysine residues

[0342] CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322, 338)

[0343] CH3 domain (at positions 360, 414, 439)

[0344] (2) Exposure of tyrosine residue

[0345] CH2 domain (at positions 278, 296, 300)

[0346] CH3 domain (at position 436)

[0347] (3) Exposure of serine residue

[0348] CH2 domain (at positions 254, 267, 298, 324)

[0349] CH3 domain (at positions 375, 400, 415, 440, 442)

[0350] (4) Exposure of threonine residue

[0351] CH2 domain (at positions 256, 289, 307)

[0352] CH3 domain (at positions 335, 359, 393, 437)

[0353] Therefore, for human IgG1 and other human IgG, when lysine residue or tyrosine residue is modified, modification at the above positions is preferred.

[0354] Preferably, for human IgG1 and other human IgG, when lysine residue, tyrosine residue, serine residue or threonine residue is modified, among the above (1) - (4) positions, it can be the lysine residue, tyrosine residue, serine residue or threonine residue existing at the following positions with high surface exposure that is modified.

[0355] (1’) Exposure of lysine residue

[0356] CH2 domain (at positions 246, 248, 274, 288, 290, 317, 320, 322)

[0357] CH3 domain (at positions 360, 414, 439)

[0358] (2’) Exposure of tyrosine residue

[0359] CH2 domain (at positions 278, 296, 300)

[0360] CH3 domain (at position 436)

[0361] (3’) Exposure of serine residue

[0362] CH2 domain (at positions 254, 267, 298)

[0363] CH3 domain (at positions 400, 415, 440)

[0364] (4’) exposes threonine residue

[0365] CH2 domain (at positions 256, 289)

[0366] CH3 domain (at positions 335, 359)

[0367] Therefore, for human IgG1 and other human IgGs, when lysine residues, tyrosine residues, serine residues or threonine residues are modified, modification at the above positions is more preferred.

[0368] More preferably, for human IgG1 and other human IgGs, when lysine residues are modified, among the positions in the above (1), the lysine residues present at the specified positions (such as positions 246, 248, 288, 290, 317) in the CH2 domain that can be effectively modified in the present invention can be modified.

[0369] In a specific embodiment, for an antibody that is a target of an affinity substance, when it contains specific amino acid residues at multiple positions as described above, it may contain 1 or more specific amino acid residues in a target region composed of 1 to 50 consecutive amino acid residues, and contain 5 or more specific amino acid residues in a non-target region other than the target region. The target region may preferably be composed of 1 to 30, more preferably 1 to 20, further preferably 1 to 10, 1 to 5 or 1 to 3 (i.e., 1, 2 or 3) amino acid residues. Particularly preferably, the target region may be a region composed of specific amino acid residues present at specific positions. Such specific positions also vary depending on the types of the target protein and the affinity substance, etc. For example, it may be a specific position in a specific region (such as CH1, CH2, CH3) in the constant region of the antibody, and preferably may be a position in the CH2 of the antibody. More specifically, the target region may be the following residues based on EU numbering in human IgG Fc:

[0370] (1) Lys248 residue (hereinafter, also simply written as "Lys248" in this specification, corresponding to the 18th residue of the human IgG CH2 region (SEQ ID NO:1)) or Lys246 residue (hereinafter, also simply written as "Lys246" in this specification, corresponding to the 16th residue of the human IgG CH2 region (SEQ ID NO:1));

[0371] (2) The Lys288 residue (hereinafter, also simply written as "Lys288" in this specification, corresponding to the 58th residue of the human IgG CH2 region (SEQ ID NO: 1)) or the Lys290 residue (hereinafter, also simply written as "Lys290" in this specification, corresponding to the 60th residue of the human IgG CH2 region (SEQ ID NO: 1));

[0372] (3) The Lys317 residue (hereinafter, also simply written as "Lys317" in this specification, corresponding to the 87th residue of the human IgG CH2 region (SEQ ID NO: 1)).

[0373] According to the present invention, specific amino acid residues in the above-mentioned target region can be modified with high site selectivity. Such site selectivity can be, for example, 30% or more, preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0374] The target region can also be a region in which, in the regions up to a (where a is any integer from 1 to 10) amino acid residues from the N-terminal side and the C-terminal side, respectively, centered on a specific position where a specific amino acid residue exists, there are no amino acid residues of the same type as the specific amino acid residue except for the specific amino acid residue existing at that specific position. a is preferably an integer from 1 to 5, more preferably an integer from 1 to 3, still more preferably 1 or 2, and particularly preferably 1.

[0375] In a preferred embodiment, the antibody is a monoclonal antibody. As isotypes of antibodies such as monoclonal antibodies, for example, IgG (such as IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY can be mentioned. The monoclonal antibody is a full-length antibody or an antibody fragment (such as F(ab’)2, Fab’, Fab, Fv, single-chain antibody), but a full-length antibody is preferred. Particularly preferably, the antibody is a human antibody, a humanized antibody, or a chimeric antibody having a human IgG (such as IgG1, IgG2, IgG3, IgG4) in the constant region.

[0376] The antibody is an antibody against any antigen. For example, such an antigen can be a component found in the above-mentioned organisms or viruses. As such an antigen, for example, proteins [including oligopeptides, polypeptides. It can be a protein modified with biomolecules such as sugars (such as glycoproteins)], sugar chains, nucleic acids, and low-molecular compounds can also be mentioned.

[0377] Preferably, the antibody can be an antibody against a protein antigen. Examples of the protein include: cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, soluble receptors.

[0378] More specifically, the protein that is the antigen of the antibody can be a disease target protein. Examples of the disease target protein include the following proteins.

[0379] (1) Cancer field

[0380] 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, Folic Acid 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, Folic Acid Receptor α, TEM-1, GM2, Glypican-3, Macrophage Inhibitory Factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, Frizzled7 Receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-Cadherin, CEA, GITR, TAM (Tumor-Associated Macrophage,tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2, CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, Tissue Factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transferrin Receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lewis Blood Group B Antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, Integrin α9, TAG-72 (CA72-4), CD70.,

[0381] (2) Autoimmune diseases - Inflammatory diseases

[0382] IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, integrin β7 (β7-Integrin), IL-4αR, HAS, eosinophil chemotactic factor 1 (Eotaxin-1), CD3, CD19, TNF-α, IL-15, CD3ε, fibronectin, IL-1β, IL-1α, IL-17, TSLP (thymic stromal lymphopoietin), LAMP (integrin α4β7), 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 ligand, IL-21, cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, integrin α9, LIFHT.

[0383] (3) Cranial nerve diseases

[0384] CGRP, CD20, β-amyloid, β-amyloid fibril, 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.

[0385] (4) Infectious diseases

[0386] Clostridium Difficile toxin B, cytomegalovirus, RS virus, LPS, S.Aureus Alpha-toxin, M2e protein, Psl, PcrV, S.Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae.

[0387] (5) Hereditary - Rare Diseases

[0388] Amyloid protein AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin.

[0389] (6) Eye Diseases

[0390] Factor D, IGF - 1R, PGDFR, Ang2, VEGF - A, CD - 105 (Endoglin), IGF - 1R, β - amyloid protein.

[0391] (7) Orthopedics - Plastic Surgery Field

[0392] Sclerostin, Myostatin, Dickkopf - 1, GDF8, RNAKL, HAS, Siglec - 15.

[0393] (8) Blood Diseases

[0394] vWF, Factor IXa, Factor X, IFNγ, C5, BMP - 6, Ferroportin, TFPI.

[0395] (9) Other Diseases

[0396] 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.

[0397] In a more preferred embodiment, the affinity substance for the antibody is an affinity substance for a monoclonal antibody. The isotype of the monoclonal antibody is the same as that of the antibody above, but preferably IgG (such as IgG1, IgG2, IgG3, IgG4). Preferably, the monoclonal antibody is a full - length monoclonal antibody.

[0398] In an even more preferred embodiment, the affinity substance for the antibody is an affinity substance for a chimeric antibody, humanized antibody, or human antibody (such as IgG1, IgG2, IgG3, IgG4, etc. IgG) that is a full - length monoclonal antibody.

[0399] In a particularly preferred embodiment, the affinity substance for the antibody is an affinity substance for the antibody, and the antibody contains any one of the following Fc region proteins selected from (A) to (C) and has antigen-binding ability:

[0400] (A) An Fc region protein containing the amino acid sequence of SEQ ID NO:1;

[0401] (B) An Fc region protein containing an amino acid sequence obtained by inserting, adding, deleting, or substituting one or more amino acid residues in the amino acid sequence of SEQ ID NO:1; or

[0402] (C) An Fc region protein containing an amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO:1.

[0403] The amino acid sequence of SEQ ID NO:1 is an Fc region protein. It is known that such an Fc region protein has the ability to secrete. Therefore, the Fc region proteins of (A) to (C) above may have the ability to secrete. In addition, an antibody containing such an Fc region protein may have antigen-binding ability. The amino acid residue at position 18 in SEQ ID NO:1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a nonpolar side chain as described later, still more preferably leucine, isoleucine, or alanine, and particularly preferably leucine or alanine. The amino acid residue at position 19 in SEQ ID NO:1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue or an acidic amino acid residue, more preferably an amino acid residue having a nonpolar side chain or an acidic amino acid residue, still more preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO:1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a nonpolar side chain, still more preferably glycine or alanine. The amino acid residue at position 140 in SEQ ID NO:1 is an arbitrary amino acid residue, but is preferably an acidic amino acid residue, more preferably glutamic acid or aspartic acid. The amino acid residue at position 142 in SEQ ID NO:1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a nonpolar side chain, still more preferably methionine, leucine, or isoleucine, and particularly preferably methionine or leucine. The amino acid residue at position 177 in SEQ ID NO:1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having an uncharged polar side chain or a nonpolar side chain as described later, still more preferably threonine, alanine, or glycine, and particularly preferably threonine or alanine.

[0404] In a preferred embodiment, the amino acid sequence of SEQ ID NO:1 may be an amino acid sequence consisting of amino acid residues at positions 220 to 449 in the amino acid sequence of SEQ ID NO:2.

[0405] In another preferred embodiment, the amino acid sequence of SEQ ID NO:1 may be an amino acid sequence consisting of amino acid residues at positions 7 to 236 in the amino acid sequence of SEQ ID NO:3.

[0406] In a specific embodiment, an antibody comprising an "Fc region protein containing the amino acid sequence as described above" may be an antibody comprising an "Fc region protein containing the amino acid sequence as described above" and a "constant region of the antibody". As the constant region of such an antibody, it may be the constant region of a chimeric antibody, a humanized antibody, or a human antibody (such as IgG1, IgG2, IgG3, IgG4, etc. of IgG).

[0407] In the Fc region protein (B), one or more amino acid residues may be altered by 1, 2, 3, or 4 mutations selected from deletion, substitution, addition, and insertion. The mutations of amino acid residues may be introduced into one region of the amino acid sequence or into multiple different regions. The term "one or more" means the number that does not significantly disrupt the protein activity. The number indicated by the term "one or more" is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 (such as 1, 2, 3, 4, or 5).

[0408] In the Fc region protein (C), the identity percentage with the amino acid sequence of SEQ ID NO: 1 may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In the present invention, the calculation of the identity percentage of a peptide or polypeptide (protein) can be performed by the algorithm blastp. More specifically, the calculation of the identity percentage of a polypeptide can be performed using the scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11 Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment) set by default in the algorithm blastp provided by the National Center for Biotechnology Information (NCBI). In addition, the calculation of the identity percentage of a polynucleotide (gene) can be performed by the algorithm blastn. More specifically, the calculation of the identity percentage of a polynucleotide can be performed using the scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) set by default in the algorithm blastn provided by NCBI.

[0409] Secretion in the secretion ability has the same meaning as the secretion of a secreted protein (so-called solubility). Therefore, "having the secretion ability" means functioning as an antibody, like a normal antibody.

[0410] As long as the antibody containing the Fc region protein maintains the target characteristics (such as secretion ability, antigen-binding ability), mutations can be introduced into specific sites. The positions of the amino acid residues where mutations can be introduced while maintaining the target characteristics are self-evident to those skilled in the art. Specifically, for those skilled in the art, 1) compare the amino acid sequences of multiple proteins with the same characteristics, 2) identify the relatively conserved regions and relatively non-conserved regions, and then 3) from the relatively conserved regions and relatively non-conserved regions, the regions that play an important role in function and the regions that do not play an important role in function can be predicted respectively, so the structure / function correlation can be recognized. Therefore, those skilled in the art can specify the positions of the amino acid residues where mutations can be introduced in the amino acid sequence of the antibody containing the Fc region protein described above.

[0411] In the case where an amino acid residue is mutated by substitution (replacement), the substitution of the amino acid residue may be a conservative substitution. As used in this specification, the term "conservative substitution" means substituting a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, as such families, amino acids having a basic side chain (e.g., lysine, arginine, histidine), amino acids having an acidic side chain (e.g., aspartic acid, glutamic acid), amino acids having an uncharged polar side chain (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having a nonpolar side chain (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having a β-branched side chain (e.g., threonine, valine, isoleucine), amino acids having an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids having a side chain containing a hydroxy group (e.g., alcoholic, phenolic) (e.g., serine, threonine, tyrosine), and amino acids having a side chain containing sulfur (e.g., cysteine, methionine) can be cited. Preferably, conservative substitutions of amino acids may be substitutions between aspartic acid and glutamic acid, substitutions between arginine, lysine, and histidine, substitutions between tryptophan and phenylalanine, substitutions between phenylalanine and valine, substitutions between leucine, isoleucine, and alanine, and substitutions between glycine and alanine.

[0412] As the antibody used in the present invention or an antibody comprising any one of the Fc regions selected from the above (A) to (C), examples include: chimeric antibodies (for example, Rituximab, Basiliximab, Infliximab, Cetuximab, Siltuximab, Dinutuximab, Altertoxaximab,(Ortatoximab)), humanized antibodies (such as Daclizumab, Palivizumab, Trastuzumab, Alemtuzumab, Omalizumab, Efalizumab, Bevacizumab, Natalizumab (IgG4), Tocilizumab, Eculizumab (IgG2), Mogamulizumab, Pertuzumab, Obinutuzumab, Vedolizumab, Pembrolizumab (IgG4), Mepolizumab, Elotuzumab, Daratumumab, Ixekizumab (IgG4), Reslizumab (IgG4), Atezolizumab)), human antibodies (such as Adalimumab, Panitumumab, Golimumab, Ustekinumab, Canakinumab, Ofatumumab, Denosumab (IgG2), Ipilimumab, Belimumab, Raxibacumab, Ramucirumab, Nivolumab (IgG4), Secukinumab, Evolocumab (IgG2), Alirocumab, Necitumumab, Brodalumab (IgG2), Olaratumab, Dupilumab (IgG4)) (when the IgG subtype is not mentioned, it means IgG1).

[0413] Examples of the affinity substances for antibodies as described above include: peptides (including oligopeptides, polypeptides, and proteins), low-molecular-weight compounds, nucleic acids, nucleic acid-peptide complexes, peptide-low-molecular-weight compound complexes, and nucleic acid-low-molecular-weight complexes.

[0414] In certain embodiments, the affinity substance for an antibody as described above may be a peptide (including oligopeptides, polypeptides, proteins, and may also be glycoproteins). As such peptides, for example, the following peptides have been reported:

[0415] (1) An IgG-binding peptide having affinity for a specific region (CH2 region) of all human IgG (i.e., human IgG1, IgG2, IgG3, and IgG4, the same hereinafter) (for example, refer to International Publication No. 2016 / 186206, International Publication No. 2013 / 027796, International Publication No. 2008 / 054030);

[0416] (2) A protein A mimetic peptide (Protein A Mimetic (PAM) peptide) having affinity for a specific region (CH2 region) of all human IgG (for example, refer to Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, Vol. 6, 564-569);

[0417] (3) EPIHRSTLTALL (SEQ ID NO: 25) having affinity for a specific region (CH2 region) of all human IgG (for example, refer to Ehrlich G.K et al., J.Biochem.Biophys.Methods, 2001, Vol. 49, 443-454);

[0418] (4) (NH2-Cys1-X1-X2-X3-X4)2-Lys-Gly-OH having affinity for a specific region (Fc region) of all human IgG (for example, refer to Ruvo M et al., ChemBioChem, 2005, Vol. 6, 1242-1253);

[0419] (5) FARLVSSIRY (SEQ ID NO: 26), FGRLVSSIRY (SEQ ID NO: 27), and TWKTSRISIF (SEQ ID NO: 28) having affinity for a specific region (Fc region) of all human IgG (for example, refer to Krook M et al., Journal of Immunological Methods, 1998, Vol. 221, 151-157);

[0420] (6) QSYP (SEQ ID NO: 29) having affinity for a specific region of all human IgG (for example, refer to Jacobs J.M. et al., Bio.Techniques, 2003, Vol. 34, 132-141);

[0421] (7) HWRGWV (SEQ ID NO:30), HYFKFD (SEQ ID NO:31), and HFRRHL (SEQ ID NO:32) that are affinity for the specific region (Fc region) of all human IgG (for example, refer to Carbonell R.G. et al., Journal of Chromatography A, 2009, Vol. 1216, 910 - 918);

[0422] (8) DAAG (SEQ ID NO:33) that is affinity for the specific region (Fc region) of all human IgG (for example, refer to Lund L.N. et al., Journal of Chromatography A, 2012, Vol. 1225, 158 - 167);

[0423] (9) Fc - I, Fc - II, and Fc - III that are affinity for the specific region (Fc region) of all human IgG (for example, refer to Warren L. Delano et al., Science, 2000, Vol. 287, 1279 - 1283; International Publication No. WO 2001 / 045746); and

[0424] (10) NARKFYKG (SEQ ID NO:3418) and NKFRGKYK (SEQ ID NO:35) that are affinity for the specific region (Fc region) of all human IgG (for example, refer to Biochemical Engineering Journal, 2013, Vol. 79, 33 - 40).

[0425] In another specific embodiment, the affinity substance for the antibody as described above can be a substance other than a peptide. As such a substance, for example, aptamers that are affinity for the specific region (CH2 region, especially the side chain of Lys340) of human IgG (such as human IgG1 - 4) have been reported [for example, aptamers containing GGUGCU and GGUGAU and other GGUG(C / A)(U / T) motifs] (for example, refer to International Publication No. WO 2007 / 004748; Nomura Y et al., Nucleic Acids Res., 2010 Nov; 38(21):7822 - 9; Miyakawa S et al., RNA., 2008 Jun; 14(6):1154 - 63).

[0426] The affinity substance for an antibody as described above can be obtained by any known method in the art. For example, the whole antibody or a partial peptide in the antibody (for example, when the exposed region on the antibody surface is clear, the partial peptide present in this region) can be used. The affinity substance can be obtained by producing an antibody (for example, the hybridoma method) or screening the affinity substance from a library from which an affinity substance can be obtained (for example, a peptide library, an antibody library, an antibody-producing cell library, an aptamer library, a phage library, an mRNA library, a cDNA library) (for example, the phage display method, the SELEX method, the mRNA display method, the ribosome display method, the cDNA display method, the yeast display method). In addition, when the affinity substance for an antibody is an affinity substance for the Fc region (soluble region) of an antibody, an affinity substance (for example, an antibody, an aptamer) that can selectively bind to any part in the Fc region of the antibody can be effectively obtained by using a partial peptide present in a specific region (for example, CH1, CH2, CH3) in the Fc regions of various antibodies (for example, IgG, IgA, IgM, IgD, IgE). Among the affinity substances obtained in this way, substances with relatively strong and weak affinity binding abilities are mixed. However, even an affinity substance with a weak affinity binding ability can make up for its affinity binding ability by using an excessive amount.

[0427] In a preferred embodiment, the affinity substance for an antibody is a peptide. As such a peptide, a peptide having the binding ability for the constant region of a monoclonal antibody is preferred, a peptide having the binding ability for the Fc region of a monoclonal antibody is more preferred, and a peptide having the binding ability for the Fc region of IgG is even more preferred. As for the length of the peptide, there is no particular limitation. For example, it is a peptide composed of 10 to 40 (for example, 10 to 20, 20 to 30, and 30 to 40) amino acid residues. As the amino acid residues constituting the peptide, 20 kinds of L-α-amino acid residues constituting natural proteins and their stereoisomers (for example, D-amino acids), and their isomers (for example, β-amino acids) can be cited.

[0428] In a specific embodiment, the affinity substance for an antibody as described above can be a peptide containing any of the following amino acid sequences.

[0429] A peptide containing the following amino acid sequence:

[0430] The amino acid sequence of (a-1-1) FNMQQQRRFYEALHD PNLNEEQRNARIRSIRDD (SEQ ID NO:11) (for example, refer to Compounds 22 to 24, 29), or

[0431] In the amino acid sequence of (a-1-2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 12) (an amino acid sequence obtained by substituting 2 Ks in the known sequence Z34C with Rs), any 1 to 3 amino acid residues in the sequence may be the same or different and are each substituted with 1 amino acid residue selected from lysine residue, aspartic acid residue, and glutamic acid residue, or

[0432] in the amino acid sequence of (a-2-1) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 13) (for example, refer to Compounds 26 to 28), or

[0433] in the amino acid sequence of (a-2-2) β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 14) (an amino acid sequence obtained by substituting 2 Ks in the known sequence Z34C with Rs and substituting the N-terminal F with β-Ala), any 1 to 3 amino acid residues in the sequence may be the same or different and are each substituted with 1 amino acid residue selected from lysine residue, aspartic acid residue, and glutamic acid residue; and

[0434] (b) has an identity of 85% or more with each of the above amino acid sequences of SEQ ID NOs: 11 to 14.

[0435] Such a peptide has the binding ability to the Fc region of a monoclonal antibody.

[0436] Due to the circumstances in the synthesis of peptide reagents, the peptide composed of the amino acid sequence of SEQ ID NO:12 is a peptide in which the two K (lysine) at the 26th and 28th positions from the N-terminus among the known affinity peptides as Z34C are changed to R (arginine). The compound of the present invention containing the peptide comprising the above amino acid sequence can be used for the site-selective modification of specific amino acid residues in human IgG Fc (for example, Lys248 residue or Lys246 residue based on Eu numbering, Lys288 or Lys290, Lys317, or other amino acid residues other than these residues). It should be noted that the amino acid sequence of the above Z34C is FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO:36) (for example, refer to Starovasnik, M.A. et al., Structural mimicry of a native protein by a minimized binding domain., Proc. Natl. Acad. Sci. USA., 94, 10080-10085 (1997)).

[0437] The affinity peptide may have an affinity for human IgG (for example, the human IgG as described above. Preferably human IgG1). In the case where the above affinity peptide contains two cysteine residues (for example, at the 5th and 34th positions), the two cysteine residues can be disulfide-bonded to form a cyclic peptide.

[0438] As the position for introducing a lysine residue, an aspartic acid residue, or a glutamic acid residue (amino acid residues that are easily modified by a crosslinking agent), any position can be used as long as it has an affinity for human IgG such as human IgG1. Such positions can be easily identified by those skilled in the art. The position for introducing a lysine residue, an aspartic acid residue, or a glutamic acid residue can be an amino acid residue other than a cysteine residue. More preferably, as the position for introducing an amino acid residue that can be easily modified by a crosslinking agent, for example, the amino acid residues at the 1st, 3rd, 6th, 7th, 13th, 20th, 24th, 31st, and 32nd positions can be cited.

[0439] Preferably, the above amino acid sequence having the characteristics of (a) and (b) further preferably has, at a specified position, one specific amino acid residue (preferably a lysine residue) selected from a lysine residue, an aspartic acid residue, and a glutamic acid residue (amino acid residues that can be easily modified by a crosslinking agent), and has mutations of the 20 common amino acid residues constituting a natural protein (preferably 17 amino acid residues other than a lysine residue, an aspartic acid residue, and a glutamic acid residue, more preferably 19 amino acid residues other than a lysine residue) at positions other than the specified position. Such a specified position is not particularly limited, and examples thereof include the 1st, 3rd, 6th, 7th, 13th, 20th, 24th, 31st, and 32nd positions. The above amino acid sequence having the characteristics of (a) and (b) maintains two cysteine residues, and these two cysteine residues can be bound by a disulfide bond. An amino acid sequence having 85% or more identity to the above amino acid sequences of SEQ ID NOs: 11 to 14 may have 1 to 3 (preferably 1 or 2, more preferably 1) amino acid residues changed by 1, 2, 3, or 4 types of mutations (preferably substitution) selected from deletion, substitution, addition, and insertion of amino acid residues. The mutations of amino acid residues may be introduced into one region of the amino acid sequence or into a plurality of different regions.

[0440] More preferably, the above amino acid sequence having the characteristics of (a) and (b) may be the following (c) or (d).

[0441] (c) An amino acid sequence selected from the following amino acid sequences (1) to (16):

[0442] (1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIKDD (SEQ ID NO: 5);

[0443] (2) FNMQQQRRFYEALHDPNLNEEQRNARIKSIRDD (SEQ ID NO: 6);

[0444] (3) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 7);

[0445] (4) FNMQQQRRFYEALHDPNLNEEQRNAKIKSIKDD (SEQ ID NO: 8);

[0446] (5) KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 37);

[0447] (6) FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:38);

[0448] (7) FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:39);

[0449] (8) FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC (SEQ ID NO:40);

[0450] (9) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO:41);

[0451] (10) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:42);

[0452] (11) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:43);

[0453] (12) FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:44);

[0454] (13) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRKDC (SEQ ID NO:45);

[0455] (14) β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSI (SEQ ID NO:97);

[0456] (15) FNMQQQRRFYEALHDPNLNKEQRNARIRSIRDD (SEQ ID NO:98); and

[0457] (16) β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSIKDD (SEQ ID NO:100); or

[0458] (d) In any one of the amino acid sequences in (1) to (16) above, having a mutation of 19 amino acid residues other than lysine residues at positions other than 1 lysine residue and 2 cysteine residues (for example, positions 1, 3, 6, 7, 13, 20, 24, 31, and 32), and having 90% or more identity to any one of the amino acid sequences in (1) to (16) above (which may be a modification of the amino acid residues in the number as described above).

[0459] It was confirmed that a peptide containing such an amino acid sequence has the binding ability to the Fc region of IgG. The affinity peptide having the above amino acid sequence in (d) is preferably a peptide having the binding ability to the constant region of a monoclonal antibody, more preferably a peptide having the binding ability to the Fc region of a monoclonal antibody, and even more preferably a peptide having the binding ability to the Fc region of IgG.

[0460] As long as the above affinity peptide has 85% or more identity to the above amino acid sequences of SEQ ID NOs: 11 to 14 or the amino acid sequences in (1) to (16) above, in addition to introducing 1 amino acid residue that is easily modified by a crosslinking agent, it may also have further mutations of amino acid residues. For the positions where further amino acid mutations can be introduced, those skilled in the art can easily identify them. For example, even the phenylalanine residue at position 1, the arginine residue at position 6, the leucine residue at position 13, the glutamate residue at position 20, the aspartate residue at position 24, or the arginine residue at position 31 (except for the positions where amino acid residues that can be easily modified by a crosslinking agent have been introduced) can be used. As the amino acids that can be introduced by further amino acid mutations, for example, 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), aspartate (D), glutamate (E), arginine (R), histidine (H), and lysine (L) can be listed. Preferably, these 19 amino acids other than lysine can be used. The amino acid can be either the L-form or the D-form, but the L-form is preferred (the amino acid residues constituting the peptide in the examples are all in the L-form).

[0461] The degree of identity % to the above amino acid sequences of SEQ ID NOs: 11 to 14 or the above amino acid sequences in (1) to (16) can be determined as described above. The degree of identity % is preferably 90% or more, 92% or more, more preferably 94% or more, even more preferably 95% or more, and particularly preferably 97% or more (that is, a sequence having only 1 amino acid residue mutation).

[0462] In another specific embodiment, the affinity substance for the antibody as described above is a peptide comprising any of the following amino acid sequences.

[0463] Formula 1-1: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-I-W-C-(X 0-3 ) b (SEQ ID NO:15)

[0464] Formula 1-2: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-V-W-C-(X 0-3 ) b (SEQ ID NO:16)

[0465] Formula 1-3: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-V-V-W-C-(X 0-3 ) b (SEQ ID NO:17)

[0466] Formula 1-4: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-A-V-W-C-(X 0-3 ) b (SEQ ID NO:18)

[0467] Formula 1-5: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-L-W-C-(X 0-3 ) b (SEQ ID NO:19)

[0468] Formula 1-6: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-I-W-C-(X 0-3 ) b (SEQ ID NO:20)

[0469] Formula 1-7: (X 0-3 ) a-C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-F-C-(X 0-3 ) b (SEQ ID NO:21)

[0470] Formula 1-8: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Q-V-W-C-(X 0-3 ) b (SEQ ID NO:22)

[0471] Formula 1-9: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-E-V-W-C-(X 0-3 ) b (SEQ ID NO:23)

[0472] [In the formula,

[0473] (X 0-3 ) a is absent, arginine residue-glycine residue-asparagine residue, glycine residue-asparagine residue, aspartic acid residue, or asparagine residue,

[0474] (X 0-3 ) b is absent, threonine residue-tyrosine residue-histidine residue, or threonine residue,

[0475] Xaa1 is alanine residue,

[0476] Xaa2 is tyrosine residue, tryptophan residue, or histidine residue,

[0477] Xaa3 is histidine residue, phenylalanine residue, tyrosine residue, tryptophan residue, arginine residue, or glycine residue,

[0478] Xaa4 is lysine residue, aspartic acid residue, or glutamic acid residue,

[0479] Xaa5 is glycine residue, serine residue, asparagine residue, glutamine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, tyrosine residue, tryptophan residue, histidine residue, threonine residue, leucine residue, alanine residue, valine residue, isoleucine residue, or arginine residue,

[0480] Xaa6 is glutamine residue, glutamic acid residue, asparagine residue, or aspartic acid residue.] Or

[0481] Formula 2-1: (X 0-3 ’) a -C-(Xaa1’)-(Xaa2’)-(Xaa3’)-(Xaa4’)-(Xaa5’)-(Xaa6’)-L-V-W-C-(X 0-3 ’) b (SEQ ID NO:24)

[0482] [Wherein,

[0483] (X 0-3 ’) a and (X 0-3 ’) b are respectively the same as the above (X 0-3 ) a and (X 0-3 ) b respectively,

[0484] Xaa1’, Xaa2’, Xaa3’, Xaa4’, Xaa5’, Xaa6’ are respectively the same as the above Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6.].

[0485] Such peptides have the binding ability to the Fc region of monoclonal antibodies.

[0486] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, (X 0-3 ) a is absent, arginine residue - glycine residue - asparagine residue, glycine residue - asparagine residue, aspartic acid residue, or asparagine residue, preferably absent, arginine residue - glycine residue - asparagine residue, aspartic acid residue, or asparagine residue.

[0487] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, Xaa2 is tyrosine residue, tryptophan residue, or histidine residue, preferably tyrosine residue or tryptophan residue.

[0488] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, Xaa3 is histidine residue, phenylalanine residue, tyrosine residue, tryptophan residue, arginine residue, or glycine residue, preferably histidine residue.

[0489] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, Xaa4 is lysine residue, aspartic acid residue, or glutamic acid residue, preferably lysine residue.

[0490] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, Xaa5 is a glycine residue, a serine residue, an asparagine residue, a glutamine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a histidine residue, a threonine residue, a leucine residue, an alanine residue, a valine residue, an isoleucine residue, or an arginine residue, preferably a glycine residue, a threonine residue, a leucine residue.

[0491] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue, more preferably a glutamine residue.

[0492] Preferably, the peptide containing any of the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1 is a peptide containing an amino acid sequence selected from the following:

[0493] (1’) RGNCAYHKGQIIWCTYH (SEQ ID NO:46);

[0494] (2’) RGNCAYHKGQIVWCTYH (SEQ ID NO:47);

[0495] (3’) RGNCAYHKGQVVWCTYH (SEQ ID NO:48);

[0496] (4’) RGNCAYHKGQAVWCTYH (SEQ ID NO:49);

[0497] (5’) RGNCAYHKGQLLWCTYH (SEQ ID NO:50);

[0498] (6’) RGNCAYHKGQLIWCTYH (SEQ ID NO:51);

[0499] (7’) DCAYHKGQIVWCT (SEQ ID NO:52);

[0500] (8’) DCAYHKGQVVWCT (SEQ ID NO:53);

[0501] (9’) DCAYHKGQAVWCT (SEQ ID NO:54);

[0502] (10’) RGNCAYHKSQIIWCTYH (SEQ ID NO:55);

[0503] (11’) RGNCAYHKNQIIWCTYH (SEQ ID NO:56);

[0504] (12’) RGNCAYHKDQIIWCTYH (SEQ ID NO:57);

[0505] (13’) RGNCAYHKQQIIWCTYH (SEQ ID NO:58);

[0506] (14’) RGNCAYHKEQIIWCTYH (SEQ ID NO:59);

[0507] (15’) RGNCAYHKFQIIWCTYH (SEQ ID NO:60);

[0508] (16’) RGNCAYHKYQIIWCTYH (SEQ ID NO:61);

[0509] (17’) RGNCAYHKWQIIWCTYH (SEQ ID NO:62);

[0510] (18’) RGNCAYHKHQIIWCTYH (SEQ ID NO:63);

[0511] (19’) RGNCAYHKTQIIWCTYH (SEQ ID NO:64);

[0512] (20’) RGNCAYHKLQIIWCTYH (SEQ ID NO:65);

[0513] (21’) CAYHKLQIVWC (SEQ ID NO:66);

[0514] (22’) CAYHKLQLIWC (SEQ ID NO:67);

[0515] (23’) CAYHKSQIVWC (SEQ ID NO:68);

[0516] (24’) RGNCAYHKGQLVFCTYH (SEQ ID NO:69);

[0517] (25’) RGNCAYHKGQQVWCTYH (SEQ ID NO:70);

[0518] (26’) RGNCAYHKGQEVWCTYH (SEQ ID NO:71);

[0519] (27’) CAYHKGQLVWC (SEQ ID NO:72);

[0520] (28’) RGNCAYHKAQLVWCTYH (SEQ ID NO:73);

[0521] (29’) RGNCAYHKVQLVWCTYH (SEQ ID NO:74);

[0522] (30’) RGNCAYHKLQLVWCTYH (SEQ ID NO:75);

[0523] (31’) RGNCAYHKIQLVWCTYH (SEQ ID NO:76);

[0524] (32’) RGNCAYHKSQLVWCTYH (SEQ ID NO:77);

[0525] (33’) RGNCAYHKTQLVWCTYH (SEQ ID NO:78);

[0526] (34’) RGNCAYHKNQLVWCTYH (SEQ ID NO:79);

[0527] (35’) RGNCAYHKDQLVWCTYH (SEQ ID NO:80);

[0528] (36’) RGNCAYHKQQLVWCTYH (SEQ ID NO:81);

[0529] (37’) RGNCAYHKEQLVWCTYH (SEQ ID NO:82);

[0530] (38’) RGNCAYHKFQLVWCTYH (SEQ ID NO:83);

[0531] (39’) RGNCAYHKRQLVWCTYH (SEQ ID NO:84);

[0532] (40’) RGNCAYHKHQLVWCTYH (SEQ ID NO:85);

[0533] (41’) RGNCAYHKWQLVWCTYH (SEQ ID NO:86);

[0534] (42’) RGNCAYHKYQLVWCTYH (SEQ ID NO:87);

[0535] (43’) RGNCAYFKGQLVWCTYH (SEQ ID NO:88);

[0536] (44’)RGNCAYYKGQLVWCTYH (SEQ ID NO:89);

[0537] (45’)RGNCAYWKGQLVWCTYH (SEQ ID NO:90);

[0538] (46’)RGNCAYRKGQLVWCTYH (SEQ ID NO:91);

[0539] (47’)RGNCAYGKGQLVWCTYH (SEQ ID NO:92);

[0540] (48’)DCAYHKGQLVWC (SEQ ID NO:93);

[0541] (49’)NCAYHKGQLVWC (SEQ ID NO:94);

[0542] (50’)CAYHKGQLVWCT (SEQ ID NO:95);

[0543] (51’)CAYHKSQLVWC (SEQ ID NO:96);

[0544] (52’)GNCAYHKGQIIWCTYH (SEQ ID NO:99); and

[0545] (53’)RGNCAYHEGQIIWCTYH (SEQ ID NO:108).

[0546] It was confirmed that the peptide containing such an amino acid sequence has the binding ability to the Fc region of IgG.

[0547] In each amino acid sequence of the above peptide, at least two separated cysteine residues can form a cyclic peptide by disulfide bonding. Alternatively, the thioether groups in the two cysteine residues in the above peptide can be linked by a carbonyl-containing linker represented below.

[0548] [Chemical formula 8]

[0549]

[0550] The dotted part of the carbonyl-containing linker represented above refers to the binding part to the thioether group. This linker is more stable than the usual disulfide bonding against reduction reactions and the like. Such a peptide can be prepared, for example, by the method described in International Publication No. 2016 / 186206.

[0551] The compounds of the present invention containing the peptides comprising the above amino acid sequences can be used for the site-selective modification of specific amino acid residues in human IgG Fc (e.g., Lys248 residue or Lys246 residue, Lys288 or Lys290, Lys317 based on Eu numbering, or other amino acid residues other than these residues). The amino acids constituting the above peptides can be either L-form or D-form, but preferably L-form (in the examples, the amino acid residues constituting the peptides are all L-form).

[0552] The above peptides can modify specific amino acid residues through crosslinking agents. As such specific amino acid residues, for example, lysine residues, aspartic acid residues, and glutamic acid residues can be mentioned, but preferably lysine residues. As crosslinking agents, for example, crosslinking agents preferably containing two or more succinimide groups such as DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate); crosslinking agents preferably containing two or more imino moieties such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2HCl), and DMS (dimethyl suberimidate·2HCl); and crosslinking agents having an SS bond such as DTBP (dimethyl 3,3’-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., International Publication No. 2016 / 186206).

[0553] When the affinity substance for an antibody is a peptide, the amino group and carboxyl group at the peptide terminus can be protected. As protecting groups for the N-terminal amino group, for example, alkylcarbonyl (acyl) (e.g., acetyl, propoxy, butoxycarbonyl such as tert-butoxycarbonyl), alkoxycarbonyl (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl, arylalkyl (aralkyl)oxycarbonyl (e.g., benzyloxycarbonyl) can be mentioned. As a protecting group for the N-terminal amino group, acetyl is preferred. As protecting groups for the C-terminal carboxyl group, for example, groups that can form esters or amides can be mentioned. As groups that can form esters or amides, for example, alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy), aryloxy (e.g., phenoxy, naphthoxy), aralkyl oxy (e.g., benzyloxy), amino can be mentioned. As a protecting group for the C-terminal carboxyl group, amino is preferred.

[0554] 1-3. Divalent group (L) containing a leaving group

[0555] In formula (I), L is a divalent group containing a leaving group.

[0556] A leaving group refers to a group having the ability to be cleaved and detached from E through a reaction between a nucleophilic group in an antibody and an electrophilic group contained in a divalent group (E) containing an electrophilic group. Such leaving groups are common general knowledge in the art (e.g., Fujishima, S. et al., J. Am. Chem. Soc, 2012, 134, 3961 - 3964. (above); Chem. Sci. 2015, 3217 - 3224.; Nature Chemistry, Volume 8, pages 542 - 548 (2016)). As the leaving group, as long as it has the ability to be cleaved from E through the above-described reaction, there is no particular limitation, and examples thereof include: (1) a group selected from -O-, -S-, -Se-, -SO2 - O-, -SO2 - N(R)-, -SO2 -, -C≡C - CH2 - O-, -N(OR)-, -N(R)-, and -O - N(R)- (here, R represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); and (2) heteroarylene.

[0557] Examples of the alkyl group having 1 to 6 carbon atoms include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, and hexyl. As the alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 4 carbon atoms is preferred.

[0558] The group selected from -O-, -S-, -Se-, -SO2 - O-, -SO2 - N(R)-, -SO2 -, -C≡C - CH2 - O-, -N(OR)-, -N(R)-, and -O - N(R)- as an example of the leaving group is the following (1) or (2):

[0559] (1) A group composed of -O-, -S-, -Se-, -SO2 - O-, -SO2 - N(R)-, -SO2 -, -C≡C - CH2 - O-, -N(OR)-, -N(R)-, or -O - N(R)-; or

[0560] (2) A group containing -O-, -S-, -Se-, -SO2 - O-, -SO2 - N(R)-, -SO2 -, -C≡C - CH2 - O-, -N(OR)-, -N(R)- or -O - N(R)- and a group that enhances its detachment ability.

[0561] A group that enhances the leaving ability of -O-, -S-, -Se-, -SO₂-O-, -SO₂-N(R)-, -SO₂-, -C≡C-CH₂-O-, -N(OR)-, -N(R)-, or -O-N(R)- means a group that, compared with the case where the group is not adjacent to -O-, -S-, -Se-, -SO₂-O-, -SO₂-N(R)-, -SO₂-, -C≡C-CH₂-O-, -N(OR)-, -N(R)-, or -O-N(R)-, enhances the ability of -O-, -S-, -Se-, -SO₂-O-, -SO₂-N(R)-, -SO₂-, -C≡C-CH₂-O-, -N(OR)-, -N(R)-, or -O-N(R)- to leave from an electrophilic group when the group is adjacent to -O-, -S-, -Se-, -SO₂-O-, -SO₂-N(R)-, -SO₂-, -C≡C-CH₂-O-, -N(OR)-, -N(R)-, or -O-N(R)-. In other words, a group that enhances the leaving ability of -O-, -S-, -Se-, -SO₂-O-, -SO₂-N(R)-, -SO₂-, -C≡C-CH₂-O-, -N(OR)-, -N(R)-, or -O-N(R)- is a group that has the ability to attract the electrons of an oxygen atom, a sulfur atom, a selenium atom, or a nitrogen atom.

[0562] Preferred examples of a group that enhances the leaving ability of -O-, -S-, -Se-, -SO₂-O-, -SO₂-N(R)-, -SO₂-, -C≡C-CH₂-O-, -N(OR)-, -N(R)-, or -O-N(R)- include: an arylene group that can be substituted with an electron-withdrawing group, a heteroarylene group that can be substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine that can be ring-condensed, a 2,6-diketopiperidine that can be ring-condensed, a 2-ketopyrrolidine that can be ring-condensed, a 2-ketopiperidine that can be ring-condensed, and 2-pyridone. The number of electron-withdrawing groups that the arylene group and the heteroarylene group can have is 1 or more (for example, 1 to 3, preferably 1 or 2). Examples of the electron-withdrawing group include: a halogen atom, an alkyl group substituted with a halogen atom (for example, trifluoromethyl), a boric acid residue, a mesyl group, a tosyl group, a trifluoromethanesulfonate, a nitro group, a cyano group, a phenyl group, and a keto group (for example, an acyl group).

[0563] As the "arylene group" in the "arylene group that can be substituted by an electron-withdrawing group", an arylene group having 6 to 24 carbon atoms is preferred, an arylene group having 6 to 18 carbon atoms is more preferred, an arylene group having 6 to 14 carbon atoms is still more preferred, and an arylene group having 6 to 10 carbon atoms is most preferred. The arylene group that can be substituted by an electron-withdrawing group may also be substituted by substituents other than the electron-withdrawing group, or may be unsubstituted. The above carbon atom numbers do not include the carbon atom numbers of the electron-withdrawing group and substituents other than it. As the arylene group, for example, phenylenes, naphthylenes, and anthracenylenes can be cited.

[0564] As the "heteroarylene group" in the "heteroarylene group that can be substituted by an electron-withdrawing group", a heteroarylene group having 1 to 21 carbon atoms is preferred, a heteroarylene group having 1 to 15 carbon atoms is more preferred, a heteroarylene group having 1 to 9 carbon atoms is still more preferred, and a heteroarylene group having 1 to 6 carbon atoms is most preferred. The heteroarylene group that can be substituted by an electron-withdrawing group may also be substituted by substituents other than the electron-withdrawing group, or may be unsubstituted. The above carbon atom numbers do not include the carbon atom numbers of the electron-withdrawing group and substituents other than it. The heteroarylene group contains 1 or more (for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3) heteroatoms selected from nitrogen atoms, oxygen atoms, and sulfur atoms as ring-constituting atoms. As the heteroarylene group, for example, pyrrolenediyls, furandiyles, thiophenediyls, pyridinediyls, pyridazinediyls, pyrimidinediyls, pyrazinediyls, triazinediyls, pyrazolediyls, imidazolediyls, thiazolediyls, isothiazolediyls, oxazolediyls, isoxazolediyls, triazolediyls, tetrazolediyls, indolediyls, purinediyls, anthraquinonediyles, carbazolediyls, fluorenediyls, quinolinediyls, isoquinolinediyls, quinazolinediyls, and phthalazinediyls can be cited.

[0565] The ring-condensable 2,5-diketopyrrolidine, ring-condensable 2,6-diketopiperidine, ring-condensable 2-ketopyrrolidine, ring-condensable 2-ketopiperidine, and 2-pyridone can be substituted by substituents such as electron-withdrawing groups, or may be unsubstituted.

[0566] The heteroarylene group as an example of the leaving group is a heteroarylene group with a low π electron density (i.e., less than 1). The heteroarylene group as the leaving group preferably contains a nitrogen atom as a ring-constituting atom. As the heteroarylene group containing a nitrogen atom as a ring-constituting atom, a heteroarylene group having 1 to 21 carbon atoms containing a nitrogen atom as a ring-constituting atom is preferred, a heteroarylene group having 1 to 15 carbon atoms containing a nitrogen atom as a ring-constituting atom is more preferred, and a heteroarylene group having 1 to 9 carbon atoms containing a nitrogen atom as a ring-constituting atom is further preferred. The heteroarylene group as the leaving group may be substituted by substituents such as electron-withdrawing groups, or may be unsubstituted. The above carbon atom numbers do not include the carbon atom numbers of the substituents. As the heteroarylene group of the leaving group containing a nitrogen atom as a ring-constituting atom, for example, imidazolediyls, triazolediyls, tetrazolediyls, 2-pyridonediyles (i.e., 2-hydroxypyridinediyls) can be cited.

[0567] Examples of groups such as "arylene which may be substituted by an electron-withdrawing group" as a group for enhancing the leaving ability of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -O-N(R)-, and "heteroarylene" as an example of a leaving group may have 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, and may also have no substituents. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the above-mentioned group has substituents, examples of such substituents are as follows:

[0568] (i) A halogen atom;

[0569] (ii) A monovalent hydrocarbon group;

[0570] (iii) An aralkyl group;

[0571] (iv) A monovalent heterocyclic group;

[0572] (v) R a -O-, R a -C(=O)-, R a -O-C(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or a monovalent hydrocarbon group.); or

[0573] (vi) NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group.);

[0574] (vii) Nitro, sulfate, sulfonic acid, cyano, and carboxyl groups.

[0575] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0576] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0577] The monovalent chain hydrocarbon group refers to a hydrocarbon group composed only of a chain structure, and the main chain does not contain a cyclic structure. However, the chain structure can be linear or branched. As the monovalent chain hydrocarbon group, for example, alkyl, alkenyl, and alkynyl can be cited. The alkyl, alkenyl, and alkynyl can be either linear or branched.

[0578] As the alkyl, an alkyl having 1 to 12 carbon atoms is preferred, an alkyl having 1 to 6 carbon atoms is more preferred, and an alkyl having 1 to 4 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the alkyl having 1 to 12 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl can be cited.

[0579] As the alkenyl, an alkenyl having 2 to 12 carbon atoms is preferred, an alkenyl having 2 to 6 carbon atoms is more preferred, and an alkenyl having 2 to 4 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the alkenyl having 2 to 12 carbon atoms, for example, vinyl, propenyl, n-butenyl can be cited.

[0580] As the alkynyl, an alkynyl having 2 to 12 carbon atoms is preferred, an alkynyl having 2 to 6 carbon atoms is more preferred, and an alkynyl having 2 to 4 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the alkynyl having 2 to 12 carbon atoms, for example, ethynyl, propynyl, n-butynyl can be cited.

[0581] As the monovalent chain hydrocarbon group, an alkyl is preferred.

[0582] The monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only an alicyclic hydrocarbon as the ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon can be either a monocyclic or polycyclic one. However, it does not need to be composed only of alicyclic hydrocarbons, and a part of it can contain a chain structure. As the monovalent alicyclic hydrocarbon group, for example, cycloalkyl, cycloalkenyl, cycloalkynyl can be cited, and these can be either monocyclic or polycyclic.

[0583] As the cycloalkyl, a cycloalkyl having 3 to 12 carbon atoms is preferred, a cycloalkyl having 3 to 6 carbon atoms is more preferred, and a cycloalkyl having 5 to 6 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the cycloalkyl having 3 to 12 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl can be cited.

[0584] As the cycloalkenyl, a cycloalkenyl having 3 to 12 carbon atoms is preferred, a cycloalkenyl having 3 to 6 carbon atoms is more preferred, and a cycloalkenyl having 5 to 6 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the cycloalkenyl having 3 to 12 carbon atoms, for example, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl can be cited.

[0585] As the cycloalkynyl group, a cycloalkynyl group having 3 to 12 carbon atoms is preferred, a cycloalkynyl group having 3 to 6 carbon atoms is more preferred, and a cycloalkynyl group having 5 to 6 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the cycloalkynyl group having 3 to 12 carbon atoms, for example, cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl can be cited.

[0586] As the monovalent alicyclic hydrocarbon group, a cycloalkyl group is preferred.

[0587] The monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to be composed only of aromatic rings, and a part thereof may contain a chain structure or an alicyclic hydrocarbon, and the aromatic ring can be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, an aryl group having 6 to 12 carbon atoms is preferred, an aryl group having 6 to 10 carbon atoms is more preferred, and an aryl group having 6 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the aryl group having 6 to 12 carbon atoms, for example, phenyl and naphthyl can be cited.

[0588] As the monovalent aromatic hydrocarbon group, a phenyl group is preferred.

[0589] Among these, as the monovalent hydrocarbon group, an alkyl group, a cycloalkyl group, and an aryl group are preferred, and an alkyl group is more preferred.

[0590] An aralkyl group refers to an arylalkyl group. The definitions, examples, and preferences of the aryl and alkyl groups in the aralkyl group are as described above. As the aralkyl group, an aralkyl group having 3 to 15 carbon atoms is preferred. As such an aralkyl group, for example, benzoyl, phenethyl, naphthylmethyl, and naphthylethyl can be cited.

[0591] The monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocyclic ring of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocyclic group, it is preferred to contain one or more selected from an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably to contain one or more selected from an oxygen atom, a sulfur atom, and a nitrogen atom.

[0592] As the monovalent aromatic heterocyclic group, a monovalent aromatic heterocyclic group having 1 to 15 carbon atoms is preferred, a monovalent aromatic heterocyclic group having 1 to 9 carbon atoms is more preferred, and a monovalent aromatic heterocyclic group having 1 to 6 carbon atoms is further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. As the monovalent aromatic heterocyclic group, for example, pyrrolyl, furyl, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinonyl, carbazolyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl can be cited.

[0593] As a monovalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferred, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferred, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is further preferred. The number of carbon atoms described above does not include the number of carbon atoms of substituents. As the monovalent non-aromatic heterocyclic group, for example, the following can be mentioned: oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuryl, tetrahydrofuryl, dioxolanyl, tetrahydrothienyl, pyrrolineyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, pyrazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0594] Among these, as the monovalent heterocyclic group, a 5- or 6-membered heterocyclic group is preferred.

[0595] Preferably, the substituent may be a group as follows:

[0596] (i’) A halogen atom;

[0597] (ii’) An alkyl group having 1 to 12 carbon atoms, a phenyl group, or a naphthyl group;

[0598] (iii’) An aralkyl group having 3 to 15 carbon atoms;

[0599] (iv’) A 5- or 6-membered heterocycle;

[0600] (v’) R a -O-, R a -C(=O)-, R a -O-C(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.); or

[0601] (vi’) NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.);

[0602] (vii’) A group the same as the group listed in the above (vii).

[0603] More preferably, the substituent may be a group as follows:

[0604] (i”) a halogen atom;

[0605] (ii”) an alkyl group having 1 to 12 carbon atoms;

[0606] (iii”) R a -O-, R a -C(=O)-, R a -O-C(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.); or

[0607] (iv”) NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c are the same or different and represent a hydrogen atom or an alkyl group having 1 to 12 carbon atoms.);

[0608] (v”) a group the same as the group listed in (vii) above.

[0609] More preferably, the substituent may be a group as follows:

[0610] (i”’) a halogen atom;

[0611] (ii”’) an alkyl group having 1 to 6 carbon atoms;

[0612] (iii”’) R a -O-, R a -C(=O)-, R a -O-C(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.); or

[0613] (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 cSame or different, and represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.);

[0614] (v”’) A group identical to the groups listed in (vii) above.

[0615] Particularly preferably, the substituent may be a group as follows:

[0616] (i””) A halogen atom;

[0617] (ii””) An alkyl group having 1 to 4 carbon atoms;

[0618] (iii””) R a -O-, R a -C(=O)-, R a -O-C(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.).; or

[0619] (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 same or different, and represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.).;

[0620] (v”’) A group identical to the groups listed in (vii) above.

[0621] More specifically, as the leaving group, any one of the following (a) to (c) can be exemplified.

[0622] (a) Ring P-Q-[Here, ring P is a group selected from arylene which may be substituted by an electron-withdrawing group, heteroarylene which may be substituted by an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, 2-pyridone, and Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0623] (b) Heteroarylene; or

[0624] (c) - Q - [where Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).].

[0625] As the leaving group, in (a), (b), and (c), (a) or (b) is preferred, and (a) is particularly preferred. Ring P in (a) is any one of an arylene group which may be substituted with an electron-withdrawing group, a heteroarylene group which may be substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine which may be ring-condensed, a 2,6-diketopiperidine which may be ring-condensed, a 2-ketopyrrolidine which may be ring-condensed, a 2-ketopiperidine which may be ring-condensed, and 2-pyridone. Among them, an arylene group substituted with an electron-withdrawing group, a heteroarylene group substituted with an electron-withdrawing group, 2,5-diketopyrrolidine, and 2,6-diketopiperidine are more preferred, and an arylene group substituted with an electron-withdrawing group, 2,5-diketopyrrolidine, and 2,6-diketopiperidine are further preferred.

[0626] Ring P in (a) is a group selected from an arylene group which may be substituted with an electron-withdrawing group, a heteroarylene group which may be substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine which may be ring-condensed, a 2,6-diketopiperidine which may be ring-condensed, a 2-ketopyrrolidine which may be ring-condensed, a 2-ketopiperidine which may be ring-condensed, and 2-pyridone. The details of these groups are the same as the groups described as preferred examples of the group for enhancing the leaving ability of -N(R)-, -N(OR)-, -O-, -S-, or -Se-.

[0627] Q in (a) is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, -O-N(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms). Among them, -O-, -S-, -SO2-O-, or -SO2-N(R)- is preferred, -O- or -S- is more preferred, and -O- is further preferred.

[0628] (b) is a heteroarylene group, preferably imidazolediyl, triazolediyl, tetrazolediyl, or 2-pyridonediy ( = 2-hydroxypyridinediyl), and imidazolediyl or 2-pyridonediy is further preferred.

[0629] Q in (c) is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms). Preferably, it is -S-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -O-N(R)-, and more preferably S, -N(OR), or -O-N(R)-.

[0630] More specifically, the preferred leaving group may be a group selected from the following structural formulas.

[0631] [Chemical formula 9]

[0632]

[0633] (Here, EWG is an electron-withdrawing group,

[0634] m is an integer from 0 to 4,

[0635] n is an integer from 0 to 3,

[0636] R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

[0637] ○ (white circle) is a bonding site to L1, and ● (black circle) is a bonding site to E1.).

[0638] Details of the electron-withdrawing group and the alkyl group having 1 to 6 carbon atoms are as described above. m is preferably an integer from 1 to 4, more preferably 1, 2, or 3. n is preferably an integer from 1 to 3, more preferably 1 or 2.

[0639] The divalent group containing the leaving group represented by L is a divalent group composed of the above leaving group or a divalent group containing other divalent groups in addition to the above leaving group. Examples of such other divalent groups include: divalent hydrocarbon groups, divalent heterocyclic groups, -C(=O)-, -NR L -(R L represents a hydrogen atom or the above substituent), -O-, -S-, -C(=S)-, and groups composed of combinations of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) of them. Divalent hydrocarbon groups and divalent heterocyclic groups may have 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may have no substituents. From the viewpoint of synthesizing compounds with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the above groups have substituents, examples and preferred examples of such substituents are the same as above.

[0640] As the divalent hydrocarbon group, it is a linear, branched or cyclic divalent hydrocarbon group, preferably a linear or branched divalent hydrocarbon group. Examples of the divalent hydrocarbon group include: alkylene, alkenylene, alkynylene, arylene.

[0641] As the alkylene, an alkylene having 1 to 12 carbon atoms is preferred, an alkylene having 1 to 6 carbon atoms is more preferred, and an alkylene having 1 to 4 carbon atoms is particularly preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. The alkylene can be any of linear, branched or cyclic, but a linear alkylene is preferred. Examples of such an alkylene include: methylene, ethylene, propylene, butylene, pentylene, hexylene.

[0642] As the alkenylene, an alkenylene having 2 to 12 carbon atoms is preferred, an alkenylene having 2 to 6 carbon atoms is more preferred, and an alkenylene having 2 to 4 carbon atoms is particularly preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. The alkenylene can be any of linear, branched or cyclic, but a linear alkenylene is preferred. Examples of such an alkenylene include: vinylidene, propenylene, butenylene, pentenylene, hexenylene.

[0643] As the alkynylene, an alkynylene having 2 to 12 carbon atoms is preferred, an alkynylene having 2 to 6 carbon atoms is more preferred, and an alkynylene having 2 to 4 carbon atoms is particularly preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. The alkynylene can be any of linear, branched or cyclic, but a linear alkynylene is preferred. Examples of such an alkynylene include: ethynylene, propynylene, butynylene, pentynylene, hexynylene.

[0644] As the arylene, an arylene having 6 to 24 carbon atoms is preferred, an arylene having 6 to 18 carbon atoms is more preferred, an arylene having 6 to 14 carbon atoms is further preferred, and an arylene having 6 to 10 carbon atoms is still further preferred. The carbon atoms in the above do not include the carbon atoms of the substituents. Examples of the arylene include: phenylene, naphthylene, anthrylene.

[0645] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably contains one or more selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom.

[0646] As the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 1 to 21 carbon atoms is preferred, a divalent aromatic heterocyclic group having 1 to 15 carbon atoms is more preferred, a divalent aromatic heterocyclic group having 1 to 9 carbon atoms is further preferred, and a divalent aromatic heterocyclic group having 1 to 6 carbon atoms is still more preferred. The carbon atom numbers described above do not include the carbon atom numbers of substituents. More specifically, examples of the divalent aromatic heterocyclic group include: pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolidiyldiyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyldiyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl.

[0647] As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 21 carbon atoms is preferred, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is more preferred, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is further preferred, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is still more preferred. The carbon atom numbers described above do not include the carbon atom numbers of substituents. More specifically, examples of the divalent non-aromatic heterocyclic group include: pyrrolidinedionediyldiyl, pyrrolinedionediyldiyl, pyrrolinediyl, ethyleneoxidediyl, aziridinediyl, azetidinediyl, oxetanediyldiyl, thietanediyldiyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyldiyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrendiyl, morpholinodiyl, thiomorpholinodiyl, pyrazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0648] In a specific embodiment, L can be represented in the form of L1-L2.

[0649] L1 is a bond or a divalent group. The definition, examples, and preferred examples of the divalent group represented by L1 are the same as the meaning of the other divalent group in the case where the divalent group containing a leaving group represented by L is a divalent group containing other divalent groups in addition to the leaving group.

[0650] L2 is a leaving group. The definition, examples, and preferred examples of the leaving group represented by L2 are the same as the meaning of the leaving group in L.

[0651] Preferably, the leaving group represented by L2 is any one of the above (a) to (c). The examples and preferred examples of the leaving group represented by L2 are also the same as the examples described in the above (a) to (c).

[0652] The length of the main chain of L (a divalent group containing a leaving group) or L1 (a bond or divalent group)-L2 (a leaving group) that connects A (an affinity substance) and E (a divalent group containing an electrophilic group) can be appropriately designed according to various factors such as the types of the antibody and the affinity substance, and the relationship between the target site of the affinity substance in the antibody and the specific amino acid residue in the antibody to be site-selectively modified. The main chain of L or L1-L2 refers to a chain-like structure composed of multiple atoms connected by covalent bonds that connects A and E, excluding hydrogen atoms, branched structural parts, and substituents. When the compound represented by formula (I) is brought into contact with an antibody, first, A associates with the antibody. Then, a nucleophilic group (such as the amino group in the side chain of a lysine residue) in the side chain of the specific amino acid residue to be modified in the antibody present near the antibody association site reacts with the electrophilic group in E, so that the nucleophilic group binds to the electrophilic group. On the other hand, the leaving group contained in L or L1 can be detached from E. At this time, when there is no other amino acid residue of the same type as the specific amino acid residue between the antibody association site and the specific amino acid residue to be modified, even if the length of the main chain is not strictly controlled, the electrophilic group in E can bind to the nucleophilic group in the side chain of the specific amino acid residue to be modified in the antibody in a site-selective manner. Of course, even when there is another amino acid residue of the same type as the specific amino acid residue in such a region, by controlling the length of the main chain, the electrophilic group in E can also bind to the specific amino acid residue in a site-selective manner.

[0653] The length of the main chain of L or L1-L2 that connects A and E only needs to be able to site-selectively modify a specific amino acid residue in the antibody, and there is no particular limitation. For example, when site-selectively modifying a specific amino acid residue in human IgG Fc (such as the Lys248 residue or Lys246 residue, Lys288 or Lys290, Lys317, or other amino acid residues other than these residues) based on Eu numbering, the length preferably consists of 20 or fewer atoms. The length of the main chain of L or L1-L2 can also be preferably 1 or more, more preferably 2 or more, still more preferably 3 or more, and particularly preferably 4 or more or 5 or more. The length of the main chain of L or L1-L2 can also be preferably 50 or less, more preferably 30 or less, still more preferably 20 or less, and particularly preferably 15 or less or 10 or less. More specifically, the length of the main chain of L or L1-L2 can be preferably 1 to 50, more preferably 1 to 30, still more preferably 1 to 20, and particularly preferably 1 to 15 or 1 to 10. Alternatively, the length of the main chain of L or L1-L2 can be preferably 2 to 30, still more preferably 3 to 20, and particularly preferably 4 to 15 or 5 to 10.

[0654] In the case where the main chain has a non-cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain-like structure (excluding the number of atoms in hydrogen atoms, branched structure portions, and substituents).

[0655] On the other hand, in the case where the main chain has a structure containing a ring structure, from the viewpoint of defining the main chain length, it is convenient to count the number of atoms in the main chain. Specifically, the number of atoms in the main chain in such a case can be determined by counting the number of atoms in the chain-like structure in the main chain that does not contain a divalent ring structure (excluding the number of atoms in hydrogen atoms, branched structure portions, and substituents), and then counting the number of atoms in the shortest path of the two bonding bonds in the connected ring structure (for example, refer to the bold paths in the following (a) to (d)).

[0656] [Chemical formula 10]

[0657]

[0658] ● represents a bonding bond.

[0659] In the case of (a), the shortest path is the bold (boldface) path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 2.

[0660] In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 3.

[0661] In the case of (c), all paths are the shortest paths (equidistant), so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.

[0662] In the case of (d), the path at the fused portion is the shortest path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.

[0663] Preferably, the main chain of L or L1-L2 can be a chain-like structure in which the "other divalent group" in L or the "divalent group" in L1 does not contain a divalent ring structure. Therefore, the "other divalent group" in L or the "divalent group" in L1 can be a divalent straight-chain or branched hydrocarbon group, -C(=O)-, -NR L -(R Lrepresents a hydrogen atom or the above-mentioned substituents), -O-, -S-, -C(=S)-, and a group composed of a combination of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) of them. The divalent straight-chain or branched hydrocarbon group may have 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may have no substituents. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the above-mentioned group has substituents, examples and preferred examples of such substituents are the same as those of the heteroarylene group that can be used as an example of a leaving group.

[0664] 1-4. Divalent group (E) containing an electrophilic group

[0665] In formula (I), L is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0666] As the nucleophilic group in the antibody, for example, NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine residue can be mentioned. As the nucleophilic group in the antibody, NH2 in the side chain of a lysine residue or OH in the side chain of a tyrosine residue is preferred, and NH2 in the side chain of a lysine residue is more preferred.

[0667] As the electrophilic group contained in E, any electrophilic group that can be connected to the leaving group and has the ability to react with the nucleophilic group in the antibody as described above can be used, but a group selected from -C(=O)-, -SO2-, and -CH2- is preferred. As the electrophilic group, depending on the electronic balance with its adjacent groups (e.g., the leaving group, L2, E2), -CH2- can also be used. For example, when using tosyl as the leaving group, -CH2- can be appropriately used as the electrophilic group (Tsukiji et al., Nature Chemical Biology, Vol. 5, No. 5, May 2009). As the electrophilic group, -C(=O)- or -SO2- is more preferred, and -C(=O)- is even more preferred.

[0668] The divalent group containing an electrophilic group represented by E is a divalent group composed of the above-mentioned electrophilic groups or a divalent group that further contains other divalent groups in addition to the above-mentioned electrophilic groups. As such other divalent groups, for example, a divalent hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -NR E -(R Erepresents a hydrogen atom or the above-mentioned substituents), -O-, -S-, -C(=S)-, and a group composed of a combination of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) of these. The divalent hydrocarbon group and the divalent heterocyclic group may have 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may have no substituents. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the above-mentioned group has substituents, examples and preferred examples of such substituents are the same as those of the heteroarylene group that can be used as an example of the leaving group. E (and E1-E2-E3 described later) can be designed not to contain a peptide moiety that has potential immunogenicity and is prone to hydrolysis in the blood.

[0669] The divalent hydrocarbon group is a straight-chain, branched-chain or cyclic divalent hydrocarbon group, preferably a straight-chain or branched-chain divalent hydrocarbon group. Examples of the divalent hydrocarbon group include: alkylene, alkenylene, alkynylene, arylene.

[0670] As the alkylene group, an alkylene group having 1 to 12 carbon atoms is preferred, an alkylene group having 1 to 6 carbon atoms is more preferred, and an alkylene group having 1 to 4 carbon atoms is particularly preferred. The carbon atom numbers mentioned above do not include the carbon atoms of the substituents. The alkylene group can be any of straight-chain, branched-chain or cyclic, but a straight-chain alkylene group is preferred. Examples of such an alkylene group include: methylene, ethylene, propylene, butylene, pentylene, hexylene.

[0671] As the alkenylene group, an alkenylene group having 2 to 12 carbon atoms is preferred, an alkenylene group having 2 to 6 carbon atoms is more preferred, and an alkenylene group having 2 to 4 carbon atoms is particularly preferred. The carbon atom numbers mentioned above do not include the carbon atoms of the substituents. The alkenylene group can be any of straight-chain, branched-chain or cyclic, but a straight-chain alkenylene group is preferred. Examples of such an alkenylene group include: vinylidene, propenylene, butenylene, pentenylene, hexenylene.

[0672] As the alkynylene group, an alkynylene group having 2 to 12 carbon atoms is preferred, an alkynylene group having 2 to 6 carbon atoms is more preferred, and an alkynylene group having 2 to 4 carbon atoms is particularly preferred. The carbon atom numbers mentioned above do not include the carbon atoms of the substituents. The alkynylene group can be any of straight-chain, branched-chain or cyclic, but a straight-chain alkynylene group is preferred. Examples of such an alkynylene group include: ethynylene, propynylene, butynylene, pentynylene, hexynylene.

[0673] As the arylene group, an arylene group having 6 to 24 carbon atoms is preferred, an arylene group having 6 to 18 carbon atoms is more preferred, an arylene group having 6 to 14 carbon atoms is further preferred, and an arylene group having 6 to 10 carbon atoms is still further preferred. The carbon atom numbers mentioned above do not include the carbon atoms of the substituents. Examples of the arylene group include: phenylene, naphthylene, anthrylene.

[0674] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it preferably contains one or more selected from an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably contains one or more selected from an oxygen atom, a sulfur atom, and a nitrogen atom.

[0675] As the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 1 to 21 carbon atoms is preferred, a divalent aromatic heterocyclic group having 1 to 15 carbon atoms is more preferred, a divalent aromatic heterocyclic group having 1 to 9 carbon atoms is further preferred, and a divalent aromatic heterocyclic group having 1 to 6 carbon atoms is still more preferred. The above carbon atom number does not include the carbon atom number of substituents. More specifically, as the divalent aromatic heterocyclic group, for example, pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazoldiyl, imidazoldiyl, thiazoldiyl, isothiazoldiyl, oxazoldiyl, isoxazoldiyl, triazoldiyl, tetrazoldiyl, indolediyl, purinediyl, anthraquinonediy, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl can be mentioned.

[0676] As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 21 carbon atoms is preferred, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is more preferred, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is further preferred, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is still more preferred. The above carbon atom number does not include the carbon atom number of substituents. More specifically, as the divalent non-aromatic heterocyclic group, for example, pyrrolidionediy, pyrrolinedionediyl, pyrrolinediyl, oxiranediy, aziridinediy, azetidinediy, oxetanediy, thietanediy, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediy, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrendiyl, morpholinediyl, thiomorpholinediyl, pyrazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl can be mentioned.

[0677] In a specific embodiment, E can be represented in the form of E1-E2-E3.

[0678] E1 is an electrophilic group that (i) is connected to the above leaving group and (ii) has the ability to react with the nucleophilic group in the above antibody. The definition, examples, and preferred examples of the electrophilic group of E1 are the same as those of the electrophilic group in E.

[0679] E2 is the following (a) or (b):

[0680] (a) -X-Y- [Here, X bonded to E1 is C(R1)(R2) (where R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or

[0681] (b) A group represented by the following formula (i):

[0682] [Chemical Formula 11]

[0683]

[0684] (Here, ring Z is: a divalent ring group in which the ring-constituting atom X' bonded to E1 and the two adjacent ring-constituting atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and the two adjacent ring-constituting atoms are carbon atoms. · represents a bonding site.)

[0685] When E2 is the above (a), the alkyl group having 1 to 6 carbon atoms in R1 to R5 is the same as the above alkyl group.

[0686] When E2 is the above (a), from the viewpoint of improving the reactivity and stability of the compound of the present invention, as X bonded to E1, C(R1)(R2), N(R3), O, S or Se is preferred, C(R1)(R2), N(R3), O or S is more preferred, C(R1)(R2), N(R3) or O is further preferred, C(R1)(R2) or N(R3) is still further preferred, and C(R1)(R2) is most preferred.

[0687] X in E2 can also be defined in relation to a leaving group (e.g., refer to L, L2). When X is an atom or group such as N(R3), O, S, or Se, not only the leaving group but also X can be detached from the electrophilic group. However, when using a compound in a specified amount represented by formula (I) in the reaction with an antibody, X in at least a part of the compound represented by formula (I) can control the reaction so as not to be detached from the electrophilic group. Therefore, the above-mentioned atoms or groups can be used as X in the present invention. Preferably, from the viewpoint of improving the efficiency of the target reaction of the compound represented by formula (I) with the antibody and further increasing the yield of the antibody having a bioorthogonal functional group, an atom or group that is more difficult to be detached than the leaving group (i.e., the pKa value of the atom or group is greater than the pKa value of the leaving group) can be used as X. Therefore, from the viewpoint of more selectively detaching the leaving group and suppressing the detachment of X in E2 to improve the efficiency of the target reaction of the compound represented by formula (I) with the antibody and further increase the yield of the antibody having a bioorthogonal functional group, X having a detachment ability equal to or lower than that of the leaving group is preferred as X in E2. As such X, it can vary depending on the type of the leaving group (e.g., -N(R)-, -N(OR)-, -O-, -S-, or -Se-, or heteroarylene), and the presence or absence and type of a group adjacent to the leaving group that contains a group for increasing the detachment ability of the leaving group, and is outlined as follows.

[0688] (1) When the leaving group is -N(R)- or -N(OR)-, as X, C(R1)(R2) or N(R3) is preferred, and C(R1)(R2) is more preferred.

[0689] (2) When the leaving group is -O-, as X, C(R1)(R2), N(R3), or O is preferred, C(R1)(R2) or N(R3) is more preferred, and C(R1)(R2) is further more preferred.

[0690] (3) When the leaving group is -S-, as X, C(R1)(R2), N(R3), O, or S is preferred, C(R1)(R2), N(R3), or O is more preferred, C(R1)(R2) or N(R3) is further more preferred, and C(R1)(R2) is still further more preferred.

[0691] (4) When the leaving group is -Se-, as X, C(R1)(R2), N(R3), O, S, or Se is preferred, C(R1)(R2), N(R3), O, or S is more preferred, C(R1)(R2), N(R3), or O is further more preferred, C(R1)(R2) or N(R3) is still further more preferred, and C(R1)(R2) is most preferred.

[0692] (5) When the leaving group is heteroarylene, as X, C(R1)(R2), N(R3), O, S or Se is preferred, C(R1)(R2), N(R3), O or S is more preferred, C(R1)(R2), N(R3) or O is further preferred, C(R1)(R2) or N(R3) is still further preferred, and C(R1)(R2) is most preferred.

[0693] When E2 is the above (b), the ring-constituting atom X' of the ring Z bonded to E1 is a carbon atom or a nitrogen atom.

[0694] When the ring-constituting atom X' of the ring bonded to E1 is a carbon atom, the ring Z is a divalent ring group in which the ring-constituting atom X' bonded to E1 and the two adjacent ring-constituting atoms are all carbon atoms. The divalent ring group may have, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may have no substituent. From the viewpoint of synthesizing compounds with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the divalent ring group has a substituent, examples and preferred examples of such substituents are the same as the substituents that the heteroarylene which is an example of the leaving group may have. Examples of such a divalent ring group include: cyclic divalent hydrocarbon groups (such as arylene, cyclic alkylene, cyclic alkenylene, cyclic alkynylene) and divalent heterocyclic groups (such as divalent aromatic heterocyclic groups, divalent non-aromatic heterocyclic groups).

[0695] As the divalent hydrocarbon group, it is a cyclic divalent hydrocarbon group. Examples of the divalent hydrocarbon group include: cyclic alkylene, cyclic alkenylene, cyclic alkynylene, arylene.

[0696] As the cyclic alkylene, an alkylene having 3 to 12 carbon atoms is preferred, an alkylene having 3 to 10 carbon atoms is more preferred, and an alkylene having 5 to 8 carbon atoms is particularly preferred. The carbon atom numbers above do not include the carbon atoms of the substituents. Examples of such an alkylene include: cyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, cyclooctylidene, cyclononylidene, cyclodecylidene.

[0697] As the cyclic alkenylene, an alkenylene having 3 to 12 carbon atoms is preferred, an alkenylene having 3 to 10 carbon atoms is more preferred, and an alkenylene having 5 to 8 carbon atoms is particularly preferred. The carbon atom numbers above do not include the carbon atoms of the substituents. Examples of such an alkenylene include: cyclopropenylene, cyclobutenylene, cyclopentenylene, cyclohexenylene, cycloheptenylene, cyclooctenylene, cyclononenylene, cyclodecenylene.

[0698] As the cyclic alkynylene group, an alkynylene group having 6 to 12 carbon atoms is preferred, an alkynylene group having 7 to 12 carbon atoms is more preferred, and an alkynylene group having 8 to 12 carbon atoms is particularly preferred. The carbon atoms in the above do not include the carbon atoms of substituents. The alkynylene group can be any of linear, branched or cyclic, and a linear alkynylene group is preferred. Examples of such an alkynylene group include: cyclohexynylene, cycloheptynylene, cyclooctynylene, cyclononynylene, cyclodecynylene, cycloundecynylene, cyclododecynylene.

[0699] As the arylene group, an arylene group having 6 to 24 carbon atoms is preferred, an arylene group having 6 to 18 carbon atoms is more preferred, an arylene group having 6 to 14 carbon atoms is further preferred, and an arylene group having 6 to 10 carbon atoms is still more preferred. The carbon atoms in the above do not include the carbon atoms of substituents. Examples of the arylene group include: phenylene, naphthylene, anthrylene.

[0700] 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 preferred to contain one or more selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to contain one or more selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom.

[0701] As the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 3 to 21 carbon atoms is preferred, a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is more preferred, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is further preferred, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is still more preferred. The carbon atoms in the above do not include the carbon atoms of substituents. More specifically, examples of the divalent aromatic heterocyclic group include: pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazoldiyl, isothiazoldiyl, isoxazoldiyl, indolediyl, anthraquinonediy, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl.

[0702] As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 21 carbon atoms is preferred, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is more preferred, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is further preferred, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is still more preferred. The carbon atoms in the above do not include the carbon atoms of substituents. More specifically, examples of the divalent non-aromatic heterocyclic group include: pyrrolidinedionediy, pyrrolinediyl, azetidinediyl, oxetanediy, thietanediy, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, tetrahydrothiophenediyl, pyrrolinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrendiyl, pyrazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0703] When the ring-constituting atom X' bonded to E1 is a nitrogen atom, ring Z is a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and the ring-constituting atoms adjacent to both sides of the nitrogen atom are carbon atoms. Such a divalent heterocyclic group is a divalent heterocyclic group containing a nitrogen atom as a ring-constituting atom. As the divalent heterocyclic group containing a nitrogen atom as a ring-constituting atom, a divalent heterocyclic group having 3 to 21 carbon atoms is preferred, a divalent heterocyclic group having 3 to 15 carbon atoms is more preferred, a divalent heterocyclic group having 3 to 9 carbon atoms is further preferred, and a divalent heterocyclic group having 3 to 6 carbon atoms is still further preferred. The divalent heterocyclic group may have, for example, 1 to 5 substituents, preferably 1 to 3 substituents, more preferably 1 or 2 substituents, or may have no substituent. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the divalent heterocyclic group has a substituent, examples and preferred examples of such substituents are the same as those of the heteroarylene that can be used as an example of the leaving group. The number of carbon atoms mentioned above does not include the carbon atoms of the substituents. As the divalent heterocyclic group containing a nitrogen atom as a ring-constituting atom, for example, a divalent aromatic heterocyclic group containing a nitrogen atom as a ring-constituting atom and a divalent non-aromatic heterocyclic group containing a nitrogen atom as a ring-constituting atom can be mentioned. As the divalent aromatic heterocyclic group containing a nitrogen atom as a ring-constituting atom, for example, pyrrolediyl, imidazolediyl, indolediyl, purinediyl, carbazolediyl can be mentioned. As the divalent non-aromatic heterocyclic group containing a nitrogen atom as a ring-constituting atom, for example, pyrrolidinedionediy, pyrrolinedionediy, pyrrolinediy, aziridinediy, azetidinediy, pyrrolidinediy, pyrrolinediy, imidazolidinediy, piperidinediy, morpholinediy, thiomorpholinediy, pyrazinediy, dihydropyrimidinediy, and tetrahydropyrimidinediy can be mentioned.

[0704] Preferably, (b) the group represented by the above formula (i) is (b') the group represented by the following formula (i'):

[0705] [Chemical formula 12]

[0706]

[0707] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom bonded to E1 and the ring-constituting atoms adjacent to both sides thereof are all carbon atoms. · represents a bonding bond.). The definition, examples and preferred examples of the divalent cyclic group of ring Z in the group represented by the above formula (i') are the same as the meaning of the divalent cyclic group of ring Z in the group represented by the above formula (i).

[0708] When E2 is -X-Y-, E3 is a divalent group, and when E2 is a group represented by formula (i), E3 is a bond or a divalent group. The divalent group represented by E3 is the same as the other divalent group in the case where the divalent group represented by E, which contains an electrophilic group, is a divalent group containing other divalent groups in addition to the electrophilic group.

[0709] The length of the main chain of E (a divalent group containing an electrophilic group) or E1 (electrophilic group)-E2 (the above (a) or (b))-E3 (bond or divalent group) that connects L (a divalent group containing a leaving group) and B (bioorthogonal functional group) is such that it cannot participate in the position-selective modification of specific amino acid residues in the antibody in the reaction between the compound represented by formula (I) and the antibody, rather than the distance between the antibody and the bioorthogonal functional group in the antibody with a bioorthogonal functional group generated after participating in the reaction. The main chain of E or E1-E2-E3 refers to a chain-like structure composed of multiple atoms connected by covalent bonds that connects L and B, excluding hydrogen atoms, branched-chain structural parts, and substituents. Therefore, from the viewpoint of adjusting this distance, the length of the main chain of E or E1-E2-E3 can be appropriately designed.

[0710] There is no particular limitation on the length of the main chain of E or E1-E2-E3 that connects L and B, and it can be a length composed of 3 or more atoms.

[0711] In the case where the main chain is a structure without a ring structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain-like structure (excluding the number of atoms in hydrogen atoms, branched-chain structural parts, and substituents).

[0712] On the other hand, in the case where the main chain is a structure containing a ring structure, from the viewpoint of defining the length of the main chain, it is convenient to count the number of atoms in the main chain. Specifically, the number of atoms in the main chain in such a case can be determined by counting the number of atoms in the chain-like structure without a divalent ring structure in the main chain (excluding the number of atoms in hydrogen atoms, branched-chain structural parts, and substituents), and then counting the number of atoms in the shortest path connecting 2 bonding bonds in the connected ring structure.

[0713] Preferably, the main chain of E or E1-E2-E3 can be a chain-like structure in which the "other divalent group" in E or the "divalent group" in E3 does not contain a divalent ring structure. Therefore, the "other divalent group" in E or the "divalent group" in E3 can be a divalent straight-chain or branched-chain hydrocarbon group, -C(=O)-, -NR E -(R Erepresents a hydrogen atom or the above-mentioned substituents), -O-, -S-, -C(=S)-, and a group composed of a combination of two or more (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4) of them. The divalent linear or branched hydrocarbon group may have 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may have no substituents. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such substituents. On the other hand, when the above-mentioned group has substituents, examples and preferred examples of such substituents are the same as those of the heteroarylene group which can be an example of a leaving group.

[0714] 1-5. Bioorthogonal functional group (B)

[0715] In formula (I), B is a bioorthogonal functional group.

[0716] A bioorthogonal functional group refers to a group that does not react with biological components (such as amino acids, nucleic acids, lipids, sugars, phosphates), or has a slow reaction rate 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 K.B. et al., Angew.Chem.Int.Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

[0717] When the target of the affinity substance is an antibody (protein), the bioorthogonal functional group is a bioorthogonal functional group for proteins. A bioorthogonal functional group for proteins refers to a group that does not react with the side chains of the natural 20 amino acid residues that make up proteins, but reacts with a specified functional group. The natural 20 amino acids that make up proteins are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Among these 20 natural amino acids, glycine without a side chain (i.e., a hydrogen atom) and alanine, isoleucine, leucine, phenylalanine, and valine with side chains that are hydrocarbon groups (i.e., the side chains do not contain heteroatoms selected from sulfur atoms, nitrogen atoms, and oxygen atoms) are inert to ordinary reactions. Therefore, a bioorthogonal functional group for proteins is a functional group that does not react with "the side chains of these amino acids with side chains that are inert to ordinary reactions" and also does not react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0718] Examples of such bioorthogonal functional groups that do not react with proteins include: azide residues, aldehyde residues, thiol residues, alkene residues (in other words, as long as it has a vinylene (ethenylene) moiety which is the smallest unit having a double bond between carbon atoms. The same applies hereinafter), alkyne residues (in other words, as long as it has an ethynylene moiety which is the smallest unit having a triple bond between carbon atoms. The same applies hereinafter), 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 (for example, a carbonyl residue having a fluorine atom, chlorine atom, bromine atom or iodine atom at the α-position. The same applies hereinafter), isonitrile residues, sydnone residues, selenium residues. The antibody can be a protein that does not contain free thiols. In a protein that does not contain free thiols, the thiol functions as a bioorthogonal functional group. Therefore, when the target of the affinity substance is an antibody, the bioorthogonal functional group includes a thiol. The thiol residue can be an unprotected thiol residue (i.e., -SH), or a protected thiol residue. Examples of the protecting group for the thiol residue in the protected thiol residue include: hydrocarbon groups [such as alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups (such as phenyl group, naphthyl group), arylalkyl groups (aralkyl groups)], acyl groups (such as acetyl group, propoxy group, butoxycarbonyl groups such as tert-butoxycarbonyl group, benzoyl group), arylalkyloxycarbonyl groups (such as fluorenylmethyloxycarbonyl group), aryloxycarbonyl groups, arylalkyl (aralkyl) oxycarbonyl groups (such as benzyloxycarbonyl group), alkylthiol groups (tert-butylthio group), arylthiol groups (such as pyridyldisulfide group). Alternatively, the protected thiol residue can be a disulfide residue. The arylalkyl in the arylalkyl (aralkyl) and arylalkyl (aralkyl) oxycarbonyl is a group in which one or more (such as 2, 3, 4, 5) aryl groups are bonded to an alkyl group. The number of carbon atoms of the protecting group for the thiol residue is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 15, still more preferably 1 to 10, and particularly preferably 1 to 6. In the present invention, the compound represented by formula (I) may contain one or two or more (such as 2, 3, 4) bioorthogonal functional groups, and preferably, it may contain one bioorthogonal functional group.

[0719] More specifically, the bioorthogonal functional group can correspond to any one of the following chemical structures.

[0720] [Chemical formula 13]

[0721]

[0722] [In the formula,

[0723] R 1f and one or more R 1gand one or more Rs 1h which are the same or different and are an atom or group selected from (a) to (g), or an electron-withdrawing group,

[0724] ● represents a bonding site.].

[0725] As the atom or group selected from (a) to (g), it is a group selected from the groups listed below:

[0726] (a) a hydrogen atom or a halogen atom;

[0727] (b) a monovalent hydrocarbon group;

[0728] (c) an aralkyl group;

[0729] (d) a monovalent heterocyclic group;

[0730] (e) R a -O-, R a -C(=O)-, R a -O-C(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or a monovalent hydrocarbon group.).; or

[0731] (f) 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 which are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group.).;

[0732] (g) a nitro group, a sulfate group, a sulfonic acid group, a cyano group, or a carboxyl group.

[0733] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, aralkyl group, monovalent heterocyclic group, and the monovalent hydrocarbon group in R a to R c are the same as the meanings in (i) to (vii) above. Particularly preferably, the atom or group selected from (a) to (g) is the atom or group of (a) or (b).

[0734] Preferably, from the viewpoint of improving reaction efficiency and the like, even among the above-mentioned bioorthogonal functional groups, the bioorthogonal functional group may be a group selected from an azide residue, a thiol residue, an alkyne residue, a maleimide residue, and a disulfide residue.

[0735] As an electron-withdrawing group, the above-mentioned groups can be exemplified, but a halogen atom, a boric acid residue, a mesyl group, a tosyl group, a trifluoromethanesulfonate or ester is preferable.

[0736] 1-6. Preferred structure of the compound represented by formula (I)

[0737] In a preferred embodiment, the compound represented by formula (I) may be a compound represented by the following formula (I-1):

[0738] A-L1-L2-E1-E2-E3-B (I-1)

[0739] [In the formula,

[0740] A and B have the same meanings as the corresponding symbols in formula (I),

[0741] L1 is a bond or a divalent group,

[0742] L2 is a leaving group,

[0743] E1 is an electrophilic group that (i) is connected to a leaving group and (ii) has the ability to react with a nucleophilic group in an antibody,

[0744] E2 is (a) -X-Y- [where X bonded to E1 is C(R1)(R2) (where R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).), N(R3) (where R3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0745] [Chemical formula 14]

[0746]

[0747] (Here, ring Z is a divalent ring group in which the ring-constituting atom X' bonded to E1 and its two adjacent ring-constituting atoms are both carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and the two adjacent ring-constituting atoms of the nitrogen atom are carbon atoms. · is a bonding bond.)

[0748] When E2 is -X-Y-, E3 is a divalent group, and when E2 is a group represented by formula (i), E3 is a bond or a divalent group,

[0749] The leaving group has the ability to be cleaved and detached from E1 through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0750] In formula (I-1), the leaving group represented by L2 is preferably any one of the above (a) to (c). Examples and preferred examples of the leaving group represented by L2 are also the same as those described in the above (a) to (c).

[0751] In a preferred specific embodiment, the compound represented by formula (I-1) may be a compound represented by the following formula (I-2):

[0752] A-L1-L2-E1-X-Y-E3-B (I-2)

[0753] [Wherein,

[0754] A, L1, X, Y and B have the same meanings as the corresponding symbols in formula (I-1),

[0755] L2 is:

[0756] (a) Ring P-Q- [Here, ring P is a group selected from arylene which may be substituted by an electron-withdrawing group, heteroarylene which may be substituted by an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, 2-pyridone; Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0757] (b) Heteroarylene; or

[0758] (c) -Q- [Here, Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0759] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0760] E3 is a divalent group.].

[0761] In another preferred specific embodiment, the compound represented by formula (I-1) may be a compound represented by the following formula (I-3):

[0762] [Chemical formula 15]

[0763]

[0764] [Wherein,

[0765] A, L1, ring Z, the ring-forming atom X', and B have the same meanings as the corresponding symbols in the above formula (I-1).

[0766] L2 is:

[0767] (a) Ring P-Q- [Here, ring P is a group selected from arylene which may be substituted by an electron-withdrawing group, heteroarylene which may be substituted by an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, and 2-pyridone; Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0768] (b) Heteroarylene; or

[0769] (c) -Q- [Here, Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0770] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-.

[0771] E3 is a bond or a divalent group.

[0772] Preferably, the compound represented by the formula (I-3) may be the compound represented by the following formula (I-4):

[0773] [Chemical formula 16]

[0774]

[0775] [In the formula,

[0776] A, L1, and B have the same meanings as the corresponding symbols in the above formula (I-1).

[0777] L2 is:

[0778] (a) Ring P-Q-[wherein ring P is a group selected from arylene which may be substituted by an electron-withdrawing group, heteroarylene which may be substituted by an electron-withdrawing group, a ring-condensable 2,5-diketopyrrolidine, a ring-condensable 2,6-diketopiperidine, a ring-condensable 2-ketopyrrolidine, a ring-condensable 2-ketopiperidine, 2-pyridone; Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0779] (b) heteroarylene; or

[0780] (c) -Q-[wherein Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0781] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0782] Ring Z is a divalent cyclic group in which the ring-constituting atom bonded to E1 and its two adjacent ring-constituting atoms are all carbon atoms,

[0783] E3 is a bond or a divalent group.].

[0784] In the compounds represented by the above formulas (I-1) to (I-4), the definitions, examples and preferred examples of A, L1, L2, E1, E2, E3 and B, and the alkyl group having 1 to 6 carbon atoms in -X-Y-, R1 to R5, and the group represented by formula (i) (such as a divalent cyclic group, a divalent heterocyclic group) are the same as the corresponding contents above. In addition, the definitions, examples and preferred examples of (a) to (c) as L2, and ring Z (such as a divalent cyclic group in which the ring-constituting atom X' bonded to E1 and its two adjacent ring-constituting atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and the two adjacent ring-constituting atoms of the nitrogen atom are carbon atoms), ring P and Q (such as the alkyl group having 1 to 6 carbon atoms in R), etc. are also the same as the corresponding contents above.

[0785] 1-7. Preparation method

[0786] A compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group can be appropriately prepared. The compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group is represented by formula (I), preferably formula (I-1), more preferably formula (I-2), (I-3) or (I-4).

[0787] As the affinity substance (A) for an antibody, a substance having an arbitrary functional group can be appropriately selected. Therefore, by using a reactive group that can react with the functional group, the affinity substance is made to react with the structural unit represented by L-E-B, and the structural unit represented by A-L-E-B can be prepared. For example, such a reaction can be carried out in a suitable reaction system, such as an organic solvent system or an aqueous solution system, at a suitable temperature (for example, about 15 to 200 °C). The reaction system may contain a suitable 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.

[0788] In the reaction system, the molar ratio (Y / X) of the structural unit (Y) represented by L-E-B to the affinity substance (X) varies depending on the types of the structural unit and the affinity substance, the number of sites in the affinity substance to be modified by the structural unit, etc., and thus is not particularly limited. For example, it is 0.1 to 50, preferably 0.5 to 40, more preferably 1 to 35, even more preferably 2 to 25, and particularly preferably 3 to 15.

[0789] The confirmation of the formation of a compound or its salt having an affinity substance for an antibody and a bioorthogonal functional group depends on the specific molecular weights of its raw materials and products, but can be carried out by, for example, electrophoresis, chromatography (such as gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, and is preferably carried out by mass spectrometry. A compound or its salt having an affinity substance for an antibody and a bioorthogonal functional group can be appropriately purified by any method such as chromatography (such as the above-mentioned chromatography and affinity chromatography).

[0790] 1-8. Others

[0791] In the inventions described later [for example, the inventions represented by formula (II), (III) and their subordinate concept formulas and partial structural formulas], the details of any symbol (such as A, L, E, B) and the terms related to the symbol (such as definitions, examples and preferred examples) are common to the inventions of the compound or its salt represented by the above formula (I) or its subordinate concept formulas. In addition, for any technical elements (such as definitions, examples and preferred examples) that can define specific groups (such as bioorthogonal functional groups, divalent groups, substituents) and specific numerical values, etc. of the inventions described later, they can also be common to the content described above. Therefore, in the absence of special instructions, these matters can be appropriately cited in the inventions described later. Similarly, the specific inventive technical elements described in the inventions described later can be appropriately cited as the technical elements of the present invention and other inventions.

[0792] 2. Method for Preparing Antibody Having Bioorthogonal Functional Group

[0793] The present invention provides a method for preparing an antibody having a bioorthogonal functional group or a salt thereof, and the preparation method includes the following steps.

[0794] (1) Reacting a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) with the antibody to generate an antibody having a bioorthogonal functional group or a salt thereof represented by the following formula (II),

[0795] A-L-E-B (I)

[0796] [In the formula,

[0797] A is an affinity substance for an antibody,

[0798] L is a divalent group containing a leaving group,

[0799] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody,

[0800] B is a bioorthogonal functional group,

[0801] The leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.];

[0802] Ab-E-B (II)

[0803] [In the formula,

[0804] E and B have the same meanings as the corresponding symbols in formula (I),

[0805] Ab is an antibody.].

[0806] The antibody used in the method for preparing an antibody having a bioorthogonal functional group is the same as the above-mentioned antibody. Preferably, the antibody is a monoclonal antibody. As the isotype of the monoclonal antibody, for example, IgG (such as IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY can be mentioned. The monoclonal antibody is a full-length antibody or an antibody fragment (such as F(ab’)2, Fab’, Fab, Fv, single-chain antibody), but a full-length antibody is preferred. Particularly preferably, the antibody is a human antibody, a humanized antibody, or a chimeric antibody having human IgG (such as IgG1, IgG2, IgG3, IgG4) in the constant region.

[0807] Ab in formula (II) is the same as the above-mentioned antibody and undergoes covalent bonding with the electrophilic group in E. As the nucleophilic group in the antibody for covalent bonding with the electrophilic group in E, for example, the following can be mentioned: NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine residue.

[0808] E in formula (II) is the same as E in the above-mentioned formula (I). E can be represented by E1-E2-E3. The meanings of E1, E2, and E3 are the same as those of the corresponding symbols described above. As described above, E and E1-E2-E3 can be designed not to contain a peptide moiety that has potential immunogenicity and is prone to hydrolysis in the blood. In this case, the antibody having a bioorthogonal functional group represented by formula (II) can be used to prepare an antibody having a functional substance without the above-mentioned problems.

[0809] The electrophilic group in E and the electrophilic group in E1 undergo covalent bonding with the nucleophilic group in the antibody. As the electrophilic group for covalent bonding with the nucleophilic group in the antibody, for example, the following can be mentioned: NH-C(=O)-, NH-SO2-, and NH-CH2- (when the nucleophilic group in the antibody for covalent bonding with E is NH2 in the side chain of a lysine residue), O-C(=O)-, O-SO2-, and O-CH2- (when the nucleophilic group in the antibody for covalent bonding with E is OH in the side chain of a tyrosine residue, a serine residue, or a threonine residue), S-C(=O)- and S-CH2- (when the nucleophilic group in the antibody for covalent bonding with E is SH in the side chain of a cysteine residue). As the electrophilic group for covalent bonding with the nucleophilic group in the antibody, NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody for covalent bonding with E is NH2 in the side chain of a lysine residue) are preferred, O-C(=O)- and O-SO2- (when the nucleophilic group in the antibody for covalent bonding with E is OH in the side chain of a tyrosine residue) are more preferred, and NH-C(=O)- (when the nucleophilic group in the antibody for covalent bonding with E is NH2 in the side chain of a lysine residue) is even more preferred.

[0810] B in formula (II) is the same as B in the above-mentioned formula (I).

[0811] Preferably, the antibody having a bioorthogonal functional group prepared by the preparation method of the present invention is an antibody having a bioorthogonal functional group in a site-selective manner. When the compound or its salt having an affinity substance for the antibody and a bioorthogonal functional group is a substance represented by the formula (I), an antibody having a bioorthogonal functional group in a site-selective manner represented by the formula (II) can be prepared. The antibody having a bioorthogonal functional group in a site-selective manner is preferably an antibody having a bioorthogonal functional group only in the constant region, and more preferably an antibody having a bioorthogonal functional group only in the Fc region.

[0812] In the present specification, "site-selective" or "site selectivity" means that although a specific amino acid residue does not unevenly exist in a specific region in the antibody, a specified structural unit capable of binding to the specific amino acid residue in the antibody unevenly exists in a specific region in the antibody. Therefore, expressions related to site selectivity such as "having in a site-selective manner", "site-selective binding", "binding in a site-selective manner", etc. mean that the binding rate or retention rate of the specified structural unit in the target region containing one or more specific amino acid residues is significantly higher than the retention rate or binding rate of the structural unit in the non-target region containing a plurality of amino acid residues of the same kind as the specific amino acid residue in the target region. Such site-selective binding or retention can be achieved by the present invention. The present invention does not randomly react a specified structural unit with a specific amino acid residue in the antibody, but by using a compound containing an affinity substance for the antibody, the specified structural unit can preferentially react with a specific amino acid residue in the target region in the antibody.

[0813] More specifically, when the antibody (Ab) contains one or more specific amino acid residues (such as lysine residue, tyrosine residue, threonine residue, serine residue, cysteine residue) in a target region composed of 1 to 50 consecutive amino acid residues [for example, a region composed of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region composed of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region composed of the amino acid residue at position 317 in the human IgG Fc region] and contains 5 or more of the above specific amino acid residues in a non-target region other than the above target region, a partial structure other than the antibody can bind to one or more specific amino acid residues contained in the target region with a site selectivity of 30% or more. The site selectivity can be preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0814] The antibody having a bioorthogonal functional group prepared by the preparation method of the present invention may have a bioorthogonal functional group (i.e., a structural unit represented by E-B) according to the number of heavy chains. An antibody having a bioorthogonal functional group at a position selectively can be prepared as follows: First, a compound (A-L-E-B) represented by formula (I) is associated with the constant region of the antibody heavy chain via an affinity substance (A) for the antibody, and then an electrophilic group in E is reacted with a nucleophilic group in the side chain of a specific amino acid residue near the association site (the constant region of the same heavy chain as the above antibody heavy chain), whereby an antibody can be obtained. Therefore, by using an antibody having a plurality of (for example, 1 to 8, preferably 1 to 4, more preferably 2) antibody heavy chains in the preparation method of the present invention, an antibody having a plurality of structural units (or a plurality of structural units of a subordinate concept thereof) represented by E-B can be prepared at a position selectively in the same target region of a plurality of antibody heavy chains. For example, by using an antibody having 2 antibody heavy chains (for example, IgG, IgD, IgE, F(ab’)2 antibody, Fc region protein, Fc fusion protein) in the preparation method of the present invention, an antibody having 2 structural units represented by E-B can be prepared at a position selectively in the same target region of 2 antibody heavy chains. That is, in the antibody having a bioorthogonal functional group, the modification mode based on the bioorthogonal functional group can be made the same between a plurality of (for example, 2) heavy chains.

[0815] The antibody having a bioorthogonal functional group may also have the same or different bioorthogonal functional groups (for example, a structural unit represented by E-B) in a plurality of (for example, 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions of 1 antibody heavy chain. In this case, in the antibody having a bioorthogonal functional group, the modification mode based on the bioorthogonal functional group can be made the same between a plurality of (for example, 2) heavy chains.

[0816] The preparation method of the present invention may further include: generating an antibody having a bioorthogonal functional group and further being modified by subjecting the generated antibody to a specific treatment. As such a specific treatment, for example, fragmentation treatment of the antibody (for example, treatment with a specific protease such as papain, pepsin, etc.) can be mentioned.

[0817] When a compound represented by formula (I) or a salt thereof is used in the preparation method of the present invention, an antibody having a bioorthogonal functional group represented by the above formula (II) can be prepared.

[0818] In a preferred embodiment, when a compound represented by formula (I-1) is used as the compound represented by formula (I) in the preparation method of the present invention, an antibody having a bioorthogonal functional group represented by the following formula (II-1) can be prepared.

[0819] Ab-E1-E2-E3-B (II-1)

[0820] [In the formula,

[0821] Ab is an antibody,

[0822] E1 is an electrophilic group linked to a nucleophilic group in the antibody,

[0823] E2 is (a) -X-Y- [wherein, X which binds to E1 is C(R1)(R2) (wherein, R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (wherein, R3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S or Se, and Y which binds to E3 is C(R4)(R5) (wherein, R4 or R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0824] [Chemical formula 17]

[0825]

[0826] (wherein, ring Z is a divalent ring group in which the ring-constituting atom X' which binds to E1 and its two adjacent ring-constituting atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' which binds to E1 is a nitrogen atom and the two adjacent ring-constituting atoms of the nitrogen atom are carbon atoms. · represents a binding bond.).

[0827] E3 is a divalent group when E2 is -X-Y-, and is a bond or a divalent group when E2 is a group represented by the formula (i).

[0828] B is a bioorthogonal functional group.].

[0829] In a preferred specific embodiment, when a compound represented by the formula (I-2) is used as the compound represented by the formula (I-1) in the preparation method of the present invention, an antibody having a bioorthogonal functional group represented by the following formula (II-2) can be prepared.

[0830] Ab-E1-X-Y-E3-B (II-2)

[0831] [In the formula,

[0832] Ab, X, Y and B have the same meanings as the corresponding symbols in the formula (II-1),

[0833] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0834] E3 is a divalent group.].

[0835] In another preferred specific embodiment, when a compound represented by formula (I-3) is used in the preparation method of the present invention as the compound represented by formula (I-1), an antibody having a bioorthogonal functional group represented by the following formula (II-3) can be prepared.

[0836] [Chemical formula 18]

[0837]

[0838] [Wherein,

[0839] Ab, the ring-constituting atom X', the ring Z and B have the same meanings as the corresponding symbols in formula (II-1),

[0840] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0841] E3 is a bond or a divalent group. ].

[0842] Preferably, when a compound represented by formula (I-4) is used in the preparation method of the present invention as the compound represented by formula (I-3), an antibody having a bioorthogonal functional group represented by the following formula (II-4) can be prepared.

[0843] [Chemical formula 19]

[0844]

[0845] [Wherein,

[0846] Ab, the ring Z and B have the same meanings as the corresponding symbols in formula (II-1),

[0847] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0848] E3 is a bond or a divalent group. ].

[0849] Details (such as definitions, examples, and preferred examples) of any symbol (such as E, E1, E2, E3, B) in formula (II), (II-1), (II-2), (II-3), or (II-4) and the terms (such as antibody, electrophilic group, bioorthogonal functional group) represented in relation to that symbol are common to the corresponding meanings in the above formula (I) or the formula of its subordinate concepts.

[0850] A compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group can react with the antibody because it has an electrophilic group in E or an electrophilic group in E1. 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 a suitable reaction system, such as in 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, more preferably 6.0 to 8.0. The buffer solution may contain a suitable 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, still more preferably 30 minutes to 8 hours. For details of such a reaction, see, for example, G.J.L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G.J.L. Bernardes et al., Chem. Asian J., 4, 630 (2009); B.G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate Chem., 25, 825 (2014).

[0851] In the reaction system, the molar ratio (Y / X) of the compound or its salt (Y) having an affinity substance for an antibody and a bioorthogonal functional group to the antibody (X) varies depending on the types of the compound or its salt having an affinity substance for an antibody and a bioorthogonal functional group and the antibody, the number of sites in the antibody to be modified by the compound or its salt having an affinity substance for an antibody and a bioorthogonal functional group (e.g., DAR), etc., and thus is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, still more preferably 2 to 50, and particularly preferably 3 to 30.

[0852] Confirmation of the production of an antibody having a bioorthogonal functional group can be carried out by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, although it also depends on the molecular weights of its specific raw materials and products, and is preferably carried out by mass spectrometry. Confirmation of position selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, it can be carried out, for example, by treatment with a protease (e.g., trypsin, chymotrypsin) and mass spectrometry. As the protease, an endoprotease is preferred. As such an endoprotease, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N can be mentioned. Confirmation of the number of bioorthogonal functional groups retained by the antibody having a bioorthogonal functional group can be carried out by, for example, electrophoresis, chromatography, or mass spectrometry, and is preferably carried out by mass spectrometry. The antibody having a bioorthogonal functional group can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0853] 3. Preparation method of an antibody having a functional substance using a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group

[0854] The present invention provides a method for preparing an antibody having a functional substance or a salt thereof, the preparation method including the following steps.

[0855] (1) React a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) with an antibody to generate an antibody having a bioorthogonal functional group or a salt thereof represented by the following formula (II).

[0856] A-L-E-B (I)

[0857] [In the formula,

[0858] A is an affinity substance for an antibody,

[0859] L is a divalent group containing a leaving group,

[0860] E is a divalent group containing an electrophilic group, the electrophilic group being: (i) connected to the leaving group and (ii) having the ability to react with a nucleophilic group in the antibody,

[0861] B is a bioorthogonal functional group,

[0862] The leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.]

[0863] Ab-E-B (II)

[0864] [In the formula,

[0865] E and B have the same meanings as the corresponding symbols in the above formula (I),

[0866] Ab is an antibody.]; and

[0867] (2) Reacting the antibody or a salt thereof having a bioorthogonal functional group represented by the above formula (II) with a functional substance via the bioorthogonal functional group to produce an antibody or a salt thereof having a functional substance represented by the following formula (III):

[0868] Ab-E-B’-F (III)

[0869] [In the formula,

[0870] Ab has the same meaning as the corresponding symbol in formula (II),

[0871] E has the same meaning as the corresponding symbol in formula (I),

[0872] B’ is a divalent group containing a part formed by the reaction between the functional substance and the bioorthogonal functional group,

[0873] F is a functional substance.].

[0874] Step (1) can be carried out in the same manner as the method for preparing an antibody having a bioorthogonal functional group.

[0875] The functional substance (F) used in step (2) is not particularly limited as long as it is a substance that imparts an arbitrary function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, but drugs or labeling substances are preferred. In addition, the functional substance may be a single functional substance, or may be a substance obtained by linking two or more functional substances.

[0876] As a drug, it can be a drug for any disease. Examples of such diseases include: cancer (e.g., lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, renal cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune-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 infectious diseases, viral infectious diseases), genetic-rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia syndrome), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), diseases in the orthopedic and plastic surgery fields (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug can be a drug for treating or preventing a specified disease, or a drug for reducing the side effects associated with the use of an antibody against a target protein for a specified disease.

[0877] More specifically, the drug is an anticancer agent. Examples of anticancer agents include: chemotherapeutic agents, toxins, radioisotopes, or substances containing them. Examples of 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, platinum compounds. Examples of toxins include: bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin). Examples of radioisotopes include: radioactive isotopes of hydrogen atoms (e.g., 3 H), radioactive isotopes of carbon atoms (e.g., 14 C), radioactive isotopes of phosphorus atoms (e.g., 32 P), radioactive isotopes of sulfur atoms (e.g., 35 S), radioactive isotopes of yttrium (e.g., 90 Y), radioactive isotopes of technetium (e.g., 99m Tc), radioactive isotopes of indium (e.g., 111 In), radioactive isotopes of iodine atoms (e.g., 123 I, 125 I, 129 I, 131 I), radioactive isotopes of samarium (e.g., 153 Sm), radioactive isotopes of rhenium (e.g., 186 Re), radioactive isotopes of astatine (e.g., 211 At), radioactive isotopes of bismuth (e.g., 212 Bi).

[0878] Examples of the labeling substance include: enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxin, aptamer), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), radioisotopes (e.g., the above-mentioned radioisotopes) or substances containing them.

[0879] In addition, the functional substance is a high molecular compound, a medium molecular compound or a low molecular compound, preferably a low molecular compound. A low molecular compound refers to a compound having a molecular weight of 1500 or less. The low molecular compound is a natural compound or a synthetic compound. The molecular weight of the low molecular compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less or 300 or less. In addition, the molecular weight of the low molecular compound may be 30 or more, 40 or more or 50 or more. The low molecular compound may be a drug or a labeling substance as described above. Examples of the low molecular compound also include: amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids and their salts.

[0880] Functional substances have various functional groups corresponding to their structures. When a functional substance has a functional group that readily reacts with a bioorthogonal functional group, the functional group of the functional substance can be appropriately reacted with the bioorthogonal functional group. The functional group that readily reacts with a bioorthogonal functional group can vary depending on the specific type of the bioorthogonal functional group. A person skilled in the art can appropriately select a suitable functional group as the functional group that readily reacts with a bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174 - 2195). As the functional group that readily reacts with a bioorthogonal functional group, for example, an azide residue can be cited when the bioorthogonal functional group is an alkyne residue, a hydrazine residue can be cited when the bioorthogonal functional group is an aldehyde residue or a ketone residue, and a maleimide residue and a disulfide residue can be cited when the bioorthogonal functional group is a thiol residue, but it is not limited to these. For example, when the bioorthogonal functional group is an alkyne residue and the functional group that readily reacts with the bioorthogonal functional group is an azide residue (or vice versa), the divalent group containing the part formed by the reaction between the functional substance and the bioorthogonal functional group can be a divalent group containing a triazole residue (which may or may not be fused with other rings); when the bioorthogonal functional group is an aldehyde residue or a ketone residue and the functional group that readily reacts with the bioorthogonal functional group is a hydrazine residue (or vice versa), the divalent group containing the part formed by the reaction between the functional substance and the bioorthogonal functional group can be a divalent group containing a hydrazone residue; when the bioorthogonal functional group is a thiol residue and the functional group that readily reacts with the bioorthogonal functional group is a maleimide residue or a disulfide residue (or vice versa), the divalent group containing the part formed by the reaction between the functional substance and the bioorthogonal functional group can be a divalent group containing a thiosuccinimide residue or a divalent group containing a disulfide residue (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174 - 2195). The divalent group containing a triazole residue (which may or may not be fused with other rings), the divalent group containing a hydrazone residue, the divalent group containing a thiosuccinimide residue, or the divalent group containing a disulfide residue are preferred examples of the divalent group containing the part formed by the reaction between the functional substance and the bioorthogonal functional group.

[0881] On the other hand, when a functional substance does not have a functional group that readily reacts with a bioorthogonal functional group, as the functional substance, a substance having a desired functional group through derivatization can be used. For example, when the functional substance is a soluble protein, a substance having a functional group that the soluble protein does not naturally have through derivatization can be used.

[0882] Derivatization is common general knowledge in the art (for example, see International Publication No. WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, and U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization can be carried out using a crosslinking agent as described above. Alternatively, derivatization can be carried out using a specific linker having the desired functional group. For example, such a linker can be a group that can separate the functional substance from the antibody by cleavage of the linker in a suitable environment (such as intracellularly or extracellularly). Examples of such linkers include: peptidyl linkers that are cleaved by specific proteases [e.g., intracellular proteases (such as proteases present in lysosomes or endosomes), extracellular proteases (such as secreted proteases)] (e.g., U.S. Patent No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), linkers that can be cleaved at locally acidic sites present in the living body (e.g., U.S. Patent No. 5,622,929, U.S. Patent No. 5,122,368, U.S. Patent No. 5,824,805). The linker can be self-immolative (e.g., International Publication No. WO 02 / 083180, International Publication No. WO 04 / 043493, International Publication No. WO 05 / 112919). In the present invention, the derivatized functional substance may also be simply referred to as "functional substance".

[0883] Ab in formula (III) is the same as the above-described antibody and undergoes covalent bonding with the electrophilic group in E. Examples of the nucleophilic group in the antibody for covalent bonding with the electrophilic group in E include: NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine residue.

[0884] E in formula (III) is the same as E in formula (I) above. E can be represented as E1-E2-E3. E1, E2, and E3 have the same meanings as the corresponding symbols described above. As described above, E and E1-E2-E3 can be designed not to include a peptide portion that has potential immunogenicity and is prone to hydrolysis in the blood. In this case, the antibody having a functional substance represented by formula (III) can be suitably used as a drug.

[0885] The electrophilic group in E and the electrophilic group in E1 form a covalent bond with the nucleophilic group in the antibody. Examples of the electrophilic group that forms a covalent bond with the nucleophilic group in the antibody include: NH-C(=O)-, NH-SO2-, and NH-CH2- (when the nucleophilic group in the antibody that covalently bonds with E is the NH2 in the side chain of a lysine residue), O-C(=O)-, O-SO2-, and O-CH2- (when the nucleophilic group in the antibody that covalently bonds with E is the OH in the side chain of a tyrosine residue, a serine residue, or a threonine residue), S-C(=O)- and S-CH2- (when the nucleophilic group in the antibody that covalently bonds with E is the SH in the side chain of a cysteine residue). As the electrophilic group that forms a covalent bond with the nucleophilic group in the antibody, NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody that covalently bonds with E is the NH2 in the side chain of a lysine residue), O-C(=O)- and O-SO2- (when the nucleophilic group in the antibody that covalently bonds with E is the OH in the side chain of a tyrosine residue) are preferred, NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody that covalently bonds with E is the NH2 in the side chain of a lysine residue) are more preferred, and NH-C(=O)- (when the nucleophilic group in the antibody that covalently bonds with E is the NH2 in the side chain of a lysine residue) is even more preferred.

[0886] In a specific embodiment, when the functional substance has a functional group that is prone to react with a bioorthogonal functional group, or when it is derivatized to have a functional group that is prone to react with a bioorthogonal functional group, the functional group that is prone to react with the bioorthogonal functional group can be a group selected from the following: azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halocarbonyl residue, isonitrile residue, sydnone residue, selenium residue.

[0887] Moreover, in a specific embodiment, when the functional substance has a functional group that is prone to react with a bioorthogonal functional group, or when it is derivatized to have a functional group that is prone to react with a bioorthogonal functional group, the functional group that is prone to react with the above bioorthogonal functional group can be a group selected from the groups represented by the following formula.

[0888] [Chemical formula 20]

[0889]

[0890] [In the formula,

[0891] R 1f one or more Rs 1g and one or more Rs 1h are the same or different and are an atom or group selected from the above (i) to (vii), or an electron-withdrawing group, and · is a bonding bond for the functional substance.

[0892] The divalent group represented by B’ in formula (III) and containing a part formed by the reaction between the functional substance and the bioorthogonal functional group may be: (1) a divalent group containing the residues mentioned in the above preferred examples, namely a triazole residue, a hydrazone residue or a succinimidyl thioester residue; or (2) not particularly limited, and may be, for example, a divalent group containing a residue selected from the following: a disulfide residue (this residue is the residue mentioned in the above preferred example), an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tert-alkyloxycarbamate residue, a silane residue, a residue containing a hydrazone, an aminophosphate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring with an electron-withdrawing group, a residue containing a coumarin, a residue containing a sulfone, a chain residue containing an unsaturated bond, a glycosyl residue.

[0893] In addition, the divalent group containing a part formed by the reaction between the functional substance and the bioorthogonal functional group is not particularly limited, and may be, for example, a divalent group containing a residue corresponding to any one of the following chemical structures:

[0894] [Chemical formula 21]

[0895]

[0896] [Here, the wavy line orthogonal to the bond represents the bond formed by the reaction,

[0897] multiple Rs 2a multiple Rs 2b and multiple Rs 2c are the same or different and are a hydrogen atom or the above substituent,

[0898] J is -CH2-, -O- or -S-,

[0899] r is any integer from 1 to 4,

[0900] ○ (white circle) represents the bond to the F side part, and ● (black circle) represents the bond to the B’ side part,

[0901] In the case where the chemical structure is asymmetric with the cleavable part as the center, it may be that ● represents the bond to the B’ side part and ○ represents the bond to the F side part.

[0902] Preferably, the antibody having a functional substance prepared by step (2) in the preparation method of the present invention is an antibody having a functional substance in a position-selective manner. When the antibody having a bioorthogonal functional group in a position-selective manner is an antibody represented by formula (II), an antibody having a functional substance in a position-selective manner represented by formula (III) can be prepared. The antibody having a functional substance in a position-selective manner is preferably an antibody having a functional substance only in the constant region, and more preferably an antibody having a functional substance only in the Fc region.

[0903] When the antibody (Ab) contains one or more specific amino acid residues (such as lysine residue, tyrosine residue, threonine residue, serine residue, cysteine residue) in a target region composed of 1 to 50 consecutive amino acid residues [for example, a region composed of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region composed of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region composed of the amino acid residue at position 317 in the human IgG Fc region] and contains five or more of the above specific amino acid residues in a non-target region other than the above target region, a partial structure other than the antibody can bind to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more. The position selectivity can be preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0904] The antibody having a functional substance prepared by the preparation method of the present invention can also have a functional substance (i.e., a structural unit represented by E-B'-F) according to the number of heavy chains. This is because: an antibody having a bioorthogonal functional group that can have a structural unit represented by E-B according to the number of heavy chains can be used to prepare an antibody having a functional substance. Therefore, by using an antibody having multiple (such as 1 to 8, preferably 1 to 4, more preferably 2) antibody heavy chains in the preparation method of the present invention, an antibody having multiple structural units (or multiple structural units of a subordinate concept thereof) represented by E-B'-F in a position-selective manner in the same target region of multiple antibody heavy chains can be prepared. For example, by using an antibody having 2 antibody heavy chains (such as IgG, IgD, IgE, F(ab')2 antibody, Fc region protein, Fc fusion protein) in the preparation method of the present invention, an antibody having 2 structural units represented by E-B'-F in a position-selective manner in the same target region of 2 antibody heavy chains can be prepared. That is, in an antibody having a functional substance, the modification mode based on the functional substance can be made the same among multiple (such as 2) heavy chains.

[0905] An antibody having a functional substance may also have the same or different functional substances (e.g., a structural unit represented by E-B’-F) in a plurality of (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions of one antibody heavy chain. In this case, in the antibody having a functional substance, the modification mode based on the functional substance can be made the same between a plurality of (e.g., 2) heavy chains.

[0906] The production method of the present invention may further include: generating an antibody having a functional substance and further modified by subjecting the generated antibody to a specific treatment. Examples of such a specific treatment include: fragmentation treatment of the antibody (e.g., treatment with specific proteases such as papain and pepsin).

[0907] When a compound represented by formula (I) or a salt thereof is used in the production method of the present invention, in step (1), an antibody having a bioorthogonal functional group represented by formula (II) above can be prepared, and then, in step (2), an antibody having a functional substance represented by formula (III) above can be prepared.

[0908] In a preferred embodiment, when a compound represented by formula (I-1) is used as the compound represented by formula (I) in the production method of the present invention, in step (1), an antibody having a bioorthogonal functional group represented by formula (II-1) above can be prepared, and then, in step (2), an antibody having a functional substance represented by formula (III-1) below can be prepared.

[0909] Ab-E1-E2-E3-B’-F (III-1)

[0910] [In the formula,

[0911] Ab is an antibody,

[0912] E1 is an electrophilic group that binds to a nucleophilic group in the antibody,

[0913] E2 is (a) -X-Y- [where, X that binds to E1 is C(R1)(R2) (where, R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where, R3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S or Se, and Y that binds to E3 is C(R4)(R5) (where, R4 or R5 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0914] [Chemical formula 22]

[0915]

[0916] (Here, ring Z is a divalent cyclic group in which the ring-forming atom X' bonded to E1 and the ring-forming atoms of the two adjacent rings of atom X' are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' bonded to E1 is a nitrogen atom and the ring-forming atoms of the two adjacent rings of the nitrogen atom are carbon atoms. · represents a bonding site.)

[0917] E3 is a divalent group when E2 is -X-Y-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0918] B' is a divalent group containing a part formed by the reaction between a functional substance and a bioorthogonal functional group.

[0919] F is a functional substance.]

[0920] In a preferred specific embodiment, when the compound represented by formula (I-2) is used as the compound represented by formula (I-1) in the preparation method of the present invention, the antibody having a bioorthogonal functional group represented by the above formula (II-2) can be prepared in step (1), and then, the antibody having a functional substance represented by the following formula (III-2) can be prepared in step (2).

[0921] Ab-E1-X-Y-E3-B'-F (III-2)

[0922] [In the formula,

[0923] Ab, X, Y, B' and F have the same meanings as the corresponding symbols in the above formula (III-1),

[0924] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-.

[0925] E3 is a divalent group.]

[0926] In another preferred specific embodiment, when the compound represented by formula (I-3) is used as the compound represented by formula (I-1) in the preparation method of the present invention, the antibody having a bioorthogonal functional group represented by the above formula (II-3) can be prepared in step (1), and then, the antibody having a functional substance represented by the following formula (III-3) can be prepared in step (2).

[0927] [Chemical formula 23]

[0928]

[0929] [In the formula,

[0930] Ab, the ring-forming atom X', ring Z, B' and F have the same meanings as the corresponding symbols in formula (III-1),

[0931] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-.

[0932] E3 is a bond or a divalent group.].

[0933] Preferably, when a compound represented by formula (I-4) is used as the compound represented by formula (I-3) in the production method of the present invention, an antibody having a bioorthogonal functional group represented by the following formula (II-4) can be prepared in step (1), and then, an antibody having a functional substance represented by the following formula (III-4) can be prepared in step (2).

[0934] [Chemical formula 24]

[0935]

[0936] [In the formula,

[0937] Ab, ring Z, B', and F have the same meanings as the corresponding symbols in formula (III-1),

[0938] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-.

[0939] E3 is a bond or a divalent group.].

[0940] Details (such as definitions, examples, and preferred examples) of any symbol (e.g., E, E1, E2, E3, B) in formula (III), (III-1), (III-2), (III-3), or (III-4) and the term (e.g., antibody, electrophilic group, bioorthogonal functional group) represented in relation to the symbol are common to the corresponding meanings in the above formula (I) or the formula of its subordinate concepts.

[0941] The antibody having a bioorthogonal functional group can react with a functional substance via the bioorthogonal functional group. Such a reaction can be appropriately carried out under the conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds) as described above.

[0942] In the reaction system, the molar ratio (Z / Y) of the functional substance (Z) to the antibody (Y) having a bioorthogonal functional group varies depending on the types of the bioorthogonal functional group, the functional substance, and the antibody, as well as the number of sites (e.g., DAR) in the antibody to be modified, and thus is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, still more preferably 2 to 50, and particularly preferably 3 to 30.

[0943] Confirmation of the production of an antibody having a functional substance can be carried out by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, although it also depends on the molecular weights of its specific raw materials and products, and is preferably carried out by mass spectrometry. Confirmation of site selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, it can be carried out, for example, by treatment with a protease (e.g., trypsin, chymotrypsin) and mass spectrometry. As the protease, an endoprotease is preferred. As such an endoprotease, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N can be mentioned. Confirmation of the number of functional substances retained by an antibody having a functional substance can be carried out by, for example, electrophoresis, chromatography or mass spectrometry, and is preferably carried out by mass spectrometry. An antibody having a functional substance can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0944] 4. An antibody or a salt thereof that selectively has a bioorthogonal functional group or a functional substance at a specific position

[0945] The present invention provides an antibody or a salt thereof that selectively has a bioorthogonal functional group or a functional substance at a specific position.

[0946] An antibody or a salt thereof that selectively has a bioorthogonal functional group at a specific position is the antibody that selectively has a bioorthogonal functional group represented by the above formula (II-1). Preferably, as the antibody that selectively has a bioorthogonal functional group represented by the above formula (II-1), an antibody that selectively has a bioorthogonal functional group represented by the above formula (II-2) or (II-3) is provided. More preferably, as the antibody that selectively has a bioorthogonal functional group represented by the above formula (II-3), an antibody that selectively has a bioorthogonal functional group represented by the above formula (II-4) is provided. The details (e.g., definitions, examples, and preferred examples) of any symbol (e.g., E, E1, E2, E3, B) in formula (II), (II-1), (II-2), (II-3), or (II-4) and the terms (e.g., antibody, electrophilic group, bioorthogonal functional group) represented in association with the symbol are common to the corresponding meanings in the above formula (I) or its subordinate concept formula.

[0947] An antibody or a salt thereof that selectively has a functional substance at a position is an antibody that selectively has a functional substance at a position represented by the above formula (III-1). Preferably, as the antibody that selectively has a functional substance at a position represented by the above formula (III-1), an antibody that selectively has a functional substance at a position represented by the above formula (III-2) or (III-3) is provided. More preferably, as the antibody that selectively has a functional substance at a position represented by the above formula (III-3), an antibody that selectively has a functional substance at a position represented by the above formula (III-4) is provided. The details (such as definitions, examples, and preferred examples) of any symbol (such as E, E1, E2, E3, B) in formula (III), (III-1), (III-2), (III-3), or (III-4) and the term represented in association with the symbol (such as antibody, electrophilic group, bioorthogonal functional group) are common to the corresponding meanings in the above formula (I) or a formula of its subordinate concept.

[0948] An antibody that selectively has a bioorthogonal functional group or a functional substance at a position is the same as the above-mentioned antibody. Preferably, such an antibody is a monoclonal antibody. Examples of the isotype of the monoclonal antibody include: IgG (such as IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. The monoclonal antibody is a full-length antibody or an antibody fragment (such as F(ab’)2, Fab’, Fab, Fv, single-chain antibody), but a full-length antibody is preferred. Particularly preferably, such an antibody is a human antibody, a humanized antibody, or a chimeric antibody having human IgG (such as IgG1, IgG2, IgG3, IgG4) in the constant region.

[0949] An antibody that selectively has a bioorthogonal functional group or a functional substance at a position is preferably an antibody that has a bioorthogonal functional group or a functional substance only in the constant region of the antibody, and more preferably an antibody that has a bioorthogonal functional group or a functional substance only in the Fc region of the antibody.

[0950] An antibody that selectively has a bioorthogonal functional group or a functional substance contains one or more specific amino acid residues (such as lysine residue, tyrosine residue, threonine residue, serine residue, cysteine residue) in a target region composed of 1 to 50 consecutive amino acid residues [for example, (a) a region composed of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region composed of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region composed of the amino acid residue at position 317 in the human IgG Fc region], and contains five or more of the above specific amino acid residues in a non-target region other than the above target region. In this case, one or more specific amino acid residues contained in the target region can have a bioorthogonal functional group or a functional substance with a positional selectivity of 30% or more. The positional selectivity is preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0951] In addition, an antibody that selectively has a bioorthogonal functional group or a functional substance can have a bioorthogonal functional group (i.e., a structural unit represented by E-B) or a functional substance (i.e., a structural unit represented by E-B'-F) according to the number of heavy chains. When an antibody that selectively has a bioorthogonal functional group or a functional substance has multiple (such as 1 to 8, preferably 1 to 4, more preferably 2) antibody heavy chains, a bioorthogonal functional group or a functional substance can be selectively present in the same target region of the multiple antibody heavy chains. That is, in an antibody that selectively has a bioorthogonal functional group or a functional substance, the modification mode based on the bioorthogonal functional group or the functional substance can be made the same among multiple (such as 2) heavy chains.

[0952] In addition, an antibody that selectively has a bioorthogonal functional group or a functional substance can have the same or different bioorthogonal functional groups (such as a structural unit represented by E-B) or the same or different functional substances (such as a structural unit represented by E-B'-F) in multiple (such as 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions of one antibody heavy chain. In this case, in an antibody that selectively has a bioorthogonal functional group or a functional substance, the modification mode based on the bioorthogonal functional group or the functional substance can be made the same among multiple (such as 2) heavy chains.

[0953] 5. A compound or a salt thereof having an affinity substance and a functional substance for an antibody

[0954] The present invention provides a compound represented by formula (IV) or a salt thereof, which has an antibody-targeting affinity substance and a functional substance.

[0955] A-L-E-F (IV)

[0956] [In the formula,

[0957] A is an antibody-targeting affinity substance,

[0958] L is a divalent group containing a leaving group,

[0959] E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody,

[0960] F is a functional substance,

[0961] The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0962] The definitions, examples, and preferred examples of the antibody-targeting affinity substance (A), the divalent group containing a leaving group (L), the divalent group containing an electrophilic group (E), and the functional substance (F) in the compound or its salt having an antibody-targeting affinity substance and a functional substance are the same as the corresponding meanings described above. Therefore, in the compound or its salt having an antibody-targeting affinity substance and a functional substance, A, L, and E can be specified in the same manner as A, L, and E in the compound or its salt having an antibody-targeting affinity substance and a bioorthogonal functional group (for example, refer to formula (I)). In addition, in the compound or its salt having an antibody-targeting affinity substance and a functional substance, F can be specified in the same manner as F in the antibody or its salt having a functional substance (for example, refer to formula (III)).

[0963] In a preferred embodiment, the compound represented by formula (IV) may be a compound represented by the following formula (IV-1):

[0964] A-L1-L2-E1-E2-E3-F (IV-1)

[0965] [In the formula,

[0966] A and F have the same meanings as the corresponding symbols in formula (IV),

[0967] L1 is a bond or a divalent group,

[0968] L2 is a leaving group,

[0969] E1 is an electrophilic group that (i) is linked to a leaving group and (ii) has the ability to react with a nucleophilic group in an antibody.

[0970] E2 is (a) -X-Y- [wherein X, which binds to E1, is C(R1)(R2) (wherein R1 and R2 are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (wherein R3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S or Se, and Y, which binds to E3, is C(R4)(R5) (wherein R4 or R5 is each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]; or (b) a group represented by the following formula (i):

[0971] [Chemical formula 25]

[0972]

[0973] (wherein ring Z is a divalent ring group in which the ring-forming atom X' that binds to E1 and the two adjacent ring-forming atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' that binds to E1 is a nitrogen atom and the two adjacent ring-forming atoms are carbon atoms. · represents a bonding site.)

[0974] E3 is a divalent group when E2 is -X-Y-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0975] The leaving group has the ability to be cleaved from and detached from E1 through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.].

[0976] In formula (IV-1), the definition, examples and preferred examples of the leaving group represented by L2 have the same meanings as those described for (a) to (c) in the above "1-3. Divalent group (L) containing a leaving group".

[0977] In a preferred specific embodiment, the compound represented by formula (IV-1) may be a compound represented by the following formula (IV-2):

[0978] A-L1-L2-E1-X-Y-E3-F (IV-2)

[0979] [In the formula,

[0980] A, L1, X, Y and F have the same meanings as the corresponding symbols in formula (IV-1),

[0981] L2 is:

[0982] (a) Ring P-Q- [Here, ring P is a group selected from arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, 2-pyridone; Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0983] (b) Heteroarylene; or

[0984] (c) -Q- [Here, Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0985] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0986] E3 is a divalent group.].

[0987] In another preferred specific embodiment, the compound represented by formula (IV-1) may be the compound represented by the following formula (IV-3):

[0988] [Chemical formula 26]

[0989]

[0990] [In the formula,

[0991] A, L1, ring Z, ring-constituting atom X' and F have the same meanings as the corresponding symbols in the above formula (IV-1),

[0992] L2 is:

[0993] (a) Ring P-Q- [Here, ring P is a group selected from arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, 2-pyridone; Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[0994] (b) heteroarylene; or

[0995] (c) -Q- [wherein, Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[0996] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0997] E3 is a bond or a divalent group.].

[0998] Preferably, the compound represented by formula (IV-3) may be a compound represented by the following formula (IV-4):

[0999] [Chemical formula 27]

[1000]

[1001] [In the formula,

[1002] A, L1 and F have the same meanings as the corresponding symbols in the above formula (IV-1),

[1003] L2 is:

[1004] (a) ring P-Q- [wherein, ring P is a group selected from arylene which may be substituted by an electron-withdrawing group, heteroarylene which may be substituted by an electron-withdrawing group, 2,5-diketopyrrolidine which may be condensed, 2,6-diketopiperidine which may be condensed, 2-ketopyrrolidine which may be condensed, 2-ketopiperidine which may be condensed, 2-pyridone, and Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).];

[1005] (b) heteroarylene; or

[1006] (c) -Q- [wherein, Q is a group selected from -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -O-N(R)- (wherein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).],

[1007] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[1008] Ring Z is a divalent cyclic group in which the ring-forming atom bonded to E1 and the ring-forming atoms adjacent to both sides thereof are all carbon atoms.

[1009] E3 is a bond or a divalent group.

[1010] In the compounds represented by the above formulas (IV-1) to (IV-4), the definitions, examples, and preferred examples of A, L1, L2, E1, E2, E3, and F, as well as the alkyl group having 1 to 6 carbon atoms in -X-Y-, R1 to R5, and the group represented by formula (i) (such as a divalent cyclic group, a divalent heterocyclic group) are the same as the corresponding meanings described above. In addition, the definitions, examples, and preferred examples of (a) to (c) as L2, and ring Z (for example, a divalent cyclic group in which the ring-forming atom X' bonded to E1 and the ring-forming atoms adjacent to both sides thereof are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' bonded to E1 is a nitrogen atom and the ring-forming atoms adjacent to both sides of the nitrogen atom are carbon atoms), ring P, and Q (for example, an alkyl group having 1 to 6 carbon atoms in R), etc. are also the same as the corresponding meanings described above.

[1011] A compound or a salt thereof having an affinity substance for an antibody and a functional substance can be appropriately prepared by reacting a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group with the above-mentioned functional substance via the bioorthogonal functional group. Such a reaction can be carried out in a suitable reaction system, such as an organic solvent system or an aqueous solution system, at a suitable temperature (for example, about 15 to 200 °C). The reaction system may contain a suitable 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.

[1012] In the reaction system, the molar ratio (Y / X) of the compound or a salt thereof (Y) having an affinity substance for an antibody and a bioorthogonal functional group to the functional substance (X) varies depending on the types of the structural unit and the affinity substance, the number of sites in the affinity substance to be modified by the structural unit, etc., and thus is not particularly limited. For example, it is 0.01 to 100, preferably 0.05 to 20, and more preferably 0.1 to 10.

[1013] The confirmation of the generation of a compound or a salt thereof having an affinity substance for an antibody and a functional substance depends on the molecular weights of its specific raw materials and products, but can be carried out by, for example, electrophoresis, chromatography (such as gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry analysis, and is preferably carried out by mass spectrometry analysis. A compound or a salt thereof having an affinity substance for an antibody and a functional substance can be appropriately purified by any method such as chromatography (such as the above-mentioned chromatography and affinity chromatography).

[1014] 6. Process for preparing an antibody having a functional substance, using a compound or a salt thereof having an affinity substance for an antibody and a functional substance

[1015] The present invention provides a process for preparing an antibody having a functional substance or a salt thereof, the process comprising the following steps.

[1016] Reacting a compound or a salt thereof having an affinity substance for an antibody and a functional substance represented by the following formula (IV) with an antibody to produce an antibody having a functional substance or a salt thereof represented by the following formula (V):

[1017] A-L-E-F (IV)

[1018] [In the formula,

[1019] A is an affinity substance for an antibody,

[1020] L is a divalent group containing a leaving group,

[1021] E is a divalent group containing an electrophilic group, the electrophilic group being: (i) connected to the above-mentioned leaving group and (ii) having the ability to react with a nucleophilic group in the above-mentioned antibody,

[1022] F is a functional substance,

[1023] The above-mentioned leaving group has the ability to be cleaved and detached from E by the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group.];

[1024] Ab-E-F (V)

[1025] [In the formula,

[1026] Ab is an antibody,

[1027] E and F have the same meanings as the corresponding symbols in the above formula (IV).].

[1028] Preferably, the antibody having a functional substance prepared by the preparation process of the present invention is an antibody that selectively has a functional substance at a specific position. In this case, an antibody that selectively has a functional substance represented by formula (V) can be prepared. The antibody that selectively has a functional substance at a specific position is preferably an antibody that has a functional substance only in the constant region, and more preferably an antibody that has a functional substance only in the Fc region.

[1029] An antibody (Ab) contains one or more specific amino acid residues (such as lysine residue, tyrosine residue, threonine residue, serine residue, cysteine residue) in a target region composed of 1 to 50 consecutive amino acid residues [for example, a region composed of amino acid residues at positions 246 - 248 in the human IgG Fc region, (b) a region composed of amino acid residues at positions 288 - 290 in the human IgG Fc region, or (c) a region composed of the amino acid residue at position 317 in the human IgG Fc region], and contains more than 5 of the above specific amino acid residues in a non-target region other than the above target region. In this case, the partial structure other than the antibody can bind to one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The positional selectivity is preferably 40% or more, more preferably 50% or more, still more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[1030] In addition, the antibody having a functional substance prepared by the preparation method of the present invention may have a functional substance (i.e., a structural unit represented by E-F) according to the number of heavy chains. Therefore, by using an antibody having multiple (such as 1 - 8, preferably 1 - 4, more preferably 2) antibody heavy chains in the preparation method of the present invention, an antibody can be prepared that selectively has multiple structural units (or multiple structural units of its subordinate concepts) represented by E-F in the same target region of multiple antibody heavy chains. For example, by using an antibody having 2 antibody heavy chains (such as IgG, IgD, IgE, F(ab’)2 antibody, Fc region protein, Fc fusion protein) in the preparation method of the present invention, an antibody can be prepared that selectively has 2 structural units represented by E-F in the same target region of 2 antibody heavy chains. That is, in an antibody having a functional substance, the modification mode based on the functional substance can be made the same among multiple (such as 2) heavy chains.

[1031] In addition, an antibody having a functional substance may have the same or different functional substances (such as a structural unit represented by E-F) in multiple (such as 2 - 5, preferably 2 - 4, more preferably 2 or 3) target regions of one antibody heavy chain. In this case, the antibody having a functional substance can make the modification mode based on the functional substance the same among multiple (such as 2) heavy chains.

[1032] The preparation method of the present invention may further include: generating an antibody having a functional substance and further modified by subjecting the generated antibody to a specific treatment. As such a specific treatment, for example, fragmentation treatment of the antibody (such as treatment with specific proteases such as papain and pepsin) can be cited.

[1033] When a compound represented by the formula (IV) or a salt thereof is used in the production method of the present invention, an antibody having a functional substance represented by the above formula (V) can be produced.

[1034] In a preferred embodiment, when a compound represented by the formula (IV-1) is used as the compound represented by the formula (IV) in the production method of the present invention, an antibody having a functional substance represented by the following formula (V-1) can be produced.

[1035] Ab-E1-E2-E3-F (V-1)

[1036] [In the formula,

[1037] Ab is an antibody,

[1038] E1, E2, E3 and F have the same meanings as the corresponding symbols in the above formula (IV-1).].

[1039] In a preferred specific embodiment, when a compound represented by the formula (IV-2) is used as the compound represented by the formula (IV-1) in the production method of the present invention, an antibody having a functional substance represented by the following formula (V-2) can be produced.

[1040] Ab-E1-X-Y-E3-F (V-2)

[1041] [In the formula,

[1042] Ab is an antibody,

[1043] E1, X, Y, E3 and F have the same meanings as the corresponding symbols in the above formula (IV-2).].

[1044] In another preferred specific embodiment, when a compound represented by the formula (IV-3) is used as the compound represented by the formula (IV-1) in the production method of the present invention, an antibody having a functional substance represented by the following formula (V-3) can be produced.

[1045] [Chemical formula 28]

[1046]

[1047] [In the formula,

[1048] Ab is an antibody,

[1049] E1, ring Z, ring-constituting atom X', E3 and F have the same meanings as the corresponding symbols in the above formula (IV-3).].

[1050] Preferably, when a compound represented by formula (IV-4) is used as the compound represented by formula (IV-3) in the preparation method of the present invention, an antibody having a functional substance represented by the following formula (V-4) can be prepared.

[1051] [Chemical formula 29]

[1052]

[1053] [In the formula,

[1054] Ab is an antibody,

[1055] E1, ring Z, E3 and F have the same meanings as the corresponding symbols in formula (IV-4) above.].

[1056] A compound or a salt thereof having an affinity substance and a functional substance for an antibody can react with the antibody because of the electrophilic group in E or the electrophilic group of E1. Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of proteins (such as cleavage of amide bonds). For example, such a reaction can be carried out in a suitable reaction system, such as in a buffer solution, at room temperature (such as about 15 to 30 °C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, more preferably 6.0 to 8.0. The buffer solution may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such a reaction, see, for example, G.J.L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G.J.L. Bernardes et al., Chem. Asian J., 4, 630 (2009); B.G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate Chem., 25, 825 (2014).

[1057] In the reaction system, the molar ratio (Y / X) of the compound or its salt (Y) having an affinity substance and a functional substance for an antibody to the antibody (X) varies depending on the types of the compound or its salt having an affinity substance and a functional substance for an antibody and the antibody, the number of sites (such as DAR) in the antibody to be modified by the compound or its salt having an affinity substance and a bioorthogonal functional group, etc., and thus is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

[1058] Confirmation of the production of an antibody having a functional substance can be carried out by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, although it also depends on the molecular weights of its specific raw materials and products, and is preferably carried out by mass spectrometry. Confirmation of position selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, it can be carried out, for example, by treatment with a protease (e.g., trypsin, chymotrypsin) and mass spectrometry. As the protease, an endoprotease is preferred. As such an endoprotease, for example, the following can be mentioned: trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. Confirmation of the number of functional substances retained by an antibody having a functional substance can be carried out by, for example, electrophoresis, chromatography or mass spectrometry, and is preferably carried out by mass spectrometry. An antibody having a functional substance can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[1059] 7. Salt

[1060] In the present invention, examples of salts include salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, 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), other metals such as zinc and aluminum, and ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkyl ethanolamine, dialkyl ethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrochloric acid) or a salt with an organic acid (e.g., trifluoroacetic acid).

[1061] 8. Use

[1062] The compound of the present invention having an affinity substance for an antibody and a bioorthogonal functional group or a salt thereof can be used, for example, for the site-selective modification of an antibody based on the bioorthogonal functional group. Therefore, the present invention provides a site-selective modification reagent for an antibody based on a bioorthogonal functional group, which comprises a compound having an affinity substance for an antibody and a bioorthogonal functional group or a salt thereof. In the site-selective modification reagent for an antibody based on a bioorthogonal functional group, the details (such as definitions, examples, and preferred examples) of the compound having an affinity substance for an antibody and a bioorthogonal functional group are the same as the corresponding meanings described above.

[1063] The compound of the present invention having an affinity substance for an antibody and a functional substance or a salt thereof can be used, for example, for the site-selective modification of an antibody based on the functional substance. Therefore, the present invention provides a site-selective modification reagent for an antibody based on a functional substance, which comprises a compound having an affinity substance for an antibody and a functional substance or a salt thereof. In the site-selective modification reagent for an antibody based on a functional substance, the details (such as definitions, examples, and preferred examples) of the compound having an affinity substance for an antibody and a functional substance are the same as the corresponding meanings described above.

[1064] In the site-selective modification reagent of the present invention, the details (such as definitions, examples, and preferred examples) of the site-selective modification of the antibody to be modified are the same as the corresponding meanings described above.

[1065] The site-selective modification reagent of the present invention can be provided in the form of a composition further containing other components. Examples of such other components include: solutions, stabilizers (such as antioxidants, preservatives). As the solution, an aqueous solution is preferred. Examples of the aqueous solution include: water (such as distilled water, sterilized distilled water, purified water, physiological saline), buffer solutions (such as aqueous phosphoric acid solution, Tris-hydrochloric acid buffer solution, carbonate-bicarbonate buffer solution, aqueous boric acid solution, glycine-sodium hydroxide buffer solution, citrate buffer solution), but a buffer solution is preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The site-selective modification reagent of the present invention can be provided in a liquid state or in a powder form (such as a lyophilized powder).

[1066] An antibody having a bioorthogonal functional group site-selectively or a salt thereof can be used, for example, as an intermediate in the preparation of an antibody having a functional substance site-selectively or a salt thereof.

[1067] An antibody or a salt thereof that selectively has a functional substance at a position can be used, for example, as a drug or a reagent (e.g., a diagnostic agent, a research reagent), and particularly as a drug. It has been reported that if the number and binding position of a drug in an antibody-drug conjugate (ADC) are changed, the pharmacokinetics, the drug release rate, and the effect will change. Based on these circumstances, for the next-generation ADCs, controlling the number and position of the conjugated drugs is required. It is generally considered that if the number and position are constant, the problems of the expected efficacy, the diversity of the conjugated agents, and the batch-to-batch variation, i.e., standardization, are solved. The antibody or a salt thereof of the present invention that selectively has a functional substance at a position can solve such a standardization problem. Therefore, the antibody or a salt thereof of the present invention that selectively has a functional substance at a position can be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the antibody or a salt thereof that selectively has a functional substance at a position, a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier 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, silica aerogel (Aerosil), talc, and sodium lauryl sulfate; flavoring agents such as citric acid, menthol, glycylglycine ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methyl p-hydroxybenzoate, and propyl p-hydroxybenzoate; 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, physiological saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene, but are not limited to these examples. The antibody or a salt thereof of the present invention that selectively has a functional substance at a position may also have any modification (e.g., PEGylation) for achieving stability.

[1068] Formulations suitable for oral administration are: liquid preparations obtained by dissolving an effective amount of a ligand in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand in solid or particulate form; suspensions in which an effective amount of an active ingredient is suspended in a suitable dispersion medium; emulsions in which a solution of an effective amount of an active ingredient is dispersed in a suitable dispersion medium and emulsified, etc.

[1069] The pharmaceutical composition is suitable for non-oral administration (parenteral administration) (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). As such a pharmaceutical composition suitable for non-oral administration, there are aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Aqueous and non-aqueous sterile suspension solutions may also be mentioned, which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, preservatives, etc.

[1070] The dosage of the pharmaceutical composition varies depending on the type / activity of the active ingredient, the severity of the disease, the type of animal as the administration subject, the drug tolerance of the administration subject, body weight, age, etc., and can be appropriately set.

[1071] Examples

[1072] Next, examples are shown to illustrate the present invention in more detail, but the present invention is not limited to the following examples.

[1073] [Example 1: Synthesis of IgG1 Fc affinity substance]

[1074] (1-1) Synthesis of affinity peptide for antibody

[1075] The peptides shown below as affinity substances for antibodies were prepared in exactly the same manner. Peptides with an N-terminal acetyl capping (Compounds 1, 2, 32 to 53) and peptides with an N-terminal 3-(triphenylmethylthio)propionic acid capping (Compounds 3, 31, 54) were prepared by solid-phase peptide synthesis using Rink amide resin based on the Fmoc method, and were stirred in a solution of trifluoroacetic acid: water: triisopropylsilane: ethanedithiol = 94:2.5:1.0:2.5 for 3 hours to cleave from the resin and deprotect. The resin was removed by filtration, diethyl ether was added for precipitation, and the diethyl ether was removed by decantation to obtain the peptide in the form of a crude crystal. It was purified by preparative HPLC to obtain the affinity peptide as the product.

[1076] Regarding Compounds 35 to 53, which are peptides having an S-S bond in the molecule (except for peptides containing methionine), the linear peptide as the precursor was obtained by the method shown above, and then synthesized by the method shown below. Dozens of mg of the obtained precursor was dissolved in 1 mL of DMSO, 100 μL of NH3 / MeOH and 10 μL of H2O2 were added, and the mixture was stirred overnight. After confirming the completion of the reaction by LCMS, it was purified by preparative HPLC to obtain the affinity peptide as the target product.

[1077] Regarding compounds 33 and 34, which are peptides with an S-S bond in the molecule (peptides containing methionine), the linear peptides as precursors were obtained by the method shown above, and then synthesized using the method shown below. Dissolve several tens of mg of the obtained precursor in 20 mL of 0.1 M Tris-HCl buffer (pH 8.00), add 5.0 eq of oxidized glutathione, and stir overnight. Confirm the completion of the reaction by LCMS, and then purify by preparative HPLC to obtain the affinity peptide as the target product.

[1078] [Chemical formula 30]

[1079]

[1080] (Amino acid sequence of the peptide part SEQ ID NO:5)

[1081] MS(ESI) m / z: z = 3 1392 [M+3H] 3+ , z = 4 1044 [M+4H] 4+

[1082] [Chemical formula 31]

[1083]

[1084] (Amino acid sequence of the peptide part SEQ ID NO:6)

[1085] MS(ESI) m / z: z = 3 1392 [M+3H] 3+ , z = 4 1044 [M+4H] 4+

[1086] [Chemical formula 32]

[1087]

[1088] (Amino acid sequence of the peptide part SEQ ID NO:7)

[1089] MS(ESI) m / z: z = 3 1478 [M+3H] 3+ , z = 4 1108 [M+4H] 4+

[1090] [Chemical formula 33]

[1091]

[1092] (Amino acid sequence of the peptide part SEQ ID NO:97)

[1093] MS(ESI) m / z: z = 2 1892 [M+2H] 2+, Z = 3 1262 [M + 3H] 3+ , Z = 4 946 [M + 4H] 4+ , z = 5757 [M + 5H] 5+

[1094] [Chemical Formula 34]

[1095]

[1096] (Amino acid sequence of the peptide part SEQ ID NO:98)

[1097] MS(ESI) m / z: z = 3 1401 [M + 3H] 3+ , Z = 4 1051 [M + 4H] 4+ , z = 5 841 [M + 5H] 5+

[1098] [Chemical Formula 35]

[1099]

[1100] (Amino acid sequence of the peptide part SEQ ID NO:41)

[1101] MS(ESI) m / z: z = 3 1426 [M + 3H] 3+ , Z = 4 1070 [M + 4H] 4+ , z = 5 859 [M + 5H] 5+

[1102] [Chemical Formula 36]

[1103]

[1104] (Amino acid sequence of the peptide part SEQ ID NO:42)

[1105] MS(ESI) m / z: z = 3 1417 [M + 3H] 3+ , Z = 4 1063 [M + 4H] 4+ , z = 5 851 [M + 5H] 5+

[1106] [Chemical Formula 37]

[1107]

[1108] (Amino acid sequence of the peptide part SEQ ID NO:43)

[1109] MS(ESI) m / z: z = 3 1425 [M + 3H] 3+ , Z = 4 1069 [M + 4H]4+ , z = 5855 [M+5H] 5+

[1110] [Chemical formula 38]

[1111]

[1112] (Amino acid sequence of the peptide part SEQ ID NO:46)

[1113] MS(ESI) m / z: z = 12090 [M+1H] + , Z = 21045 [M+2H] 2+ , Z = 3697 [M+3H] 3+

[1114] [Chemical formula 39]

[1115]

[1116] (Amino acid sequence of the peptide part SEQ ID NO:47)

[1117] MS(ESI) m / z: z = 12074 [M+1H] + , Z = 21037 [M+2H] 2+ , Z = 3692 [M+3H] 3+

[1118] [Chemical formula 40]

[1119]

[1120] (Amino acid sequence of the peptide part SEQ ID NO:48)

[1121] MS(ESI) m / z: z = 12061 [M+1H] + , Z = 21031 [M+2H] 2+ , Z = 3687 [M+3H] 3+

[1122] [Chemical formula 41]

[1123]

[1124] (Amino acid sequence of the peptide part SEQ ID NO:49)

[1125] MS(ESI) m / z: z = 12032 [M+1H] + , Z = 21016 [M+2H] 2+ , Z = 3678 [M+3H] 3+

[1126] [Chemical Formula 42]

[1127]

[1128] (Amino acid sequence of the peptide moiety SEQ ID NO:50)

[1129] MS(ESI) m / z: z = 1 2089 [M+1H] + , Z = 2 1044 [M+2H] 2+ , Z = 3 696 [M+3H] 3+

[1130] [Chemical Formula 43]

[1131]

[1132] (Amino acid sequence of the peptide moiety SEQ ID NO:51)

[1133] MS(ESI) m / z: z = 1 2088 [M+1H] + , Z = 2 1044 [M+2H] 2+ , Z = 3 696 [M+3H] 3+

[1134] [Chemical Formula 44]

[1135]

[1136] (Amino acid sequence of the peptide moiety SEQ ID NO:52)

[1137] MS(ESI) m / z: z = 1 1561 [M+H] + , Z = 2 781 [M+2H] 2+

[1138] [Chemical Formula 45]

[1139]

[1140] (Amino acid sequence of the peptide moiety SEQ ID NO:53)

[1141] MS(ESI) m / z: z = 1 1548 [M+1H] + , Z = 2 774 [M+2H] 2+

[1142] [Chemical Formula 46]

[1143]

[1144] (Amino acid sequence of the peptide moiety SEQ ID NO:55)

[1145] MS(ESI) m / z: z = 2 1059 [M+2H] 2+ , Z = 3 706 [M+3H] 3+

[1146] [Chemical formula 47]

[1147]

[1148] (Amino acid sequence of the peptide moiety SEQ ID NO:57)

[1149] MS(ESI) m / z: z = 2 1074 [M+2H] 2+ , Z = 3 716 [M+3H] 3+

[1150] [Chemical formula 48]

[1151]

[1152] (Amino acid sequence of the peptide moiety SEQ ID NO:59)

[1153] MS(ESI) m / z: z = 2 1081 [M+2H] 2+ , Z = 3 721 [M+3H] 3+ , Z = 4 541 [M+4H] 4+

[1154] [Chemical formula 49]

[1155]

[1156] (Amino acid sequence of the peptide moiety SEQ ID NO:63)

[1157] MS(ESI) m / z: z = 2 1085 [M+2H] 2+ , Z = 3 723 [M+3H] 3+ , Z = 4 543 [M+4H] 4+

[1158] [Chemical formula 50]

[1159]

[1160] (Amino acid sequence of the peptide moiety SEQ ID NO:71)

[1161] MS(ESI) m / z: z = 2 1045 [M+2H] 2+, Z = 3697 [M + 3H] 3+

[1162] [Chemical Formula 51]

[1163]

[1164] (Amino acid sequence of the peptide part SEQ ID NO:72)

[1165] MS(ESI) m / z: z = 11345 [M + 1H] + , Z = 2673 [M + 2H] 2+

[1166] [Chemical Formula 52]

[1167]

[1168] (Amino acid sequence of the peptide part SEQ ID NO:77)

[1169] MS(ESI) m / z: z = 21052 [M + 2H] 2+ , Z = 3702 [M + 3H] 3+

[1170] [Chemical Formula 53]

[1171]

[1172] (Amino acid sequence of the peptide part SEQ ID NO:81)

[1173] MS(ESI) m / z: z = 21073 [M + 2H] 2+ , Z = 3715 [M + 3H] 3+

[1174] [Chemical Formula 54]

[1175]

[1176] (Amino acid sequence of the peptide part SEQ ID NO:82)

[1177] MS(ESI) m / z: z = 21073 [M + 2H] 2+ , Z = 3716 [M + 3H] 3+

[1178] [Chemical Formula 55]

[1179]

[1180] (Amino acid sequence of the peptide part SEQ ID NO:99)

[1181] MS(ESI) m / z: z = 2966 [M + 2H] 2+ , Z = 3644 [M + 3H] 3+

[1182] [Chemical Formula 56]

[1183]

[1184] (Amino acid sequence of the peptide part SEQ ID NO: 100)

[1185] MS(ESI) m / z: z = 31381 [M + 3H] 3+ , Z = 41036 [M + 4H] 4+ , z = 5829 [M + 5H] 5+

[1186] (1 - 2) Synthesis of the affinity peptide azide adduct against the antibody

[1187] The following peptide - azide adducts (Compound 4, 5) were synthesized as follows. Subsequently, amino acids with only the functionalized residue Lys protected with the mtt group were used, and a peptide with an acetyl - capped N - terminus was prepared by solid - phase peptide synthesis using Rink amide resin based on the Fmoc method. It was stirred in a solution of dichloromethane:trifluoroacetic acid:triisopropylsilane = 90:5:5 for 1 hour to deprotect only the mtt group. The resin was washed with DMF, and then dissolved in 50 molar equivalents of triethylamine, 10 molar equivalents of NHS ester of azidoacetic acid, and 4 mL of DMF for addition, and stirred for 16 hours. After removing the solution, it was stirred in a solution of trifluoroacetic acid:water:triisopropylsilane = 95:2.5:2.5 for 1 hour to cleave from the resin and deprotect. The resin was removed by filtration, diethyl ether was added for precipitation, and the diethyl ether was removed by decantation to obtain the peptide in the form of a crude crystal. It was purified by preparative HPLC to obtain the affinity peptide azide adduct as the product.

[1188] [Chemical Formula 57]

[1189]

[1190] (Amino acid sequence of the peptide part SEQ ID NO: 8)

[1191] MS(ESI) m / z: z = 31401 [M + 3H] 3+ , z = 41051 [M + 4H] 4+

[1192] [Chemical Formula 58]

[1193]

[1194] (Amino acid sequence SEQ ID NO:6 of the peptide moiety)

[1195] MS(ESI) m / z: z = 3 1420 [M+3H] 3+ , z = 4 1065 [M+4H] 4+

[1196] [Example 2: Synthesis of an antibody-modified linker and ligation with an IgG1 Fc affinity peptide azide adduct]

[1197] (2-1) Synthesis of imidazolylcarbonyl compounds

[1198] (2-1-1) Synthesis of imidazolylcarbonyl compound (Compound 6)

[1199] [Chemical formula 59]

[1200]

[1201] Dissolve 0.100 g (1.32 mmol) of 3-butyn-1-ol and 263 mg (1.62 mmol) of carbonyldiimidazole in THF solvent and stir at room temperature for 1 hour. Dilute the reaction solution with ethyl acetate, wash with water and brine, add sodium sulfate, and let stand for 5 minutes. Filter off the sodium sulfate and concentrate under reduced pressure to obtain a crude product, which is then purified by silica gel column chromatography. Recover the fraction containing the product and concentrate under reduced pressure to obtain 0.180 g (1.10 mmol) of 1H-imidazole-1-carboxylic acid 3-butynyl ester corresponding to Compound 6.

[1202] 1 1H NMR (400 MHz, chloroform-d) δ 2.08 (t, J = 2.7 Hz, 1H), 2.73 (td, J = 6.6, 2.7 Hz, 2H), 4.54 (t, J = 6.6 Hz, 2H), 7.11 (s, 1H), 7.43 (s, 1H), 8.18 (s, 1H). MS(ESI) m / z: 165 [M+Na] +

[1203] (2-1-2) Synthesis of imidazolylcarbonyl compound (Compound 7)

[1204] [Chemical formula 60]

[1205]

[1206] Dissolve 0.100 g (0.745 mmol) of 9-decyn-1-ol and 148 mg (0.916 mmol) of carbonyldiimidazole in THF solvent and stir at room temperature for 1 hour. Dilute the reaction solution with ethyl acetate, wash with water and brine, add sodium sulfate, and let stand for 5 minutes. Filter off the sodium sulfate and concentrate under reduced pressure to obtain a crude product, which is then purified by silica gel column chromatography. Recover the fraction containing the product and concentrate under reduced pressure to obtain 0.152 g (0.612 mmol) of 1H-imidazole-1-carboxylic acid 9-butynyl ester corresponding to Compound 7.

[1207] 1 H NMR (400 MHz, chloroform-d) δ 1.33 - 1.50 (m, 10H), 1.82 (m, 2H), 1.96 (t, J = 2.6 Hz, 1H) 2.21 (td, J = 6.8, 2.6 Hz, 2H), 4.43 (t, J = 6.8 Hz, 2H) 7.10 (s, 1H), 7.45 (brs, 1H), 8.16 (s, 1H).

[1208] MS (ESI) m / z: 271 [M+Na] +

[1209] (2-1-3) Synthesis of imidazolylcarbonyl compound (Compound 9)

[1210] [Chemical Formula 61]

[1211]

[1212] Dissolve 113 mg (0.546 mmol) of N,N'-dicyclohexylcarbodiimide, 78.1 mg (0.546 mmol) of 1-hydroxybenzotriazole monohydrate, and 53.6 mg (0.546 mmol) of 4-pentynoic acid in dichloromethane solvent, stir at room temperature for 1 hour, then add 0.100 g (0.497 mmol) of 12-amino-dodecanol and stir at room temperature for 4 hours. Dilute the reaction solution with ethyl acetate, wash with water and brine, add sodium sulfate, and let stand for 5 minutes. Filter off the sodium sulfate and concentrate under reduced pressure to obtain 0.102 g (0.363 mmol) of 12-(pent-4-yn-1-oxo)aminododecan-1-ol corresponding to Compound 8.

[1213] MS (ESI) m / z: 281 [M+H] +

[1214] [Chemical Formula 62]

[1215]

[1216] 25.0 mg (0.089 mmol) of 12-(pent-4-ynamide)dodecan-1-ol and 29.1 mg (0.178 mmol) of carbonyldiimidazole were dissolved in THF solvent and stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate, washed with water and brine, and then sodium sulfate was added. After standing for 5 minutes, sodium sulfate was removed by filtration and concentrated under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain 12.0 mg (0.032 mmol) of 1H-imidazole-1-carboxylic acid 12-(pent-4-yn-1-yloxy)aminododecyl ester corresponding to Compound 9.

[1217] 1 H NMR (400 MHz, chloroform-d) δ 1.23 - 1.50 (m, 20H), 1.81 (t, J = 2.6 Hz, 1H), 2.36 (m, 2H), 2.53 (td, J = 6.8, 2.6 Hz, 2H), 3.23 (t, J = 6.8 Hz, 2H), 4.41 (t, J = 6.8 Hz, 2H), 5.64 (brs, 1H), 7.07 (s, 1H), 7.43 (brs, 1H), 8.13 (s, 1H). MS (ESI) m / z: 398 [M+Na] +

[1218] (2-2) Synthesis of imidazolylcarbonyl compounds and conjugation with affinity peptide azides

[1219] The affinity peptide azide (Compound 4) synthesized in Example 1 and the imidazolylcarbonyl compounds (Compounds 6, 7, 9) synthesized in Example 2 were conjugated by exactly the same method. The peptide azide was dissolved in 100 mM phosphate buffer (pH 7.0), and 20 molar equivalents of aminoguanidine hydrochloride, 20 molar equivalents of sodium ascorbate, and 3 molar equivalents of a dimethyl sulfoxide solution of the imidazolylcarbonyl compound at 100 mg / mL were added. An aqueous solution of 4 molar equivalents of copper(II) sulfate monohydrate and 20 molar equivalents of tris(3-hydroxypropyltriazolylmethyl)amine, which was freshly prepared, was added to the reaction mixture and stirred. The reaction was monitored by LC-MS. After confirming the disappearance of the starting materials, it was concentrated and diluted with water 4 times repeatedly by ultrafiltration (Amicon Ultra, 3K MWCO), and the resulting aqueous solution was lyophilized to obtain the peptide-imidazolylcarbonyl compounds (Compounds 10, 11, 12) shown below.

[1220] [Chemical formula 63]

[1221]

[1222] (Amino acid sequence of the peptide part SEQ ID NO: 8)

[1223] MS(ESI) m / z: z = 3 1455 [M+3H] 3+ , z = 4 1092 [M+4H] 4+

[1224] [Chemical formula 64]

[1225]

[1226] (Amino acid sequence of the peptide part SEQ ID NO:8)

[1227] MS(ESI) m / z: z = 3 1483 [M+3H] 3+ , z = 4 1113 [M+4H] 4+

[1228] [Chemical formula 65]

[1229]

[1230] (Amino acid sequence of the peptide part SEQ ID NO:8)

[1231] MS(ESI) m / z: z = 3 1525 [M+3H] 3+ , z = 4 1144 [M+4H] 4+

[1232] [Reference Example 1: Verification of the difference in reactivity caused by the difference in the number of atoms of the affinity peptide - antibody modification group (electrophilic group) through model experiments]

[1233] (1-1) Synthesis of IgG antibody trastuzumab - peptide complex

[1234] Dissolve 20 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) in 2.0 μL of 100 mM HEPES buffer (pH 7.2). Add 20 molar equivalents of the peptide - imidazolylcarbonyl compounds (Compounds 10, 11, 12) synthesized in (2-2) of Example 2 to the antibody, and stir at 37 °C for 4 hours. Perform water replacement on the reaction solution by ultrafiltration (Amicon Ultra, 3K MWCO) to remove the peptide reagent and stop the reaction, obtaining the IgG antibody trastuzumab - peptide complex. (1-2) Analysis of IgG antibody trastuzumab - peptide complex based on SDS-PAGE

[1235] Three kinds of IgG antibody trastuzumab-peptide complexes obtained in (1-1) were analyzed by SDS-PAGE (Mini-PROTEAN TGX gel, 4-20%, Bio-RAD; under reducing conditions; stained with Coomassie Brilliant Blue G-250 dye) using three peptide-imidazolylcarbonyl compounds (Compound 10, 11, 12). The results are shown in Figure 2 . From the results, it can be seen that the antibody modification reaction was carried out when using Compound 10 and 11, while the reaction did not proceed when using Compound 12. [Example 3: Synthesis of Antibody Affinity Reagent with Maleimide Attached]

[1236] (3-1) Synthesis of Protected Thiol Compound

[1237] (3-1-1) Synthesis of Protected Thiol Compound (Compound 14)

[1238] [Chemical Formula 66]

[1239]

[1240] 100 mg (0.287 mmol) of 3-(triphenylmethylthio)propanoic acid was dissolved in THF, and 42.7 μL (0.316 mmol) of isobutyl chloroformate and 69.4 μL (0.631 mmol) of N-methylmorpholine were added at 0 °C, and the mixture was stirred for 30 minutes to prepare the corresponding mixed anhydride. 33.6 mg (0.287 mmol) of 5-aminopentanoic acid was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, and then the THF solution of the above mixed anhydride was added dropwise at room temperature. After stirring at room temperature for 16 hours, the reaction solution was washed with water and ethyl acetate, and the aqueous phase was recovered. 6 M hydrochloric acid aqueous solution was added to the aqueous phase to adjust the pH in the system to 3.0, and then liquid-liquid extraction was carried out with ethyl acetate. After that, the organic phase was washed with brine, and then anhydrous magnesium sulfate was added and allowed to stand for 5 minutes. Magnesium sulfate was removed by filtration, and the solution was concentrated under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain 110 mg (0.246 mmol) of 5-[3-(triphenylmethyl)thioalkyl-propyl-1-oxo]amino-hexanoic acid corresponding to Compound 13.

[1241] 1 H NMR (400 MHz, chloroform-d) δ 1.66 (m, 3H), 2.03 (t, J = 7.3 Hz, 2H), 2.39 (t, J = 7.3 Hz, 2H), 2.52 (t, J = 7.3 Hz, 2H), 3.22 (q, J = 6.6 Hz, 2H), 5.37 (s, 1H), 7.19-7.36 (m, 9H), 7.40-7.49 (m, 6H).

[1242] MS(ESI) m / z: 470 [M+Na] +

[1243] [Chemical formula 67]

[1244]

[1245] Dissolve 20.2 mg (0.045 mmol) of 5-(3-tritylpropyl-1-oxo)aminohexanoic acid in THF, add 6.70 μL (0.050 mmol) of isobutyl chloroformate and 10.9 μL (0.0991 mmol) of N-methylmorpholine at 0 °C, and stir for 30 minutes to prepare the corresponding mixed anhydride. Dissolve 16.6 μL (0.226 mmol) of propargylamine in 1 M aqueous sodium hydroxide solution at room temperature, and then dropwise add the THF solution of the above mixed anhydride at room temperature. Stir at room temperature for 16 hours, then wash the reaction solution with water and ethyl acetate, and recover the aqueous phase. Add 6 M aqueous hydrochloric acid solution to the aqueous phase, adjust the pH in the system to 3.0, then perform liquid-liquid extraction with ethyl acetate, wash the organic phase with brine, then add anhydrous magnesium sulfate, and let stand for 5 minutes. Filter off the magnesium sulfate and concentrate under reduced pressure to obtain the crude product, which is then purified by silica gel column chromatography. Recover the fraction containing the product and concentrate under reduced pressure to obtain 20.0 mg (0.041 mmol) of N-propynyl-5-(3-tritylthiopropyl-1-oxo)amino-hexanamide corresponding to Compound 14.

[1246] 1 H NMR (400 MHz, chloroform-d) δ 1.52 (dt, J = 8.2, 6.5 Hz, 2H), 1.59 - 1.72 (m, 2H), 2.02 - 2.09 (m, 2H), 2.17 - 2.28 (m, 2H) 2.52 (t, J = 7.2 Hz, 2H), 3.21 (brs, 2H), 4.01 (dd, J = 5.3, 2.6 Hz, 2H), 6.04 (s, 1H), 7.19 - 7.35 (m, 9H), 7.40 - 7.49 (m, 6H).

[1247] MS(ESI) m / z: 507 [M+H] +

[1248] (3-1-2) Synthesis of protected thiol compound (Compound 15)

[1249] [Chemical formula 68]

[1250]

[1251] 100 mg (0.287 mmol) of 3-(triphenylmethylthio)propanoic acid was dissolved in THF, and 42.7 μL (0.316 mmol) of isobutyl chloroformate and 69.4 μL (0.631 mmol) of N-methylmorpholine were added thereto at 0 °C, followed by stirring for 30 minutes to prepare the corresponding mixed anhydride. 105 μL (1.43 mmol) of propargylamine was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, and the THF solution of the above-mentioned mixed anhydride was added dropwise thereto at room temperature. After stirring at room temperature for 16 hours, the reaction solution was washed with water and ethyl acetate, and the aqueous phase was recovered. 6 M aqueous hydrochloric acid solution was added to the aqueous phase to adjust the pH in the system to 3.0, followed by liquid-liquid extraction with ethyl acetate. Then, the organic phase was washed with brine, anhydrous magnesium sulfate was added, and the mixture was allowed to stand for 5 minutes. Magnesium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain 110 mg (0.286 mmol) of N-propynyl-3-triphenylmethylthio-propionamide corresponding to Compound 15.

[1252] 1 H NMR (400 MHz, chloroform-d) δ 2.01 (t, J = 7.1 Hz, 2H), 2.24 (t, J = 2.8 Hz, 1H), 2.53 (t, J = 7.6 Hz, 2H), 3.99 (m, 2H), 5.40 (brs, 1H), 7.19 - 7.35 (m, 9H), 7.40 - 7.49 (m, 6H).

[1253] MS (ESI) m / z: 408 [M+Na] +

[1254] (3-2) Ligation of the Affinity Peptide with the Protected Thiol Compound

[1255] (3-2-1) Synthesis of the Peptide-Protected Thiol Conjugate (Compound 16)

[1256] [Chemical Formula 69]

[1257]

[1258] (Amino Acid Sequence of the Peptide Moiety SEQ ID NO: 6)

[1259] The compound 5 synthesized in Example 1 was dissolved in N,N'-dimethylformamide, and 5 molar equivalents of 3-(triphenylmethylthio)propanoic acid, 15 molar equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 15 molar equivalents of 1-hydroxybenzotriazole were dissolved in N,N'-dimethylformamide and added to the system. After stirring at room temperature for 12 hours, purification was performed by preparative HPLC. After confirming the fraction containing the target compound by ESI-MS, freeze-drying was carried out to obtain the peptide-protected thiol conjugate (Compound 16) as the product.

[1260] MS(ESI) m / z: z = 3 1502 [M+3H] 3+ , z = 4 1125 [M+4H] 4+

[1261] Synthesis of (3-2-2) Peptide-Protected Thiol Linkers (Compounds 17, 18)

[1262] The affinity peptide azide (Compound 5) synthesized in Example 1 and the protected thiol compounds (Compounds 17, 18) synthesized in (4-1-1) and (4-1-2) were linked in the same manner. The peptide azide was dissolved in 100 mM phosphate buffer (pH 7.0), and 20 molar equivalents of aminoguanidine hydrochloride, 20 molar equivalents of sodium ascorbate, and 3 molar equivalents of a dimethyl sulfoxide solution of the protected thiol compound at 100 mg / mL were added. An aqueous solution of 4 molar equivalents of copper(II) sulfate monohydrate and 20 molar equivalents of tris(3-hydroxypropyltriazolylmethyl)amine, freshly prepared, was added to the reaction mixture and stirred. The reaction was monitored by LC-MS. After confirming the disappearance of the starting materials, the mixture was concentrated and diluted with water 4 times repeatedly by ultrafiltration (Amicon Ultra-4, 3K MWCO), and the resulting aqueous solution was lyophilized to obtain the peptide-protected thiol linkers (Compounds 17, 18).

[1263] [Chemical Formula 70]

[1264]

[1265]

[1266] (Amino acid sequence of the peptide part SEQ ID NO: 6)

[1267] MS(ESI) m / z: z = 3 1548 [M+3H] 3+ , z = 4 1161 [M+4H] 4+

[1268] [Chemical Formula 71]

[1269]

[1270] (Amino acid sequence of the peptide part SEQ ID NO: 6)

[1271] MS(ESI) m / z: z = 3 1581 [M+3H] 3+ , z = 4 1186 [M+4H] 4+

[1272] (3 - 3) Synthesis of Peptide-Protected Thiol Linkers (Compounds 19, 20, 21)

[1273] The affinity peptide-protected thiol linkers (Compounds 16, 17, 18) synthesized in (3 - 2) were stirred in a 1:1 solution of trifluoroacetic acid:dichloromethane for 1 hour to remove the trityl group. After confirming the completion of the reaction by LC-MS, purification was carried out by preparative HPLC. After confirming the fraction containing the target compound by ESI-MS, lyophilization was performed to obtain the thiol-peptide linkers (Compounds 19, 20, 21) as the product.

[1274] [Chemical Formula 72]

[1275]

[1276] (Amino acid sequence of the peptide part SEQ ID NO:6)

[1277] MS(ESI) m / z: z = 3 1454 [M+3H] 3+ , z = 4 1091 [M+4H] 4+

[1278] [Chemical Formula 73]

[1279]

[1280]

[1281] (Amino acid sequence of the peptide part SEQ ID NO:6)

[1282] MS(ESI) m / z: z = 3 1468 [M+3H] 3+ , z = 4 1111 [M+4H] 4+

[1283] [Chemical Formula 74]

[1284]

[1285] (Amino acid sequence of the peptide part SEQ ID NO:6)

[1286] m / z: z = 3 1501 [M+3H] 3+ , z = 4 1126 [M+4H] 4+

[1287] (3 - 4) Synthesis of Antibody Affinity Peptide Reagents Loaded with Maleimide (1) (Compounds 22, 23, 24)

[1288] The thiol-peptide linkers (Compounds 19, 20, 21) synthesized in (3-3) were dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of 20 molar equivalents of N-hydroxysuccinimide was added, followed by stirring for 1 hour. After confirming the disappearance of the starting materials by LC-MS, 40 molar equivalents of trans-4-[(2,5-dihydro-1H-pyrrol-1-yl)methyl)]cyclohexanecarboxylic acid, 30 molar equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 30 molar equivalents of 1-hydroxybenzotriazole were added, and the mixture was stirred for 4 hours. After confirming the completion of the reaction by LC-MS, purification was carried out by preparative HPLC. After confirming the fraction containing the target compound by ESI-MS, it was freeze-dried to obtain the maleimide-affinity peptide reagents (Compounds 22, 23, 24) as products.

[1289] [Chemical formula 75]

[1290]

[1291] (Amino acid sequence of the peptide part SEQ ID NO: 6)

[1292] MS(ESI) m / z: z = 3 1532 [M+3H] 3+ , z = 4 1150 [M+4H] 4+

[1293] [Chemical formula 76]

[1294]

[1295] (Amino acid sequence of the peptide part SEQ ID NO: 6)

[1296] MS(ESI) m / z: z = 3 1578 [M+3H] 3+ , z = 4 1184 [M+4H] 4+

[1297] [Chemical formula 77]

[1298]

[1299] (Amino acid sequence of the peptide part SEQ ID NO: 6)

[1300] MS(ESI) m / z: z = 3 1611 [M+3H] 3+ , z = 4 1208 [M+4H] 4+

[1301] (3-5) Synthesis of antibody-affinity peptide reagents bearing maleimide (2) (Compounds 56, 58)

[1302] (3-5-1) Synthesis of N-Hydroxysuccinimide-Peptide Conjugate (Compound 55)

[1303] Dissolve the compound 3 synthesized in (1-1) in dimethyl sulfoxide, add 1 equivalent of triethylamine and a dimethyl sulfoxide solution of 1.2 equivalents of N-hydroxymaleimide, and stir at room temperature for 1 hour. Confirm the completion of the reaction by LC-MS, and then purify by preparative HPLC. After confirming the fraction containing the target compound by ESI-MS, perform lyophilization to obtain the N-hydroxysuccinimide-peptide conjugate (Compound 55) as the product.

[1304] [Chemical Formula 78]

[1305]

[1306] (Amino acid sequence of the peptide part SEQ ID NO:7)

[1307] MS(ESI) m / z: z = 3 1429 [M+3H] 3+ , Z = 4 1072 [M+4H] 4+ , z = 5 857 [M+5H] 5+ , z = 6 715 [M+6H] 6+

[1308] (3-5-2) Synthesis of Antibody-Affinity Peptide Reagent with Maleimide (Compound 56)

[1309] Dissolve the N-hydroxysuccinimide-peptide conjugate obtained in (3-5-1) and 20 equivalents of N-succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate in dimethyl sulfoxide, and add 8 equivalents of triethylamine. Stir at room temperature for 2 hours, and then confirm the progress of the reaction by...

Claims

1. A compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I): A-L-E-B (I) In the formula, A is an affinity substance for an antibody, L is a divalent group containing a leaving group, E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group, and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody, B is a bioorthogonal functional group, The above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group, The above-mentioned affinity substance is a peptide having the ability to bind to the Fc region of IgG, The above-mentioned peptide contains any one of the following amino acid sequences: (1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIKDD (SEQ ID NO:5), (2) FNMQQQRRFYEALHDPNLNEEQRNARIKSIRDD (SEQ ID NO:6), (3) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO:7), (4) FNMQQQRRFYEALHDPNLNEEQRNAKIKSIKDD (SEQ ID NO:8), (5) FARLVSSIRY (SEQ ID NO:26), (6) FGRLVSSIRY (SEQ ID NO:27), (7) TWKTSRISIF (SEQ ID NO:28), (8) HWRGWV (SEQ ID NO:30), (9) HYFKFD (SEQ ID NO:31), (10) HFRRHL (SEQ ID NO:32), (11) DAAG (SEQ ID NO:33), (12) NARKFYKG (SEQ ID NO:34), (13) NKFRGKYK (SEQ ID NO:35), (14) FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO:36), (15) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO:41), (16) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:42), (17) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:43), (18) RGNCAYHKGQIIWCTYH (SEQ ID NO:46), (19) RGNCAYHKGQIVWCTYH (SEQ ID NO:47), (20)RGNCAYHKGQVVWCTYH (SEQ ID NO:48), (21)RGNCAYHKGQAVWCTYH (SEQ ID NO:49), (22)RGNCAYHKGQLLWCTYH (SEQ ID NO:50), (23)RGNCAYHKGQLIWCTYH (SEQ ID NO:51), (24)DCAYHKGQIVWCT (SEQ ID NO:52), (25)DCAYHKGQVVWCT (SEQ ID NO:53), (26)RGNCAYHKSQIIWCTYH (SEQ ID NO:55), (27)RGNCAYHKDQIIWCTYH (SEQ ID NO:57), (28)RGNCAYHKEQIIWCTYH (SEQ ID NO:59), (29)RGNCAYHKHQIIWCTYH (SEQ ID NO:63), (30)RGNCAYHKGQEVWCTYH (SEQ ID NO:71), (31)CAYHKGQLVWC (SEQ ID NO:72), (32)RGNCAYHKSQLVWCTYH (SEQ ID NO:77), (33)RGNCAYHKQQLVWCTYH (SEQ ID NO:81), (34)RGNCAYHKEQLVWCTYH (SEQ ID NO:82), (35)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSI (SEQ ID NO:97), (36)FNMQQQRRFYEALHDPNLNKEQRNARIRSIRDD (SEQ ID NO:98), (37)GNCAYHKGQIIWCTYH (SEQ ID NO:99), (38)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSIKDD (SEQ ID NO:100), or (39)RGNCAYHEGQIIWCTYH (SEQ ID NO:108), Among them, The compound represented by the above formula (I) is the compound represented by the following formula (I-1): A-L1-L2-E1-E2-E3-B (I-1) In the formula, A and B have the same meanings as the corresponding symbols in the above formula (I), L1 is a bond or a divalent group, L2 is a leaving group, E1 is an electrophilic group that (i) is connected to the above-mentioned leaving group and (ii) has the ability to react with a nucleophilic group in the above-mentioned antibody, E2 is a group represented by the following formula (i), Here, ring Z is a divalent cyclic group in which the ring-forming atom X' bonded to E1 and the ring-forming atoms of the two adjacent rings are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' bonded to E1 is a nitrogen atom and the ring-forming atoms of the two adjacent rings are carbon atoms, · represents a bonding bond. E3 is a bond or a divalent group. The above leaving group has the ability to be cleaved and detached from E1 through the reaction between the above nucleophilic group and the above electrophilic group. The above electrophilic group is -C(=O)-. The above bioorthogonal functional group is a group selected from the following: azide residue, thiol residue, maleimide residue, and thioester residue. The above L2 is a group selected from the following structural formulas: Here, ○ (white circle) is the bonding bond with L1, and ● (black circle) is the bonding bond with E1.

2. The compound or its salt according to claim 1, wherein, The above nucleophilic group is a group selected from the following: NH2 in the side chain of lysine residue, OH in the side chain of tyrosine residue, OH in the side chain of serine residue, OH in the side chain of threonine residue, and SH in the side chain of cysteine residue.

3. The compound or its salt according to claim 1, wherein, The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

4. The compound or its salt according to claim 1, wherein, E3 is a divalent group.

5. The compound or its salt according to claim 1, wherein, E3 is a bond.

6. Use of the compound or its salt having an affinity substance for an antibody and a bioorthogonal functional group according to claim 1 in the position-selective modification of an antibody based on the bioorthogonal functional group.

7. The application according to claim 6, wherein, The above nucleophilic group is a group selected from the following: NH2 in the side chain of lysine residue, OH in the side chain of tyrosine residue, OH in the side chain of serine residue, OH in the side chain of threonine residue, and SH in the side chain of cysteine residue.

8. The application according to claim 6, wherein, The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

9. The application according to claim 6, wherein E3 is a divalent group.

10. The use according to claim 6, wherein E3 is a bond.

11. A method for preparing an antibody or its salt having a bioorthogonal functional group, the preparation method comprising: Reacting a compound or its salt having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) with an antibody to generate an antibody or its salt having a bioorthogonal functional group represented by the following formula (II). A-L-E-B (I) In the formula, A is an affinity substance for an antibody. L is a divalent group containing a leaving group. E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above leaving group and (ii) having the ability to react with the nucleophilic group in the above antibody. B is a bioorthogonal functional group. The above leaving group has the ability to be cleaved and detached from E through the reaction between the above nucleophilic group and the above electrophilic group. The above affinity substance is a peptide having the ability to bind to the Fc region of IgG. The above peptide contains any one of the following amino acid sequences: (1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIKDD (SEQ ID NO:5) (2) FNMQQQRRFYEALHDPNLNEEQRNARIKSIRDD (SEQ ID NO:6) (3) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO:7), (4) FNMQQQRRFYEALHDPNLNEEQRNAKIKSIKDD (SEQ ID NO:8), (5) FARLVSSIRY (SEQ ID NO:26), (6) FGRLVSSIRY (SEQ ID NO:27), (7) TWKTSRISIF (SEQ ID NO:28), (8) HWRGWV (SEQ ID NO:30), (9) HYFKFD (SEQ ID NO:31), (10) HFRRHL (SEQ ID NO:32), (11) DAAG (SEQ ID NO:33), (12) NARKFYKG (SEQ ID NO:34), (13) NKFRGKYK (SEQ ID NO:35), (14) FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO:36), (15) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO:41), (16) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:42), (17) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:43), (18) RGNCAYHKGQIIWCTYH (SEQ ID NO:46), (19) RGNCAYHKGQIVWCTYH (SEQ ID NO:47), (20) RGNCAYHKGQVVWCTYH (SEQ ID NO:48), (21) RGNCAYHKGQAVWCTYH (SEQ ID NO:49), (22) RGNCAYHKGQLLWCTYH (SEQ ID NO:50), (23) RGNCAYHKGQLIWCTYH (SEQ ID NO:51), (24) DCAYHKGQIVWCT (SEQ ID NO:52), (25) DCAYHKGQVVWCT (SEQ ID NO:53), (26) RGNCAYHKSQIIWCTYH (SEQ ID NO:55), (27) RGNCAYHKDQIIWCTYH (SEQ ID NO:57), (28) RGNCAYHKEQIIWCTYH (SEQ ID NO:59), (29) RGNCAYHKHQIIWCTYH (SEQ ID NO:63), (30)RGNCAYHKGQEVWCTYH (SEQ ID NO:71), (31)CAYHKGQLVWC (SEQ ID NO:72), (32)RGNCAYHKSQLVWCTYH (SEQ ID NO:77), (33)RGNCAYHKQQLVWCTYH (SEQ ID NO:81), (34)RGNCAYHKEQLVWCTYH (SEQ ID NO:82), (35)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSI (SEQ ID NO:97), (36)FNMQQQRRFYEALHDPNLNKEQRNARIRSIRDD (SEQ ID NO:98), (37)GNCAYHKGQIIWCTYH (SEQ ID NO:99), (38)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSIKDD (SEQ ID NO:100), or (39)RGNCAYHEGQIIWCTYH (SEQ ID NO:108), wherein the compound represented by the above formula (I) is the compound represented by the following formula (I-1): A-L1-L2-E1-E2-E3-B (I-1) In the formula, A and B have the same meanings as the corresponding symbols in the above formula (I), L1 is a bond or a divalent group, L2 is a leaving group, E1 is (i) an electrophilic group that is connected to the above-mentioned leaving group and (ii) has the ability to react with a nucleophilic group in the above-mentioned antibody, E2 is a group represented by the following formula (i), wherein ring Z is a divalent ring group in which the ring-forming atom X' bonded to E1 and the two adjacent ring-forming atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' bonded to E1 is a nitrogen atom and the two adjacent ring-forming atoms are carbon atoms, · is a bonding bond, E3 is a bond or a divalent group, the above-mentioned leaving group has the ability to be cleaved and detached from E1 through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group, the above-mentioned electrophilic group is -C(=O)-, the above-mentioned bioorthogonal functional group is a group selected from the following: azide residue, thiol residue, maleimide residue, and thioester residue, the above-mentioned L2 is a group selected from the following structural formulas: wherein, ○ (white circle) is the bonding bond with L1, ● (black circle) is the bonding bond with E1; Ab-E-B (II) In the formula, E and B have the same meanings as the corresponding symbols in the above formula (I), Ab is an antibody.

12. The method according to claim 11, wherein, the above-mentioned nucleophilic group is a group selected from the following: NH2 in the side chain of lysine residue, OH in the side chain of tyrosine residue, OH in the side chain of serine residue, OH in the side chain of threonine residue, and SH in the side chain of cysteine residue.

13. The method according to claim 11, wherein The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

14. The method according to claim 11, wherein, E3 is a divalent group.

15. The method according to claim 11, wherein, E3 is a bond.

16. A method for preparing an antibody or a salt thereof having a functional substance, the method comprising: (1) reacting a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) with the antibody to produce an antibody or a salt thereof having a bioorthogonal functional group represented by the following formula (II), A-L-E-B (I) In the formula, A is an affinity substance for an antibody, L is a divalent group containing a leaving group, E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above-mentioned leaving group and (ii) capable of reacting with a nucleophilic group in the above-mentioned antibody, B is a bioorthogonal functional group, the above-mentioned leaving group has the ability to be cleaved and detached from E through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group, the above-mentioned affinity substance is a peptide having the ability to bind to the Fc region of IgG, the above-mentioned peptide contains any one of the following amino acid sequences: (1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIKDD (SEQ ID NO:5), (2) FNMQQQRRFYEALHDPNLNEEQRNARIKSIRDD (SEQ ID NO:6), (3) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO:7), (4) FNMQQQRRFYEALHDPNLNEEQRNAKIKSIKDD (SEQ ID NO:8), (5) FARLVSSIRY (SEQ ID NO:26), (6) FGRLVSSIRY (SEQ ID NO:27), (7) TWKTSRISIF (SEQ ID NO:28), (8) HWRGWV (SEQ ID NO:30), (9) HYFKFD (SEQ ID NO:31), (10) HFRRHL (SEQ ID NO:32), (11) DAAG (SEQ ID NO:33), (12) NARKFYKG (SEQ ID NO:34), (13) NKFRGKYK (SEQ ID NO:35), (14) FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO:36), (15) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO:41), (16) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:42), (17) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:43), (18) RGNCAYHKGQIIWCTYH (SEQ ID NO:46), (19)RGNCAYHKGQIVWCTYH (SEQ ID NO:47), (20)RGNCAYHKGQVVWCTYH (SEQ ID NO:48), (21)RGNCAYHKGQAVWCTYH (SEQ ID NO:49), (22)RGNCAYHKGQLLWCTYH (SEQ ID NO:50), (23)RGNCAYHKGQLIWCTYH (SEQ ID NO:51), (24)DCAYHKGQIVWCT (SEQ ID NO:52), (25)DCAYHKGQVVWCT (SEQ ID NO:53), (26)RGNCAYHKSQIIWCTYH (SEQ ID NO:55), (27)RGNCAYHKDQIIWCTYH (SEQ ID NO:57), (28)RGNCAYHKEQIIWCTYH (SEQ ID NO:59), (29)RGNCAYHKHQIIWCTYH (SEQ ID NO:63), (30)RGNCAYHKGQEVWCTYH (SEQ ID NO:71), (31)CAYHKGQLVWC (SEQ ID NO:72), (32)RGNCAYHKSQLVWCTYH (SEQ ID NO:77), (33)RGNCAYHKQQLVWCTYH (SEQ ID NO:81), (34)RGNCAYHKEQLVWCTYH (SEQ ID NO:82), (35)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSI (SEQ ID NO:97), (36)FNMQQQRRFYEALHDPNLNKEQRNARIRSIRDD (SEQ ID NO:98), (37)GNCAYHKGQIIWCTYH (SEQ ID NO:99), (38)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSIKDD (SEQ ID NO:100), or (39)RGNCAYHEGQIIWCTYH (SEQ ID NO:108), wherein, the compound represented by the above formula (I) is the compound represented by the following formula (I-1): A-L1-L2-E1-E2-E3-B (I-1) In the formula, A and B have the same meanings as the corresponding symbols in the above formula (I), L1 is a bond or a divalent group, L2 is a leaving group, E1 is an electrophilic group that (i) is connected to the above leaving group and (ii) has the ability to react with the nucleophilic group in the above antibody, E2 is a group represented by the following formula (i), Here, ring Z is a divalent cyclic group in which the ring atom X' bonded to E1 and the ring atoms forming the two adjacent rings are all carbon atoms, or a divalent heterocyclic group in which the ring atom X' bonded to E1 is a nitrogen atom and the ring atoms forming the two adjacent rings are carbon atoms, · represents a bonding bond. E3 is a bond or a divalent group. The above leaving group has the ability to be cleaved and detached from E1 through the reaction between the above nucleophilic group and the above electrophilic group. The above electrophilic group is -C(=O)-. The above bioorthogonal functional group is a group selected from the following: azide residue, thiol residue, maleimide residue, and thioester residue. The above L2 is a group selected from the following structural formulas: Here, ○ (white circle) is the bonding bond with L1, and ● (black circle) is the bonding bond with E1; Ab-E-B (II) In the formula, E and B have the same meanings as the corresponding symbols in the above formula (I). Ab is an antibody; and (2) Reacting the antibody or its salt having a bioorthogonal functional group represented by the above formula (II) with a functional substance via the bioorthogonal functional group to produce an antibody or its salt having a functional substance represented by the following formula (III): Ab-E-B'-F (III) In the formula, Ab has the same meaning as the corresponding symbol in the above formula (II). E has the same meaning as the corresponding symbol in the above formula (I). B' is a divalent group containing a part formed by the reaction between the functional substance and the bioorthogonal functional group. F is a functional substance.

17. The method according to claim 16, wherein, The above nucleophilic group is a group selected from the following: NH2 in the side chain of lysine residue, OH in the side chain of tyrosine residue, OH in the side chain of serine residue, OH in the side chain of threonine residue, and SH in the side chain of cysteine residue.

18. The method according to claim 16, wherein The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

19. The method according to claim 16, wherein E3 is a divalent group. The method according to claim 16, wherein, E3 is a bond.

21. A compound or its salt having an affinity substance for an antibody and a functional substance represented by the following formula (IV): A-L-E-F (IV) In the formula, A is an affinity substance for an antibody. L is a divalent group containing a leaving group. E is a divalent group containing an electrophilic group, and the electrophilic group is: (i) connected to the above leaving group and (ii) having the ability to react with the nucleophilic group in the above antibody. F is a functional substance. The above leaving group has the ability to be cleaved and detached from E through the reaction between the above nucleophilic group and the above electrophilic group. The above affinity substance is a peptide having the ability to bind to the Fc region of IgG. The above peptide contains any one of the following amino acid sequences: (1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIKDD (SEQ ID NO:5), (2) FNMQQQRRFYEALHDPNLNEEQRNARIKSIRDD (SEQ ID NO:6), (3) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO:7), (4) FNMQQQRRFYEALHDPNLNEEQRNAKIKSIKDD (SEQ ID NO:8), (5) FARLVSSIRY (SEQ ID NO:26), (6) FGRLVSSIRY (SEQ ID NO:27), (7) TWKTSRISIF (SEQ ID NO:28), (8) HWRGWV (SEQ ID NO:30), (9) HYFKFD (SEQ ID NO:31), (10) HFRRHL (SEQ ID NO:32), (11) DAAG (SEQ ID NO:33), (12) NARKFYKG (SEQ ID NO:34), (13) NKFRGKYK (SEQ ID NO:35), (14) FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO:36), (15) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO:41), (16) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:42), (17) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:43), (18) RGNCAYHKGQIIWCTYH (SEQ ID NO:46), (19) RGNCAYHKGQIVWCTYH (SEQ ID NO:47), (20) RGNCAYHKGQVVWCTYH (SEQ ID NO:48), (21) RGNCAYHKGQAVWCTYH (SEQ ID NO:49), (22) RGNCAYHKGQLLWCTYH (SEQ ID NO:50), (23) RGNCAYHKGQLIWCTYH (SEQ ID NO:51), (24) DCAYHKGQIVWCT (SEQ ID NO:52), (25) DCAYHKGQVVWCT (SEQ ID NO:53), (26) RGNCAYHKSQIIWCTYH (SEQ ID NO:55), (27) RGNCAYHKDQIIWCTYH (SEQ ID NO:57), (28) RGNCAYHKEQIIWCTYH (SEQ ID NO:59), (29) RGNCAYHKHQIIWCTYH (SEQ ID NO:63), (30) RGNCAYHKGQEVWCTYH (SEQ ID NO:71), (31) CAYHKGQLVWC (SEQ ID NO:72), (32) RGNCAYHKSQLVWCTYH (SEQ ID NO:77), (33) RGNCAYHKQQLVWCTYH (SEQ ID NO:81), (34) RGNCAYHKEQLVWCTYH (SEQ ID NO:82), (35) β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSI (SEQ ID NO:97), (36) FNMQQQRRFYEALHDPNLNKEQRNARIRSIRDD (SEQ ID NO:98), (37) GNCAYHKGQIIWCTYH (SEQ ID NO:99), (38) β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSIKDD (SEQ ID NO:100), or (39) RGNCAYHEGQIIWCTYH (SEQ ID NO:108), Among them, The compound represented by the above formula (IV) is the compound represented by the following formula (IV-1): A-L1-L2-E1-E2-E3-F (IV-1) [In the formula, A and F have the same meanings as the corresponding symbols in the above formula (IV), L1 is a bond or a divalent group, L2 is a leaving group, E1 is an electrophilic group that (i) is connected to the above-mentioned leaving group and (ii) has the ability to react with a nucleophilic group in the above-mentioned antibody, E2 is a group represented by the following formula (i), Here, ring Z is a divalent ring group in which the ring-forming atom X' bonded to E1 and its two adjacent ring-forming atoms are all carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X' bonded to E1 is a nitrogen atom and the two adjacent ring-forming atoms of the nitrogen atom are carbon atoms, · is a bonding bond, E3 is a bond or a divalent group, The above-mentioned leaving group has the ability to be cleaved and detached from E1 through the reaction between the above-mentioned nucleophilic group and the above-mentioned electrophilic group, The above-mentioned electrophilic group is -C(=O)-, The above-mentioned L2 is a group selected from the following structural formulas: Here, ○ (white circle) is the bonding bond with L1, and ● (black circle) is the bonding bond with E1.

22. The compound or its salt according to claim 21, wherein, The above-mentioned nucleophilic group is a group selected from the following: NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine residue.

23. The compound or its salt according to claim 21, wherein, The above-mentioned functional substance is selected from drugs, labeling substances, and stabilizers.

24. The compound or its salt according to claim 21, wherein, The above-mentioned functional substance is an anticancer agent.

25. The compound or its salt according to claim 21, wherein, The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

26. The compound or its salt according to claim 21, wherein, E3 is a divalent group.

27. The compound or its salt according to claim 21, wherein, E3 is a bond.

28. Use of the compound or its salt having an affinity substance and a functional substance for an antibody according to claim 21 in the position-selective modification of an antibody based on the functional substance.

29. The application according to claim 28, wherein, The above nucleophilic group is a group selected from the following: NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine residue.

30. The application according to claim 28, wherein, The above functional substance is selected from a drug, a labeling substance, and a stabilizer.

31. The application according to claim 28, wherein, The above functional substance is an anticancer agent.

32. The application according to claim 28, wherein, The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms.

33. The application according to claim 28, wherein E3 is a divalent group.

34. The application according to claim 28, wherein, E3 is a bond. A method for preparing an antibody or a salt thereof having a functional substance position-selectively, The above antibody or a salt thereof is represented by the following formula (III-1): Ab-E1-E2-E3-B’-F (III-1) In the formula, Ab is an antibody, E1 is an electrophilic group connected to the nucleophilic group in the antibody, E2 is a group represented by the following formula (i), Here, ring Z is a divalent ring group in which the ring-forming atom X’ bonded to E1 and its two adjacent ring-forming atoms are both carbon atoms, or a divalent heterocyclic group in which the ring-forming atom X’ bonded to E1 is a nitrogen atom and the two adjacent ring-forming atoms of the nitrogen atom are carbon atoms, · is a bonding bond, E3 is a bond or a divalent group, B’ is a divalent group containing a part formed by the reaction between a functional substance and a bioorthogonal functional group, F is a functional substance, The preparation method includes: Reacting the compound or a salt thereof having an affinity substance for an antibody and a functional substance according to claim 21 with the antibody to generate an antibody or a salt thereof having a functional substance represented by the following formula (III): Ab-E-F (III) In the formula, Ab is an antibody, The meanings of E and F are the same as those of the corresponding symbols in the above formula (IV).

36. The method according to claim 35, wherein, The above nucleophilic group is a group selected from the following: NH2 in the side chain of a lysine residue, OH in the side chain of a tyrosine residue, OH in the side chain of a serine residue, OH in the side chain of a threonine residue, and SH in the side chain of a cysteine residue.

37. The method according to claim 35, wherein, The above functional substance is selected from a drug, a labeling substance, and a stabilizer.

38. The method according to claim 35, wherein, The above functional substance is an anticancer agent.

39. The method according to claim 35, wherein, The main chain of L or L1-L2 connecting A and E is composed of 20 or fewer atoms. The method according to claim 35, wherein, E3 is a divalent group.

41. The method according to claim 35, wherein, E3 is a bond.

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