Compound having affinity substance for antibody and bio-orthogonal functional group, or salt thereof

Abstract

Provided is a technique with which it is possible to modify an antibody, in particular to position-selective modification of an antibody. More specifically, the present invention provides a compound having an affinity for an antibody and a bio-orthogonal functional group represented by formula (I): A-L-E-B (I) [in the formula, A is an affinity for an antibody, L is a divalent group containing a leaving group, E is a divalent group containing an electrophilic group, and B is a bivalent group containing a leaving group; a is a leaving group having the ability to cleave and separate from E by a reaction between the leaving group and the electrophilic group, and (ii) has the ability to react with a nucleophilic group in the antibody, B is a bioorthogonality functional group, and the leaving group has the ability to cleave and separate from E by the reaction between the nucleophilic group and the electrophilic group. ].

Description

[0001] This application is a divisional application of a patent application with the original application date of June 14, 2019, application number 201980039638.7 (international application number PCT / JP2019 / 023779), and invention name “Compounds or their salts having affinity substances for antibodies and bioorthogonal functional groups”. Technical Field

[0002] The present invention relates to a compound having an affinity substance for an antibody and a bioorthogonal functional group, or a salt thereof. Background Art

[0003] In recent years, research and development of antibody-drug complexes (ADCs) have been actively underway. As the name suggests, ADCs are drugs (e.g., anticancer agents) conjugated to antibodies, and they exhibit direct cytotoxic activity against cancer cells. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)) (Non-Patent Documents 1-3).

[0004] ADCs, such as T-DM1, have been plagued by heterogeneity issues since their early stages of development. Specifically, because low-molecular-weight drugs are randomly reacted with the approximately 70 to 80 Lys residues present in antibodies, the drug-antibody ratio (DAR) and conjugation position are uncertain. It is generally known that with such random conjugation methods, the DAR ranges from 0 to 8, resulting in a variety of agents with varying numbers of drug conjugates. In recent years, it has been reported that altering the number and position of drug conjugates in an ADC can alter in vivo kinetics, drug release rate, and efficacy. Based on these circumstances, control over the number and position of conjugated drugs is required for next-generation ADCs. It is generally believed that determining the number and position of conjugated drugs will resolve issues such as desired efficacy, diversity of conjugated drugs, batch variations, and so-called regulation (Non-Patent Document 4).

[0005] Research is underway globally on methods for position-selective antibody modification, but nearly all involve genetic engineering or enzyme-based modification. While genetic engineering modification methods can control positional and number selectivity, they have been cited as problematic, such as reduced antibody expression efficiency (reduced overall yield during ADC preparation). Furthermore, the time required to construct antibody expression systems is a significant challenge (Non-Patent Documents 5-7).

[0006] In recent years, methods have been reported for chemically modifying proteins using small molecule probes in the complex environment of cells. This method is used for receptor identification in imaging and the relocalization of low-molecular-weight drugs. Furthermore, in the field of biochemistry, organic chemical protein modification methods using synthetic small molecule probes are attracting attention (Non-Patent Documents 8-11).

[0007] Recently, the CCAP (Chemical Conjugation by Affinity Peptide) method has been developed. This method successfully achieves position-selective modification of antibodies by reacting a peptide reagent obtained by linking an NHS active ester and a drug to an affinity peptide (i.e., a method for preparing ADCs via a linker containing a peptide moiety). This method is the first in the world to successfully modify the Fc region of an antibody positionally with a drug using chemical synthesis, and has also demonstrated excellent results in actual use (reaction time of 30 minutes, yield of 70% (in the case of DAR 1), and position selectivity of 100%). It has been demonstrated that the DAR can be controlled to 2 by adding approximately 5 equivalents of the peptide reagent, and that the modification position can also be controlled, which is of epoch-making significance in this regard (Patent Document 1).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. 2016 / 186206;

[0011] Non-patent literature

[0012] Non-patent document 1: Reichert JM et al., Nat Biotechnol 2005; 23: 1073-8;

[0013] Non-patent document 2: Kubota T et al., Cancer Sci 2009; 100: 1566-72;

[0014] Non-patent document 3: Wu AM et al., Nat Biotechnol 2005; 23: 1137-46;

[0015] Non-patent document 4: Junutula JR et al., Nat Biotechnol 2008; 26: 925-32;

[0016] Non-patent document 5: Shen BQ et al., Nat Biotechnol 2012; 30: 184-9;

[0017] Non-patent document 6: Hofer T et al., Biochemistry 2009; 48: 12047-57;

[0018] Non-patent document 7: Liu W et al., Nat Methods 2007; 4: 239-44;

[0019] Non-patent document 8: STLaughlin et al., Science 2008; 320, 664;

[0020] Non-patent literature 9: AE Spears et al., ChemBioChem 2004; 5, 41;

[0021] 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

[0022] Problems to be solved by the invention

[0023] The object of the present invention is to develop a technology that enables modification of antibodies, particularly position-selective modification of antibodies.

[0024] Means for solving problems

[0025] The present inventors have conducted intensive research and have discovered that compounds or salts thereof having a structural unit such as ALE (wherein 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, wherein the electrophilic group is (i) linked to the aforementioned leaving group and (ii) capable of reacting with a nucleophilic group in the aforementioned antibody) can be used for position-specific modification of antibodies. For example, it has been discovered that a predetermined compound having an affinity substance for an antibody and a bioorthogonal functional group represented by formula (I) can be used for position-specific modification of antibodies (e.g., Figure 1, various embodiments). It has also been discovered that a compound represented by formula (IV) having an affinity substance for an antibody and a functional substance or a salt thereof can be used for position-specific modification of an antibody (for example, Examples 13 and 14). The present inventors have further discovered that by using such a compound, antibodies (antibody-drug complexes (ADCs)) that do not contain a peptide portion as a linker and that selectively contain a functional substance (such as a drug) can be prepared. Avoiding the use of linkers containing peptide portions that are potentially immunogenic and easily hydrolyzed in the blood is desirable in the clinical application of ADCs. That is, according to the method developed by the present inventors, the Fc region of an antibody can be selectively modified with a drug by chemical synthesis without using a linker containing a peptide portion.

[0026] That is, the present invention is as follows.

[0027] [1] A compound having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) or a salt thereof:

[0028] ALEB(I)

[0029] [Where,

[0030] A is an affinity substance for antibodies,

[0031] L is a divalent group containing a leaving group,

[0032] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0033] B is a bioorthogonal functional group,

[0034] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group. ]

[0035] [2] The compound or salt thereof according to [1], wherein the affinity substance is a peptide.

[0036] [3] The compound or salt thereof according to [2], wherein the peptide is a peptide capable of binding to the constant region of a monoclonal antibody.

[0037] [4] The compound or salt thereof according to [2] or [3], wherein the peptide is a peptide capable of binding to the Fc region of a monoclonal antibody.

[0038] [5] The compound or salt thereof according to [4], wherein the peptide is a peptide capable of binding to the Fc region of IgG.

[0039] [6] The compound or salt thereof according to any one of [2] to [5], wherein the peptide has 10 to 40 amino acid residues.

[0040] [7] The compound or salt thereof according to any one of [2] to [6], wherein the peptide comprises the following amino acid sequence:

[0041] (a) the amino acid sequence of (a-1-1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 11), or

[0042] (a-1-2) in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 12),

[0043] Any 1 to 3 amino acid residues in the sequence may be the same or different, and are substituted with one amino acid residue each selected from a lysine residue, an aspartic acid residue, and a glutamic acid residue,

[0044] in the amino acid sequence of (a-2-1)β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 13), or

[0045] (a-2-2) in the amino acid sequence of β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 14),

[0046] Any 1 to 3 amino acid residues in the sequence may be the same or different, and are substituted by one amino acid residue each selected from a lysine residue, an aspartic acid residue, and a glutamic acid residue; and

[0047] (b) having 85% or greater identity with each of the amino acid sequences of SEQ ID NOs: 11 to 14.

[0048] [8] The compound or salt thereof according to any one of [2] to [6], wherein the peptide comprises any one of the following amino acid sequences:

[0049] Formula 1-1: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X 0-3 ) b (SEQ ID NO: 15)

[0050] 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)

[0051] 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)

[0052] Formula 1-4: (X 0-3 ) a 2]-C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-A-V-W-C-(X 0-3 ) b (SEQ ID NO:18)

[0053] Formula 1-5: (X<ooo0017>) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-L-W-C-(X 0-3 ) b (SEQ ID NO:19)

[0054] Formula 1-6: (X<0om0021>) '' a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-I-W-C-(X 0-3 ) b ' (SEQ ID NO:20)

[0055] Formula 1-7: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-F-C-(X<000002i>) b (SEQ ID NO:21)

[0056] 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)

[0057] Formula 1-9: (X 0-3 ) It should be noted that there may be some inaccuracies in the translation due to the unclear or potentially incorrect original text in some parts (such as "ooo0017" and "om0021" which seem to be typos in the original). Please double-check the original for more accurate translation.a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-EVWC-(X 0-3 ) b (SEQ ID NO:23)

[0058] [Where,

[0059] (X 0-3 ) a is absent, an arginine residue-glycine residue-asparagine residue, a glycine residue-asparagine residue, an aspartic acid residue, or an asparagine residue,

[0060] (X 0-3 ) b is absent, a threonine residue-tyrosine residue-histidine residue, or a threonine residue,

[0061] Xaa1 is an alanine residue,

[0062] Xaa2 is a tyrosine residue, a tryptophan residue, or a histidine residue,

[0063] Xaa3 is a histidine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an arginine residue, or a glycine residue,

[0064] Xaa4 is a lysine residue, an aspartic acid residue, or a glutamic acid residue,

[0065] 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,

[0066] Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. ]; or

[0067] Formula 2-1: (X 0-3 ') a -C-(Xaa1')-(Xaa2')-(Xaa3')-(Xaa4')-(Xaa5')-(Xaa6')-LVWC-(X 0-3 ') b (SEQ ID NO:24)

[0068] [Where,

[0069] (X 0-3 ') a and (X 0-3 ') bRespectively with the above (X 0-3 ) a and (X 0-3 ) b same,

[0070] Xaa1', Xaa2', Xaa3', Xaa4', Xaa5', and Xaa6' are the same as Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6 described above, respectively. ]

[0071] [9] The compound or salt thereof according to any one of [1] to [8], 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 -ON(R)- (where R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); or (2) a heteroarylene group.

[0072]

[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.

[0073]

[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-.

[0074]

[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.

[0075]

[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.

[0076]

[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):

[0077] A-L1-L2-E1-E2-E3-B (I-1)

[0078] [Where,

[0079] A and B have the same meanings as the corresponding symbols in the above formula (I),

[0080] L1 is a bond or a divalent group,

[0081] L2 is a leaving group,

[0082] 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,

[0083] E2 is (a) -XY- [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):

[0084] [Chemical Formula 1]

[0085]

[0086] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms adjacent to it are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. · is a bonding bond.)

[0087] E3 is a divalent group when E2 is -XY-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0088] The leaving group has the ability to be cleaved and separated from E1 by the reaction between the nucleophilic group and the electrophilic group. ]

[0089]

[15]

[14] or a salt thereof, wherein the above L2 is:

[0090] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.];

[0091] (b) heteroarylene; or

[0092] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]

[0093]

[16]

[14] or

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

[0094] [Chemical Formula 2]

[0095]

[0096] (Here, EWG is an electron-withdrawing group,

[0097] m is an integer from 0 to 4,

[0098] n is an integer from 0 to 3,

[0099] R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,

[0100] ○ (white circle) indicates the bond with L1, and ● (black circle) indicates the bond with E1. ).

[0101]

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

[16] , wherein the main chain of L or L1-L2 connecting A and E consists of 20 atoms or less.

[0102]

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

[14] to

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

[0103] A-L1-L2-E1-XY-E3-B(I-2)

[0104] [Where,

[0105] A, L1, X, Y and B have the same meanings as the corresponding symbols in the above formula (I-1),

[0106] L2 is:

[0107] (a) Ring PQ- [herein, Ring P is 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a group of 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.];

[0108] (b) heteroarylene; or

[0109] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]

[0110] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0111] E3 is a divalent group. ].

[0112]

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

[14] to

[17] , wherein the compound represented by the above formula (I-1) is a compound represented by the following formula (I-3):

[0113] [Chemical Formula 3]

[0114]

[0115] [Where,

[0116] A, L1, ring Z and B have the same meanings as the corresponding symbols in the above formula (I-1),

[0117] L2 is:

[0118] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.];

[0119] (b) heteroarylene; or

[0120] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]

[0121] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0122] E3 is a bond or a divalent group. ].

[0123]

[20] A reagent for selectively modifying an antibody based on a bioorthogonal functional group, the reagent comprising a compound having an affinity for an antibody and a bioorthogonal functional group represented by the following formula (I) or a salt thereof:

[0124] ALEB(I)

[0125] [Where,

[0126] A is an affinity substance for antibodies,

[0127] L is a divalent group containing a leaving group,

[0128] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0129] B is a bioorthogonal functional group,

[0130] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group. ]

[0131]

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

[0132] A compound having an affinity substance for an antibody and a bioorthogonal functional group represented by the following formula (I) or a salt thereof is reacted with an antibody to produce an antibody having a bioorthogonal functional group represented by the following formula (II) or a salt thereof,

[0133] ALEB (I)

[0134] [Where,

[0135] A is an affinity substance for antibodies,

[0136] L is a divalent group containing a leaving group,

[0137] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0138] B is a bioorthogonal functional group,

[0139] The leaving group has the ability to be cleaved and separated from E through the reaction between the nucleophilic group and the electrophilic group.];

[0140] Ab-EB (II)

[0141] [Where,

[0142] E and B have the same meanings as the corresponding symbols in the above formula (I),

[0143] Ab is an antibody. ].

[0144]

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

[0145] (1) reacting a compound having an affinity for an antibody and a bioorthogonal functional group represented by the following formula (I) or a salt thereof with an antibody to produce an antibody having a bioorthogonal functional group represented by the following formula (II) or a salt thereof;

[0146] ALEB (I)

[0147] [Where,

[0148] A is an affinity substance for antibodies,

[0149] L is a divalent group containing a leaving group,

[0150] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0151] B is a bioorthogonal functional group,

[0152] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group.

[0153] Ab-EB (II)

[0154] [Where,

[0155] E and B have the same meanings as the corresponding symbols in the above formula (I),

[0156] Ab is antibody.]; and

[0157] (2) The antibody or salt thereof having a bioorthogonal functional group represented by the above formula (II) is reacted with a functional substance via the bioorthogonal functional group to produce an antibody or salt thereof having a functional substance represented by the following formula (III):

[0158] Ab-E-B'-F (III)

[0159] [Where,

[0160] Ab has the same meaning as the corresponding symbols in the above formula (II),

[0161] E has the same meaning as the corresponding symbol in the above formula (I),

[0162] B' is a divalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0163] F is a functional substance. ].

[0164]

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

[0165] Ab-E1-E2-E3-B (II-1)

[0166] [Where,

[0167] Ab is antibody,

[0168] E1 is an electrophilic group connected to the nucleophilic group in the antibody,

[0169] E2 is (a) -XY- [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):

[0170] [Chemical Formula 4]

[0171]

[0172] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms adjacent to it are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. · is a bonding bond.)

[0173] E3 is a divalent group when E2 is -XY-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0174] B is a bioorthogonal functional group. ].

[0175]

[24] The antibody or salt thereof of

[23] , wherein the antibody has a bioorthogonal functional group only in the constant region of a monoclonal antibody.

[0176]

[25] The antibody or salt thereof of

[23] or

[24] , wherein the antibody has a bioorthogonal functional group only in the Fc region of a monoclonal antibody.

[0177]

[26] The antibody or salt thereof according to any one of

[23] to

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

[0178]

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

[23] to

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

[0179] Ab-E1-XY-E3-B (II-2)

[0180] [Where,

[0181] Ab, X, Y and B have the same meanings as the corresponding symbols in the above formula (II-1),

[0182] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0183] E3 is a divalent group. ].

[0184]

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

[23] to

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

[0185] [Chemical Formula 5]

[0186]

[0187] [Where,

[0188] Ab, ring-constituting atoms X', ring Z and B have the same meanings as the corresponding symbols in the above formula (II-1),

[0189] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0190] E3 is a bond or a divalent group. ].

[0191]

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

[0192] Ab-E1-E2-E3-B'-F (III-1)

[0193] [Where,

[0194] Ab is antibody,

[0195] E1 is an electrophilic group connected to the nucleophilic group in the antibody,

[0196] E2 is (a) -XY- [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):

[0197] [Chemical Formula 6]

[0198]

[0199] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms adjacent to it are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. · is a bonding bond.)

[0200] E3 is a divalent group when E2 is -XY-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0201] B' is a divalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0202] F is a functional substance. ].

[0203]

[30]

[29] The antibody or a salt thereof, wherein the antibody has a bioorthogonal functional group only in the constant region of a monoclonal antibody.

[0204]

[31]

[29] or

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

[0205]

[32] The antibody or salt thereof according to any one of

[29] to

[31] , wherein the antibody is a human IgG having a functional substance positionally selectively in the region consisting of amino acid residues 246 to 248 or 288 to 290 in the human IgG Fc region.

[0206]

[33] The antibody or salt thereof according to any one of

[29] to

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

[0207] Ab-E1-XY-E3-B'-F (III-2)

[0208] [Where,

[0209] Ab, X, Y, B' and F have the same meanings as the corresponding symbols in the above formula (III-1),

[0210] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0211] E3 is a divalent group].

[0212]

[34] The antibody or salt thereof according to any one of

[29] to

[32] , wherein the antibody represented by the above formula (III-1) is an antibody having a functional substance selectively located at a position represented by the following formula (III-3):

[0213] [Chemical Formula 7]

[0214]

[0215] [Where,

[0216] Ab, ring-constituting atoms X', ring Z, B' and F have the same meanings as the corresponding symbols in the above formula (III-1),

[0217] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0218] E3 is a bond or a divalent group. ].

[0219]

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

[0220] ALEF (IV)

[0221] [Where,

[0222] A is an affinity substance for antibodies,

[0223] L is a divalent group containing a leaving group,

[0224] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0225] F is a functional substance,

[0226] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group. ]

[0227]

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

[0228] ALEF (IV)

[0229] [Where,

[0230] A is an affinity substance for antibodies,

[0231] L is a divalent group containing a leaving group,

[0232] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0233] F is a functional substance,

[0234] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group. ]

[0235]

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

[0236] A compound having an affinity for an antibody and a functional substance represented by the following formula (IV) or a salt thereof is reacted with an antibody to produce an antibody having a functional substance represented by the following formula (III) or a salt thereof:

[0237] ALEF (IV)

[0238] [Where,

[0239] A is an affinity substance for antibodies,

[0240] L is a divalent group containing a leaving group,

[0241] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0242] F is a functional substance,

[0243] The leaving group has the ability to be cleaved and separated from E through the reaction between the nucleophilic group and the electrophilic group.];

[0244] Ab-EF (III)

[0245] [Where,

[0246] Ab is antibody,

[0247] E and F have the same meanings as the corresponding symbols in the above formula (IV).].

[0248] Effects of the Invention

[0249] The compound of the present invention or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group or functional substance can be used, for example, for position-selective modification of an antibody.

[0250] The antibody or salt thereof of the present invention having a bioorthogonal functional group selectively at the position thereof can be used as an intermediate in the preparation of an antibody or salt thereof having a functional substance selectively at the position thereof.

[0251] The antibody of the present invention or a salt thereof that selectively possesses a functional substance can be used as, for example, a drug or a reagent (eg, a diagnostic drug, a research reagent). BRIEF DESCRIPTION OF THE DRAWINGS

[0252] [ Figure 1 ] Figure 1 It is a schematic diagram showing the outline of the present invention.

[0253] [ Figure 2 ] Figure 2 This figure shows the results of SDS-PAGE analysis of the synthesis of the IgG antibody trastuzumab-peptide complex. Lanes 1, 3, 6, and 8: Molecular weight markers; Lane 2: Unreacted IgG antibody trastuzumab (control, the band around a molecular weight of 50,000 indicates the heavy chain, and the band around a molecular weight of 25,000 indicates the light chain); Lane 4: Complex formed by the IgG antibody trastuzumab and compound 10 (the band above a molecular weight of 50,000 indicates that compound 10 is coupled to the heavy chain of trastuzumab. The band below a molecular weight of 50,000 indicates the unreacted heavy chain, and the band around a molecular weight of 25,000 indicates the unreacted light chain); Lane 5: Complex formed by the IgG antibody trastuzumab and compound 10 (the band above a molecular weight of 50,000 indicates that compound 10 is coupled to the heavy chain of trastuzumab. The band below a molecular weight of 50,000 indicates the unreacted heavy chain, and the band around a molecular weight of 25,000 indicates the unreacted light chain). Lane G: complex formed by antibody trastuzumab and compound 11 (the band above the molecular weight of 50,000 shows that compound 11 is coupled to the heavy chain of trastuzumab. The band below the molecular weight of 50,000 shows the unreacted heavy chain, and the band around the molecular weight of 25,000 shows the unreacted light chain); Lane 7: reaction mixture after coupling of IgG antibody trastuzumab and compound 12 (the band around the molecular weight of 50,000 shows the unreacted heavy chain, and the band around the molecular weight of 25,000 shows the unreacted light chain. No band coupled to compound 12 is seen).

[0254] [ Figure 3 ] Figure 3This figure shows the results of SDS-PAGE analysis of a trastuzumab-peptide complex synthesized by position-specifically introducing a maleimide into the IgG antibody trastuzumab, followed by coupling with a thiol-containing peptide reagent. Lanes 1 and 9: Molecular weight markers. Lane 2: Complex obtained by treating the IgG antibody trastuzumab with compound 22 (10 molar equivalents relative to the antibody), then regioselectively introducing a maleimide, and then coupling with compound 25. Bands above a molecular weight of approximately 50,000 indicate maleimide introduction into the heavy chain of trastuzumab and coupling with compound 25. Bands below a molecular weight of approximately 50,000 indicate unreacted heavy chains, and bands around a molecular weight of approximately 25,000 indicate unreacted light chains. Lane 3: Complex obtained by treating the IgG antibody trastuzumab with compound 22 (20 molar equivalents relative to the antibody), then regioselectively introducing a maleimide, and then coupling with compound 25. Bands above a molecular weight of approximately 50,000 indicate the introduction of a maleimide into the heavy chain of trastuzumab, followed by coupling with compound 25. Bands below a molecular weight of approximately 50,000 indicate unreacted heavy chains, and bands around a molecular weight of 25,000 indicate unreacted light chains. Lane 4: Complex obtained by treating IgG trastuzumab with compound 22 (40 molar equivalents relative to the antibody), regioselectively introducing a maleimide, and then coupling with compound 25. Bands above a molecular weight of approximately 50,000 indicate the introduction of a maleimide into the heavy chain of trastuzumab, followed by coupling with compound 25. Bands below a molecular weight of approximately 50,000 indicate unreacted heavy chains, and bands around a molecular weight of 25,000 indicate unreacted light chains. Lane 5: Complex obtained by treating IgG trastuzumab with compound 23 (10 molar equivalents relative to the antibody), regioselectively introducing a maleimide, and then coupling with compound 25. Bands above a molecular weight of approximately 50,000 indicate the introduction of a maleimide into the heavy chain of trastuzumab, followed by coupling with compound 25. Bands below a molecular weight of approximately 50,000 indicate unreacted heavy chains, and bands around a molecular weight of approximately 25,000 indicate unreacted light chains. Lane 6: Complex obtained by treating IgG trastuzumab with compound 23 (20 molar equivalents relative to the antibody), regioselectively introducing a maleimide, and then coupling with compound 25. Bands above a molecular weight of approximately 50,000 indicate the introduction of a maleimide into the heavy chain of trastuzumab, followed by coupling with compound 25. Bands below a molecular weight of approximately 50,000 indicate unreacted heavy chains, and bands around a molecular weight of approximately 25,000 indicate unreacted light chains. Lane 7: Complex obtained by treating IgG trastuzumab with compound 23 (40 molar equivalents relative to the antibody), regioselectively introducing a maleimide, and then coupling with compound 25. The band with a molecular weight of approximately 50,000 or more indicates that maleimide was introduced into the heavy chain of trastuzumab and coupled with compound 25.Bands below a molecular weight of 50,000 indicate unreacted heavy chains, and bands around a molecular weight of 25,000 indicate unreacted light chains. Lanes 8 and 10: Unreacted IgG trastuzumab (control, bands around a molecular weight of 50,000 indicate heavy chains, and bands around a molecular weight of 25,000 indicate light chains). Lane 11: Reaction mixture obtained by treating IgG trastuzumab with compound 24 (10 molar equivalents relative to the antibody) and then adding compound 25. Bands around a molecular weight of 50,000 indicate unreacted heavy chains, and bands around a molecular weight of 25,000 indicate unreacted light chains. Lane 12: Reaction mixture obtained by treating IgG trastuzumab with compound 24 (20 molar equivalents relative to the antibody) and then adding compound 25. Bands around a molecular weight of 50,000 indicate unreacted heavy chains, and bands around a molecular weight of 25,000 indicate 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. The band around molecular weight 50,000 indicates unreacted heavy chain, and the band around molecular weight 25,000 indicates unreacted light chain.

[0255] [ Figure 4 ] Figure 4 This is a graph showing ESI-TOFMS analysis of the trastuzumab-maleimide modified form synthesized in (4-5-1) under reduced conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0256] [ Figure 5 ] Figure 5 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form (azide-modified antibody 1) synthesized in (6-1-1) under post-reduction conditions. The lower panel shows the measurement results for unreacted trastuzumab, and the upper panel shows the modified form.

[0257] [ Figure 6 ] Figure 6 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form (azide-modified antibody 3) synthesized in (6-1-2) under post-reduction conditions. The lower panel shows the measurement results for unreacted trastuzumab, and the upper panel shows the modified form.

[0258] [ Figure 7 ] Figure 7 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form (azide-modified antibody 6) synthesized in (6-1-3) under post-reduction conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0259] [ Figure 8 ] Figure 8This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form (azide-modified antibody 8) synthesized in (6-1-3) under post-reduction conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0260] [ Figure 9 ] Figure 9 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form (azide-modified antibody 10) synthesized in (6-1-3) under post-reduction conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0261] [ Figure 10 ] Figure 10 This is a graph showing ESI-TOFMS analysis of the adalimumab-azide modified form (azide-modified antibody 28) synthesized in (6-1-4) under post-reduction conditions. The upper panel shows the measurement results for unreacted adalimumab, and the lower panel shows the modified form.

[0262] [ Figure 11 ] Figure 11 This is a graph showing ESI-TOFMS analysis of the denosumab-azide-modified antibody (azide-modified antibody 29) synthesized in (6-1-4) under post-reduction conditions. The upper panel shows the measurement results for unreacted denosumab, and the lower panel shows the modified form.

[0263] [ Figure 12 ] Figure 12 This is a graph showing ESI-TOFMS analysis of the Dupilumab-azide-modified antibody (azide-modified antibody 30) synthesized in (6-1-4) under post-reduction conditions. The upper panel shows the measurement results for unreacted Dupilumab, and the lower panel shows the modified form.

[0264] [ Figure 13 ] Figure 13 This is a graph showing ESI-TOFMS analysis of the rituximab-azide-modified antibody (azide-modified antibody 31) synthesized in (6-1-4) under post-reduction conditions. The lower panel shows the measurement results for unreacted rituximab, and the upper panel shows the modified form.

[0265] [ Figure 14 ] Figure 14 This is a graph showing ESI-TOFMS analysis of the trastuzumab-protected thiol modified form synthesized in (8-1-1) under reduced conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0266] [ Figure 15 ] Figure 15This is a graph showing ESI-TOFMS analysis of the deprotected trastuzumab-thiol modified form in (8-3-1) under reduced conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0267] [ Figure 16 ] Figure 16 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form synthesized in (9-1-1) under reduced conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0268] [ Figure 17 ] Figure 17 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form synthesized in (9-1-5) under reduced conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0269] [ Figure 18 ] Figure 18 This is a graph showing ESI-TOFMS analysis of the trastuzumab-azide modified form synthesized in (9-2-2) under reduced conditions. The upper panel shows the measurement results for unreacted trastuzumab, and the lower panel shows the modified form.

[0270] [ Figure 19 ] Figure 19 This is an ESI-TOFMS image showing the trastuzumab-Cy3 complex synthesized in (10-1-1). The lower panel shows the measurement results for unreacted trastuzumab, the middle panel shows the measurement results for the trastuzumab-azide modified form synthesized in (6-1-1), and the upper panel shows the trastuzumab-Cy3 complex.

[0271] [ Figure 20 ] Figure 20 This graph shows the ESI-TOFMS analysis of the trastuzumab-Cy3 complex treated in (10-1-2) under reducing conditions. The lower panel shows the measurement results for unreacted trastuzumab, the middle panel shows the measurement results for the trastuzumab-azide-modified form synthesized in (6-1-1), and the upper panel shows the trastuzumab-Cy3 complex.

[0272] [ Figure 21 ] Figure 21 This is an ESI-TOFMS image showing the trastuzumab-peptide complex synthesized in (10-2-2). The lower panel shows the measurement results for unreacted trastuzumab, the middle panel shows the measurement results for the trastuzumab-azide-modified form synthesized in (6-1-1), and the upper panel shows the trastuzumab-Cy3 complex.

[0273] [ Figure 22 ] Figure 22This image shows the ESI-TOFMS analysis of the trastuzumab-Cy3 complex treated in (10-2-3) under reducing conditions. The lower panel shows the measurement results for unreacted trastuzumab, the middle panel shows the measurement results for the trastuzumab-azide-modified form synthesized in (6-1-1), and the upper panel shows the trastuzumab-peptide complex.

[0274] [ Figure 23 ] Figure 23 This is a graph showing the ESI-TOFMS analysis of the trastuzumab-maleimide compound complex treated in (10-3-1) under reducing conditions. The upper panel shows the measurement results of the thiol-introduced form of trastuzumab, and the lower panel shows the trastuzumab-maleimide compound complex.

[0275] [ Figure 24 ] Figure 24 This is a graph showing the ESI-TOFMS analysis of the reaction product treated in (10-3-2) under reducing conditions. The upper panel shows the measurement results of the thiol-introduced form of trastuzumab, and the lower panel shows the trastuzumab-maleimide compound complex.

[0276] [ Figure 25 ] Figure 25 It is a diagram showing (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 2) and (2) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 4).

[0277] [ Figure 26 ] Figure 26 It is a diagram showing (1) the amino acid sequence of the heavy chain of denosumab (SEQ ID NO: 104) and (2) the amino acid sequence of the light chain of denosumab (SEQ ID NO: 105).

[0278] [ Figure 27 ] Figure 27 The diagram shows (1) the amino acid sequence of the heavy chain of dupilumab (SEQ ID NO: 106) and (2) the amino acid sequence of the light chain of dupilumab (SEQ ID NO: 107).

[0279] [ Figure 28 ] Figure 28 The figure shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0280] [ Figure 29 ] Figure 29The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0281] [ Figure 30 ] Figure 30 The figure shows the MS spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0282] [ Figure 31 ] Figure 31 The diagram shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue (aminobenzoic acid introducer (+119.037 Da)) of azidobenzoic acid-modified trastuzumab, digested with trypsin.

[0283] [ Figure 32 ] Figure 32 The diagram shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of maleimide-modified trastuzumab to which mercaptopropionic acid was added, digested with trypsin.

[0284] [ Figure 33 ] Figure 33 The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of maleimide MPA-modified trastuzumab digested with trypsin.

[0285] [ Figure 34 ] Figure 34 The figure shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of alkylazide-modified trastuzumab digested with trypsin.

[0286] [ Figure 35 ] Figure 35The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of trastuzumab modified with alkylazide, digested with trypsin.

[0287] [ Figure 36 ] Figure 36 The figure shows the MS spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 101) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0288] [ Figure 37 ] Figure 37 The figure shows the CID spectrum of a peptide fragment of azidobenzoic acid-modified trastuzumab containing the modification site VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 101) at a lysine residue, digested with trypsin.

[0289] [ Figure 38 ] Figure 38 This figure shows the results of analysis using BioPharma Finder, which demonstrates that the lysine residue at position 317 of azidobenzoic acid-modified trastuzumab is highly selectively modified.

[0290] [ Figure 39 ] Figure 39 The figure shows the MS spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0291] [ Figure 40 ] Figure 40 The figure shows the CID spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0292] [ Figure 41 ] Figure 41 The figure shows the analysis results using BioPharma Finder, which show that the lysine residue at position 288 or 290 of azidobenzoic acid-modified trastuzumab is highly selectively modified.

[0293] [ Figure 42 ] Figure 42The figure shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0294] [ Figure 43 ] Figure 43 The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0295] [ Figure 44 ] Figure 44 The figure shows the analysis results using BioPharma Finder, which show that the lysine residue at position 246 or 248 of azidobenzoic acid-modified trastuzumab is highly selectively modified.

[0296] [ Figure 45 ] Figure 45 The graph shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of trastuzumab modified with acetylthiol, digested with trypsin.

[0297] [ Figure 46 ] Figure 46 The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of trastuzumab modified with acetylthiol, digested with trypsin.

[0298] [ Figure 47 ] Figure 47 The figure shows the analysis results using BioPharma Finder, which show that the lysine residue at position 246 or 248 of acetylthiol-modified trastuzumab is highly selectively modified.

[0299] [ Figure 48 ] Figure 48 The figure shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0300] [ Figure 49 ] Figure 49The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0301] [ Figure 50 ] Figure 50 The figure shows the MS spectrum of a peptide fragment of FNWYVDGVEVHN AKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue of azidobenzoic acid-modified trastuzumab digested with trypsin.

[0302] [ Figure 51 ] Figure 51 The figure shows the CID spectrum of a peptide fragment of azidobenzoic acid-modified trastuzumab FNWYVDGVEVHNAKT KPR (SEQ ID NO: 12) containing a modification site on a lysine residue, digested with trypsin.

[0303] [ Figure 52 ] Figure 52 The figure shows the MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of trastuzumab modified with acetylthiol and azidobenzoic acid, digested with trypsin.

[0304] [ Figure 53 ] Figure 53 The figure shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11) containing a modification site on a lysine residue of trastuzumab modified with acetylthiol and azidobenzoic acid, digested with trypsin.

[0305] [ Figure 54 ] Figure 54 The figure shows the MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue of trastuzumab modified with acetylthiol and azidobenzoic acid, digested with trypsin.

[0306] [ Figure 55 ] Figure 55 The figure shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12) containing a modification site on a lysine residue of trastuzumab modified with acetylthiol and azidobenzoic acid, digested with trypsin.

[0307] [ Figure 56 ] Figure 56 The figure shows the results of analysis using BioPharma Finder, which shows that lysine residues at positions 246 or 248 and 288 or 290 of trastuzumab modified with acetylthiol and azidobenzoic acid are highly selectively modified.

[0308] [ Figure 57 ] Figure 57 The figure shows the MS spectrum of a peptide fragment of CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR (SEQ ID NO: 102) containing a modification site on a lysine residue of benzoic acid-modified denosumab digested with trypsin.

[0309] [ Figure 58 ] Figure 58 The figure shows the CID spectrum of a peptide fragment of CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR (SEQ ID NO: 102) containing a modification site on a lysine residue of benzoic acid-modified denosumab digested with trypsin.

[0310] [ Figure 59 ] Figure 59 The figure shows the analysis results using BioPharma Finder, which show that the lysine residue at position 247 or 249 of benzoic acid-modified denosumab is highly selectively modified.

[0311] [ Figure 60 ] Figure 60 The figure shows the MS spectrum of a peptide fragment of YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 103) containing a modification site on a lysine residue of benzoic acid-modified pilumab digested with trypsin.

[0312] [ Figure 61 ] Figure 61 It is a diagram showing the CID spectrum of a peptide fragment of YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 103) containing a modification site on a lysine residue of benzoic acid-modified pilumab digested with trypsin.

[0313] [ Figure 62 ] Figure 62 This is a graph showing the results of analysis using BioPharma Finder, which shows that the lysine residue at position 251 or position 253 of benzoic acid-modified pilolumab is highly selectively modified.

[0314] [ Figure 63 ] Figure 63It is a graph showing the analysis results of the trastuzumab-DM1 conjugate synthesized in (12-1-1) by ESI-TOFMS (under non-reducing conditions).

[0315] [ Figure 64 ] Figure 64 It is a graph showing the analysis results of the trastuzumab-DM1 conjugate synthesized in (12-1-1) by ESI-TOFMS (under reducing conditions).

[0316] [ Figure 65 ] Figure 65 It is a graph showing the analysis results of the trastuzumab-MMAE conjugate synthesized in (12-2-1) by ESI-TOFMS (under non-reducing conditions).

[0317] [ Figure 66 ] Figure 66 It is a graph showing the analysis results of the trastuzumab-MMAE conjugate synthesized in (12-2-1) by ESI-TOFMS (under reducing conditions).

[0318] [ Figure 67 ] Figure 67 It is a graph showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-3-1) by ESI-TOFMS (under non-reducing conditions).

[0319] [ Figure 68 ] Figure 68 It is a graph showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-3-1) by ESI-TOFMS (under reducing conditions).

[0320] [ Figure 69 ] Figure 69 It is a graph showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-4-1) by ESI-TOFMS (under non-reducing conditions).

[0321] [ Figure 70 ] Figure 70 It is a graph showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-4-1) by ESI-TOFMS (under reducing conditions).

[0322] [ Figure 71 ] Figure 71 The diagram shows (1) the consensus amino acid sequence of the Fc region in the heavy chain of trastuzumab and the IgG1 Fc region (SEQ ID NO: 1), and (2) the amino acid sequence of the IgG1 Fc region (SEQ ID NO: 3). DETAILED DESCRIPTION

[0323] 1. Compounds having an affinity substance for antibodies and a bioorthogonal functional group or salts thereof 1-1. Overview

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

[0325] ALEB(I)

[0326] [Where,

[0327] A is an affinity substance for antibodies,

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

[0329] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0330] B is a bioorthogonal functional group,

[0331] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group. ]

[0332] In the writing of formula (I) and other formulae shown in connection with the present invention, - (hyphen) indicates that two units present 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 having an affinity substance for antibodies and a bioorthogonal functional group represented by formula (I) or a salt thereof shows that the affinity substance (A) for antibodies contains a structural unit having LEB via a covalent bond between A and L. Therefore, in formula (I), the affinity substance (A) for antibodies may have one structural unit having LEB or a plurality of (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) structural units (homologous or heterologous) having LEB.

[0333] Other formulas also show that the affinity substance (A) or antibody (Ab) for antibodies contains the specific structural unit (structural unit other than A or Ab) in the formula via a covalent bond. Therefore, in other formulas, the affinity substance (A) or antibody (Ab) for antibodies may have one specific structural unit or multiple (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) specific structural units (of the same or different species).

[0334] 1-2. Affinity substances for antibodies (A)

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

[0336] The affinity substance used in the present invention targets antibodies. Antibodies can be antibodies modified with biomolecules (such as sugars) or antibodies that are not modified with biomolecules. As antibodies, any antibodies against any components such as components from organisms, components from viruses, and components visible in the environment can be used, but antibodies against components from organisms or components from viruses are preferred. As components from organisms, for example, components (such as proteins) from animals such as mammals, birds (such as chickens), insects, microorganisms, plants, fungi, and fish can be listed. Preferably, the components from organisms are components from mammals. 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 cattle, pigs, goats), and draft animals (such as horses and sheep) can be listed. Components from organisms are more preferably components (such as proteins) from primates or rodents, and from the perspective of the clinical application of the present invention, they are further preferably components (such as proteins) from humans. Examples of virus-derived components include components (eg, proteins) derived from influenza viruses (eg, avian influenza viruses, swine influenza viruses), HIV, Ebola viruses, and bacteriophage viruses.

[0337] In addition, the antibody is a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. As a monoclonal antibody, for example, a chimeric antibody, a humanized antibody, a human antibody, an antibody to which a prescribed sugar chain is attached (for example, an antibody modified in the manner of a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), a bispecific antibody, a scFv antibody, a Fab antibody, a F(ab')2 antibody, a VHH antibody, an Fc region protein, an Fc fusion protein can be listed. The antibody can also be a bivalent antibody (for example, IgG, IgD, IgE) or a tetravalent or higher antibody (for example, an IgA antibody, an IgM antibody).

[0338] The antibody serving as the target of the affinity substance may also be composed of any amino acid residues, preferably the 20 naturally occurring L-α-amino acid residues that generally 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), L-lysine (K), and glycine (G) (hereinafter, L is omitted). The antibody may be composed of, for example, 100 or more amino acid residues, preferably 120 or more, more preferably 150 or more, even more preferably 180 or more, and particularly preferably 200 or more amino acid residues. The antibody may also have, for example, 1000 or fewer, preferably 900 or fewer, more preferably 800 or fewer, even more preferably 700 or fewer, and particularly preferably 600 or fewer. More specifically, the antibody may be composed of, for example, 100 to 1000 amino acid residues, preferably 120 to 900, more preferably 150 to 800, even more preferably 180 to 700 or more, and particularly preferably 200 to 600 amino acid residues. When the antibody is an antibody (e.g., the monoclonal antibody described above), the above number of amino acid residues may correspond to the amino acid residues of the antibody heavy chain.

[0339] The antibody as the target of the affinity substance can also be a protein containing a side chain or terminal (N-terminal and / or C-terminal) that can react with a bioorthogonal functional group as described later, preferably a specific amino acid residue of a side chain at one position or multiple positions (preferably multiple positions). As such a specific amino acid residue, for example, an amino acid residue as described later can be cited, preferably an amino acid residue selected from lysine residues, tyrosine residues, serine residues, threonine residues and cysteine ​​residues. If it is considered that the compound of the present invention can selectively modify the antibody at a position, it is preferred that an antibody containing such a specific amino acid residue at multiple positions is included. As multiple positions, as long as it is more than 2 positions, there is no particular limitation, for example, it can be more than 3 positions, preferably more than 5 positions, more preferably more than 10 positions, further preferably more than 20 positions, and particularly preferably more than 30 positions. Multiple positions, for example, can also be less than 200 positions, preferably less than 180 positions, more preferably less than 150 positions, further preferably less than 120 positions, and particularly preferably less than 100 positions. More specifically, the plurality of positions may be, for example, 3 to 200 positions, preferably 5 to 180 positions, more preferably 10 to 150 positions, further preferably 20 to 120 positions, and particularly preferably 30 to 100 positions. Even for antibodies containing such specific amino acid residues at multiple positions, the compounds of the present invention can selectively modify one or two specific amino acid residues present in a specific region. For example, the number of lysine residues in human IgG1, although it also depends on the amino acid composition in the variable region, is generally believed to be around 70 to 90. In the present invention, position-selective modification of one or two lysine residues present in such a specific region of human IgG1 was successfully achieved.

[0340] More specifically, in the present invention, from the perspective of modifying amino acid residues at specific target sites in the antibody while maintaining antibody function (i.e., maintaining the native folding without denaturing the antibody), position-selective modification of amino acid residues exposed on the antibody surface is preferred. For example, in human IgG1 and other human IgGs, exposed lysine residues and exposed tyrosine residues are present at the following positions (based on EU numbering, see 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 (positions 360, 414, and 439)

[0344] (2) Exposed tyrosine residues

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

[0346] CH3 domain (position 436)

[0347] (3) Exposed serine residues

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

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

[0350] (4) Exposed threonine residues

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

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

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

[0354] Preferably, when a lysine residue, tyrosine residue, serine residue or threonine residue is modified in human IgG1 or other human IgG, the modified lysine residue, tyrosine residue, serine residue or threonine residue at the following positions with high exposure to the surface among the above-mentioned positions (1) to (4) may be used.

[0355] (1') exposed lysine residues

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

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

[0358] (2') exposed tyrosine residues

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

[0360] CH3 domain (position 436)

[0361] (3') exposed serine residue

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

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

[0364] (4') exposed threonine residue

[0365] CH2 domain (positions 256 and 289)

[0366] CH3 domain (positions 335, 359)

[0367] Therefore, when human IgG1 or other human IgG is modified at a lysine residue, a tyrosine residue, a serine residue or a threonine residue, modification at the above position is more preferred.

[0368] More preferably, when a lysine residue in human IgG such as human IgG1 is modified, among the positions in (1) above, the lysine residue that is modified may be a specified position (e.g., position 246, position 248, position 288, position 290, position 317) in the CH2 domain that can be effectively modified in the present invention.

[0369] In a specific embodiment, the antibody as the target of the affinity substance, when containing specific amino acid residues at multiple positions as described above, may be a target region consisting of 1 to 50 consecutive amino acid residues containing one or more specific amino acid residues and a non-target region other than the target region containing 5 or more specific amino acid residues. 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. It is particularly preferred that the target region be a region consisting of specific amino acid residues present at a specific position. Such a specific position also varies according to the type of target protein and affinity substance, for example, it may be a specific position in a specific region (e.g., CH1, CH2, CH3) in the constant region of the antibody, preferably 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 referred to simply as "Lys248" in this specification, corresponding to the 18th residue in the CH2 region of human IgG (SEQ ID NO: 1)) or Lys246 residue (hereinafter, also referred to simply as "Lys246" in this specification, corresponding to the 16th residue in the CH2 region of human IgG (SEQ ID NO: 1));

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

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

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

[0374] The target region may also be a region from a specific amino acid residue present at a specific position to a position a (where a is an integer of 1 to 10) amino acid residues from the N-terminus and C-terminus, centered at the specific position, and containing no amino acid residues of the same type as the specific amino acid residue other than the specific amino acid residue present at the specific position. a is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, further preferably 1 or 2, and particularly preferably 1.

[0375] In a preferred embodiment, the antibody is a monoclonal antibody. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Monoclonal antibodies are full-length antibodies or antibody fragments (e.g., F(ab')2, Fab', Fab, Fv, single-chain antibodies), with full-length antibodies being preferred. Particularly preferably, the antibody is a human antibody, humanized antibody, or chimeric antibody having a human IgG constant region (e.g., IgG1, IgG2, IgG3, IgG4).

[0376] Antibodies are antibodies directed against any antigen. For example, such antigens may be components found in organisms or viruses as described above. Examples of such antigens include proteins (including oligopeptides and polypeptides, including proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low molecular weight compounds.

[0377] Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.

[0378] More specifically, the protein serving as an antigen for the antibody may be a disease target protein. Examples of disease target proteins 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, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endothelial growth factor), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (insulin-like growth factor factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, Glypican-3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, tissue factor Factor), IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor-associated macrophages,tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, FucosylGM1, IGF-1, Angiopoietin 2 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, immunosuppressive protein (ImmuneSuppressor Protein), NaPi2b, Lewis blood group B antigen, A34, lysyl 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, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin) 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) Brain and nerve diseases

[0384] CGRP, CD20, β-amyloid protein, β-amyloid fibrils, 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 pneumoniae, and Pneumococcus.

[0387] (5) Hereditary-rare diseases

[0388] Amyloid 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, and β-amyloid protein.

[0391] (7) Orthopedics and Plastic Surgery

[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 described above for the antibody, but is preferably IgG (e.g., IgG1, IgG2, IgG3, IgG4). Preferably, the monoclonal antibody is a full-length monoclonal antibody.

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

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

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

[0401] (B) an Fc region protein comprising an amino acid sequence in which one or more amino acid residues are inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or

[0402] (C) An Fc region protein comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 1.

[0403] The amino acid sequence of SEQ ID NO:1 represents an Fc region protein. Such Fc region proteins are known to have secretory ability. Therefore, the Fc region proteins (A) to (C) described above may have secretory ability. In addition, antibodies comprising such Fc region proteins may have antigen-binding ability. The amino acid residue at position 18 in SEQ ID NO:1 is any amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a nonpolar side chain as described below, further preferably leucine, isoleucine, or alanine, and particularly preferably leucine or alanine. The amino acid residue at position 19 in SEQ ID NO:1 is any 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, further preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO:1 is any amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a nonpolar side chain, further preferably glycine or alanine. The amino acid residue at position 140 in SEQ ID NO: 1 is any 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 any amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, further preferably methionine, leucine, or isoleucine, and particularly preferably methionine or leucine. The amino acid residue at position 177 in SEQ ID NO: 1 is any amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having an uncharged polar side chain or an amino acid residue having a non-polar side chain as described below, further 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 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 comprising the above-described amino acid sequence" may be an antibody comprising an "Fc region protein comprising the above-described amino acid sequence" and an "antibody constant region." Such an antibody constant region may be a chimeric antibody, a humanized antibody, or a human antibody (e.g., IgG1, IgG2, IgG3, IgG4, etc.).

[0407] In the Fc region protein (B), one or more amino acid residues can be changed by one, two, three or four mutations selected from deletion, substitution, addition and insertion. The mutation of the amino acid residue can be introduced into one region in the amino acid sequence, or it can be introduced into multiple different regions. The term "one or more" means the number that does not significantly destroy the activity of the protein. The number shown in 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 (for example, 1, 2, 3, 4 or 5).

[0408] In the Fc region protein (C), the % identity with the amino acid sequence of SEQ ID NO: 1 can 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 % identity of a peptide or polypeptide (protein) can be calculated by the algorithm blastp. More specifically, the % identity of a polypeptide can be calculated 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 % identity of a polynucleotide (gene) can be calculated by the algorithm blastn. More specifically, the calculation of the % identity of the polynucleotide can be performed using the default scoring parameters (Match / Mismatch Scores=1, -2; Gap Costs=Linear) in the blastn algorithm provided by NCBI.

[0409] The term "secretion" in the secretory capacity has the same meaning as the secretion of secretory proteins (so-called solubility). Therefore, "having secretory capacity" means that the protein functions as an antibody in the same manner as a normal antibody.

[0410] As long as the above-mentioned antibody comprising the Fc region protein maintains the characteristics of the target (such as secretory ability, antigen binding ability), mutations can be introduced into specific sites. The position of the amino acid residue that can be introduced into the mutation that can maintain the target characteristics is self-evident to those skilled in the art. Specifically, for those skilled in the art, 1) compare the amino acid sequences of various proteins with the same characteristics, 2) clarify the relatively preserved region and the relatively unpreserved region, and then 3) the relatively preserved region and the relatively unpreserved region can respectively predict the region that can play an important role for the function and the region that cannot play an important role for the function, so the correlation of structure / function can be identified. Therefore, those skilled in the art can specify the position of the amino acid residue that can be introduced into the amino acid sequence of the above-mentioned antibody comprising the Fc region protein.

[0411] In the case where an amino acid residue is mutated by substitution (replacement), the substitution of the amino acid residue can be a conservative substitution. In the case used in this specification, the term "conservative substitution" refers to replacing a specified amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such a family includes amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids having side chains containing hydroxyl groups (e.g., alcoholic, phenolic) (e.g., serine, threonine, tyrosine), and amino acids having sulfur-containing side chains (e.g., cysteine, methionine). Preferably, the 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] Examples of the antibody used in the present invention or the antibody comprising any one of the Fc regions selected from (A) to (C) above include chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoxaximab,オルタトキサシマブ), humanized antibodies (e.g., Daclizumab, Palivizumab, Trastuzumab, Alemtuzumab, Omalizumab, Efalizumab, Bevacizumab, Natalizumab (IgG4), Tocilizumab, Eculizumab ) (IgG2), Mogamulizumab, Pertuzumab, Obinutuzumab, Vedolizumab, Pembrolizumab (IgG4), Mepolizumab, Elotuzumab, Daratumumab, Ixekizumab (IgG4), Reslizumab mab (IgG4), Atezolizumab), human antibodies (e.g., Adalimumab, Panitumumab, Golimumab, Ustekinumab, Canakinumab, Ofatumumab, Denosumab (IgG2), Ipirimumab, Belimumab, Raxicumab axibacumab), Ramucirumab, Nivolumab (IgG4), Secukinumab, Evolocumab (IgG2), Alirocumab, Necitumumab, Brodalumab (IgG2), Olaratumab, Dupilumab (IgG4) (If the IgG isotype is not mentioned, it is IgG1).

[0413] Examples of the substance having affinity for the antibody 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 a specific embodiment, the affinity substance for the antibody as described above can be a peptide (including oligopeptides, polypeptides, proteins, or glycoproteins). As such peptides, for example, the following peptides have been reported:

[0415] (1) IgG-binding peptides having affinity for a specific region (CH2 region) of all human IgGs (i.e., human IgG1, IgG2, IgG3, and IgG4, hereinafter the same) (see, for example, International Publication Nos. 2016 / 186206, 2013 / 027796, and 2008 / 054030);

[0416] (2) Protein A mimetic peptides (PAM) that have affinity for the specific region (CH2 region) of all human IgG (for example, see Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, Vol. 6, pp. 564-569);

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

[0418] (4) (NH2-Cys1-X1-X2-X3-X4)2-Lys-Gly-OH, which has affinity for the specific region (Fc region) of all human IgG (see, for example, 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) that have affinity for the specific region (Fc region) of all human IgG (see, for example, 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 (e.g., see 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 have affinity for the specific region (Fc region) of all human IgG (see, for example, Carbonell RG et al., Journal of Chromatography A, 2009, Vol. 1216, 910-918);

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

[0423] (9) Fc-I, Fc-II, and Fc-III that have affinity for all human IgG specific regions (Fc regions) (e.g., see Warren L. Delano et al., Science, 2000, Vol. 287, 1279-1283; International Publication No. 2001 / 045746); and

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

[0425] In another specific embodiment, the affinity substance for the antibody described above may be a substance other than a peptide. As such a substance, for example, aptamers having affinity for a specific region (CH2 region, particularly the side chain of Lys340) of human IgG (e.g., human IgG1-4) [e.g., aptamers containing a GGUG(C / A)(U / T) motif such as GGUGCU and GGUGAU] have been reported (e.g., see International Publication No. 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] Affinity substances for antibodies as described above can be obtained by any known method in the field. For example, partial peptides in the entire antibody or antibody (such as partial peptides present in the region where the antibody surface is exposed) can be used, by making antibodies (such as hybridoma methods), or by screening affinity substances (such as peptide libraries, antibody libraries, antibody-producing cell libraries, aptamer libraries, phage libraries, mRNA libraries, cDNA libraries) from libraries where affinity substances can be obtained (such as phage display methods, SELEX methods, mRNA display methods, ribosome display methods, cDNA display methods, yeast display methods), to obtain. In addition, when the affinity substance for an antibody is an affinity substance for the antibody Fc region (soluble region), it is possible to efficiently obtain an affinity substance (e.g., antibody, aptamer) that can selectively bind to any part of the antibody Fc region by using partial peptides present in specific regions (e.g., CH1, CH2, CH3) of the Fc region of various antibodies (e.g., IgG, IgA, IgM, IgD, IgE). Among the affinity substances obtained in this way, there are substances with relatively strong affinity binding ability and substances with relatively weak affinity binding ability. However, even an affinity substance with relatively weak affinity binding ability can be supplemented by using an excess amount of it.

[0427] In a preferred embodiment, the affinity substance for the antibody is a peptide. As such a peptide, a peptide having a binding ability to the constant region of a monoclonal antibody is preferred, a peptide having a binding ability to the Fc region of a monoclonal antibody is more preferred, and a peptide having a binding ability to the Fc region of an IgG is further preferred. The length of the peptide is not particularly limited, and for example, a peptide consisting of 10 to 40 (e.g., 10 to 20, 20 to 30, and 30 to 40) amino acid residues is preferred. As amino acid residues constituting the peptide, 20 kinds of L-α-amino acid residues and their stereoisomers (e.g., D-amino acids) and their isomers (e.g., β-amino acids) that constitute natural proteins can be listed.

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

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

[0430] In the amino acid sequence of (a-1-1) FNMQQQRRFYEALHD PNLNEEQRNARIRSIRDD (SEQ ID NO: 11) (for example, reference compounds 22 to 24, 29), or

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

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

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

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

[0435] Such peptides have the ability to bind to the Fc region of a monoclonal antibody.

[0436] Due to circumstances in peptide reagent synthesis, the peptide consisting of the amino acid sequence of SEQ ID NO: 12 is a peptide in which the two K (lysine) residues at positions 26 and 28 from the N-terminus are changed to R (arginine) in the affinity peptide known as Z34C. The compounds of the present invention containing the peptide comprising the above amino acid sequence can be used for position-selective modification of specific amino acid residues in human IgG Fc (e.g., Lys248 or Lys246, Lys288 or Lys290, Lys317, or other amino acid residues other than these residues based on Eu numbering). It should be noted that the amino acid sequence of the above Z34C is FNMQCQRRFYEALHDPNLNEEQRNA KIKSIRDDC (SEQ ID NO: 36) (see, for example, Starovasnik, MA 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 affinity for human IgG (eg, human IgG as described above, preferably human IgG1). When the affinity peptide contains two cysteine ​​residues (eg, at positions 5 and 34), the two cysteine ​​residues may 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 (an amino acid residue that can be easily modified by a cross-linking agent), any position can be used as long as it has affinity for human IgG such as human IgG1. For such a position, as long as one skilled in the art can easily identify it. 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 cross-linking agent, for example, amino acid residues at positions 1, 3, 6, 7, 13, 20, 24, 31 and 32 can be listed.

[0439] Preferably, the amino acid sequence having the characteristics of (a) and (b) also preferably has a specific amino acid residue (preferably a lysine residue) selected from lysine residues, aspartic acid residues, and glutamic acid residues (amino acid residues that can be easily modified by a cross-linking agent) at a specified position, and has mutations of the usual 20 amino acid residues constituting natural proteins (preferably 17 amino acid residues other than lysine residues, aspartic acid residues, and glutamic acid residues, more preferably 19 amino acid residues other than lysine residues) at positions other than the specified position. Such a specified position is not particularly limited, and for example, position 1, position 3, position 6, position 7, position 13, position 20, position 24, position 31, and position 32 can be cited. The amino acid sequence having the characteristics of (a) and (b) maintains two cysteine ​​residues, and these two cysteine ​​residues can be combined by disulfide bonding. The amino acid sequences having 85% or greater identity to the 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 altered by 1, 2, 3, or 4 mutations (preferably substitutions) selected from deletion, substitution, addition, and insertion of amino acid residues. The amino acid residue mutations may be introduced into a single region within the amino acid sequence or into multiple different regions.

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

[0441] (c) an amino acid sequence selected from the group consisting of 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) An amino acid sequence having 90% or more identity with any of the amino acid sequences described above (1) to (16), which has mutations in 19 types of amino acid residues other than a lysine residue at positions other than one lysine residue and two cysteine ​​residues (e.g., positions 1, 3, 6, 7, 13, 20, 24, 31, and 32), and which has the above-mentioned number of amino acid residues as the number of modifications, other than a lysine residue.

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

[0460] The affinity peptides may have a mutation in further amino acid residues in addition to the introduction of one amino acid residue that is easily modified by a cross-linking agent, as long as they have an identity of 85% or more to the amino acid sequences of SEQ ID NOs: 11 to 14 or the amino acid sequences of (1) to (16) above. Positions where further amino acid mutations can be introduced can be easily identified by those skilled in the art. For example, even positions such as the phenylalanine residue at position 1, the arginine residue at position 6, the leucine residue at position 13, the glutamic acid residue at position 20, the aspartic acid residue at position 24, or the arginine residue at position 31 (excluding positions where amino acid residues that can be easily modified by a cross-linking agent have already been introduced) can be used. As amino acids that can be introduced by further amino acid mutation, 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), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Preferably, these 19 amino acids except lysine can be utilized. The amino acid may be either L- or D-individual, but the L-individual is preferred (the amino acid residues constituting the peptide in the examples are all L-individual).

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

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

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

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

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

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

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

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

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

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

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

[0472] [Where,

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

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

[0475] Xaa1 is an alanine residue,

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

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

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

[0479] 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,

[0480] Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. ]; or

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

[0482] [Where,

[0483] (X 0-3 ') a and (X 0-3 ') b Respectively with the above (X 0-3 ) a and (X 0-3 ) b same,

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

[0485] Such peptides have the ability to bind to the Fc region of a monoclonal antibody.

[0486] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and 2-1, (X 0-3 ) a It 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 formulae 1-1 to 1-9 and 2-1, Xaa2 is a tyrosine residue, a tryptophan residue, or a histidine residue, preferably a tyrosine residue or a tryptophan residue.

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

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

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

[0492] Preferably, the peptide comprising any one of the amino acid sequences represented by the above formulae 1-1 to 1-9 and 2-1 is a peptide comprising 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);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0504] <h2 style=";text-align:left;direction:ltr"> (12')RGNCAYHKDQIIWCTYH(SEQ ID NO:57);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0505] <h2 style=";text-align:left;direction:ltr"> (13')RGNCAYHKQQIIWCTYH(SEQ ID NO:58);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0506] <h2 style=";text-align:left;direction:ltr"> (14')RGNCAYHKEQIIWCTYH(SEQ ID NO:59);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0507] <h2 style=";text-align:left;direction:ltr"> (15')RGNCAYHKFQIIWCTYH(SEQ ID NO:60);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0508] <h2 style=";text-align:left;direction:ltr"> (16')RGNCAYHKYQIIWCTYH(SEQ ID NO:61);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0509] <h2 style=";text-align:left;direction:ltr"> (17')RGNCAYHKWQIIWCTYH(SEQ ID NO:62);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0510] <h2 style=";text-align:left;direction:ltr"> (18')RGNCAYHKHQIIWCTYH(SEQ ID NO:63);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0511] <h2 style=";text-align:left;direction:ltr"> (19')RGNCAYHKTQIIWCTYH(SEQ ID NO:64);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0512] <h2 style=";text-align:left;direction:ltr"> (20')RGNCAYHKLQIIWCTYH(SEQ ID NO:65);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0513] <h2 style=";text-align:left;direction:ltr"> (21')CAYHKLQIVWC(SEQ ID NO:66);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0514] <h2 style=";text-align:left;direction:ltr"> (22')CAYHKLQLIWC(SEQ ID NO:67);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0515] <h2 style=";text-align:left;direction:ltr"> (23')CAYHKSQIVWC(SEQ ID NO:68);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0516] <h2 style=";text-align:left;direction:ltr"> (24')RGNCAYHKGQLVFCTYH(SEQ ID NO:69);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0517] <h2 style=";text-align:left;direction:ltr"> (25')RGNCAYHKGQQVWCTYH(SEQ ID NO:70);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0518] <h2 style=";text-align:left;direction:ltr"> (26')RGNCAYHKGQEVWCTYH(SEQ ID NO:71);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0519] <h2 style=";text-align:left;direction:ltr"> (27')CAYHKGQLVWC(SEQ ID NO:72);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0520] <h2 style=";text-align:left;direction:ltr"> (28')RGNCAYHKAQLVWCTYH(SEQ ID NO:73);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0521] <h2 style=";text-align:left;direction:ltr"> (29')RGNCAYHKVQLVWCTYH(SEQ ID NO:74);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0522] <h2 style=";text-align:left;direction:ltr"> (30')RGNCAYHKLQLVWCTYH(SEQ ID NO:75);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0523] <h2 style=";text-align:left;direction:ltr"> (31')RGNCAYHKIQLVWCTYH(SEQ ID NO:76);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0524] <h2 style=";text-align:left;direction:ltr"> (32')RGNCAYHKSQLVWCTYH(SEQ ID NO:77);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0525] <h2 style=";text-align:left;direction:ltr"> (33')RGNCAYHKTQLVWCTYH(SEQ ID NO:78);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0526] <h2 style=";text-align:left;direction:ltr"> (34')RGNCAYHKNQLVWCTYH(SEQ ID NO:79);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0527] <h2 style=";text-align:left;direction:ltr"> (35')RGNCAYHKDQLVWCTYH(SEQ ID NO:80);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0528] <h2 style=";text-align:left;direction:ltr"> (36')RGNCAYHKQQLVWCTYH(SEQ ID NO:81);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0529] <h2 style=";text-align:left;direction:ltr"> (37')RGNCAYHKEQLVWCTYH(SEQ ID NO:82);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0530] <h2 style=";text-align:left;direction:ltr"> (38')RGNCAYHKFQLVWCTYH(SEQ ID NO:83);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0531] <h2 style=";text-align:left;direction:ltr"> (39')RGNCAYHKRQLVWCTYH(SEQ ID NO:84);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0532] <h2 style=";text-align:left;direction:ltr"> (40')RGNCAYHKHQLVWCTYH(SEQ ID NO:85);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0533] <h2 style=";text-align:left;direction:ltr"> (41')RGNCAYHKWQLVWCTYH(SEQ ID NO:86);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0534] <h2 style=";text-align:left;direction:ltr"> (42')RGNCAYHKYQLVWCTYH(SEQ ID NO:87);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0535] <h2 style=";text-align:left;direction:ltr"> (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 a peptide containing such an amino acid sequence has the ability to bind to the Fc region of IgG.

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

[0548] [Chemical Formula 8]

[0549]

[0550] The dotted portion of the carbonyl-containing linker shown above represents the portion that binds to the thioether group. This linker is more stable to reduction reactions, etc., than a conventional disulfide bond. Such a peptide can be prepared, for example, using the method described in International Publication No. 2016 / 186206.

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

[0552] The above-mentioned peptides can be modified with specific amino acid residues using a cross-linking agent. Examples of such specific amino acid residues include lysine residues, aspartic acid residues, and glutamic acid residues, with lysine residues being preferred. Examples of cross-linking agents include DSG (disuccinimidyl glutarate, disuccinimidyl glutarate), DSS (disuccinimidyl suberate, disuccinimidyl suberate), and preferably a cross-linking agent containing two or more succinimide groups; DMA (dimethyl adipimidate · 2HCl, dimethyl pimelimidate dihydrochloride), DMP (dimethyl pimelimidate · 2HCl, dimethyl pimelimidate dihydrochloride), and DMS (dimethylsuberimidate · 2HCl, dimethyl suberimidate dihydrochloride) and the like, preferably a cross-linking agent containing two or more imidic acid moieties; and DTBP (dimethyl3,3'-dithiobispropionimidate · Cross-linking agents having an SS bond, such as 2HCl (dimethyl 3,3'-dithiobispropionate dihydrochloride) and DSP (dithiobis(succinimidyl propionate)) (for example, International Publication No. 2016 / 186206).

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

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

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

[0556] A leaving group is a group that has the ability to cleave and dissociate from E by reacting 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 technical knowledge in this field (e.g., Fujishima, S. et al., J. Am. Chem. Soc, 2012, 134, 3961-3964. (supra); Chem. Sci. 2015, 3217-3224.; Nature Chemistry, Vol. 8, pp. 542-548 (2016)). The leaving group is not particularly limited as long as it has the ability to be cleaved from E by the reaction as described above, 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 -ON(R)- (where R represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); and (2) a heteroarylene group.

[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. Alkyl groups having 1 to 6 carbon atoms are preferably those having 1 to 4 carbon atoms.

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

[0559] (1) a group consisting of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(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 -ON(R)- and a group that improves its ability to leave.

[0561] The group that improves the ability to leave -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- is a group that improves the ability to leave -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- compared to 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 -ON(R)-. This group improves the ability of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- to dissociate from the 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 -ON(R)-. In other words, the group that improves the ability of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- is a group that has the ability to attract electrons from an oxygen atom, a sulfur atom, a selenium atom, or a nitrogen atom.

[0562] Preferred examples of groups that enhance the ability to leave -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- include: arylene groups that may be substituted with electron-withdrawing groups, heteroarylene groups that may be substituted with electron-withdrawing groups, 2,5-diketopyrrolidines that may be substituted with rings, 2,6-diketopiperidines that may be substituted with rings, 2-ketopyrrolidines that may be substituted with rings, 2-ketopiperidines that may be substituted with rings, and 2-pyridones. The number of electron-withdrawing groups that an arylene group or a heteroarylene group may have is 1 or more (e.g., 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 (e.g., a trifluoromethyl group), a boronic acid residue, a mesyl group, a tosyl group, a trifluoromethanesulfonate group, a nitro group, a cyano group, a phenyl group, and a keto group (e.g., an acyl group).

[0563] The "arylene group" in the "arylene group which may be substituted with an electron-withdrawing group" is preferably an arylene group having 6 to 24 carbon atoms, more preferably an arylene group having 6 to 18 carbon atoms, even more preferably an arylene group having 6 to 14 carbon atoms, and most preferably an arylene group having 6 to 10 carbon atoms. The arylene group which may be substituted with an electron-withdrawing group may be substituted with a substituent other than the electron-withdrawing group, or may be unsubstituted. The carbon atom counts above do not include the carbon atom counts of the electron-withdrawing group and substituents other than the electron-withdrawing group. Examples of the arylene group include phenylene, naphthylene, and anthracene.

[0564] As the "heteroarylene group" in the "heteroarylene group which may be substituted with 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 further preferred, and a heteroarylene group having 1 to 6 carbon atoms is most preferred. The heteroarylene group which may be substituted with an electron-withdrawing group may be substituted with a substituent other than the electron-withdrawing group, or may be unsubstituted. The above-mentioned number of carbon atoms does not include the number of carbon atoms of the electron-withdrawing group and the substituents other than the electron-withdrawing group. The heteroarylene group contains one 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. Examples of the heteroarylene group include a pyrrolediyl group, a furandiyl group, a thiophenediyl group, a pyridinediyl group, a pyridazinediyl group, a pyrimidinediyl group, a pyrazinediyl group, a triazinediyl group, a pyrazolediyl group, an imidazolediyl group, a thiazolediyl group, an isothiazolediyl group, an oxazolediyl group, an isoxazolediyl group, a triazolediyl group, a tetrazolediyl group, an indolediyl group, a purinediyl group, an anthraquinonediyl group, a carbazolediyl group, a fluorenediyl group, a quinolinediyl group, an isoquinolinediyl group, a quinazolinediyl group, and a phthalazinediyl group.

[0565] The ring-contractable 2,5-diketopyrrolidine, the ring-contractable 2,6-diketopiperidine, the ring-contractable 2-ketopyrrolidine, the ring-contractable 2-ketopiperidine, and the 2-pyridone may be substituted with a substituent such as an electron-withdrawing group, or may be unsubstituted.

[0566] The heteroarylene group as an example of a leaving group is a heteroarylene group having a low π electron density (i.e., less than 1). The heteroarylene group as a leaving group is preferably a heteroarylene group containing 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 a leaving group may be substituted with a substituent such as an electron-withdrawing group, or may be unsubstituted. The above-mentioned number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of heteroarylene groups containing a nitrogen atom as a leaving group include imidazolediyl, triazolediyl, tetrazolyldiyl, and 2-pyridonediyl (i.e., 2-hydroxypyridinediyl).

[0567] As an example of a group that improves the ability of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- to leave, a group such as an "arylene group that may be substituted with an electron-withdrawing group" and an example of a leaving group such as a "heteroarylene group" may have 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may not have a substituent. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such a substituent. On the other hand, when the group as described above has a substituent, such a substituent may be exemplified as follows:

[0568] (i) halogen atoms;

[0569] (ii) a monovalent hydrocarbon group;

[0570] (iii) aralkyl;

[0571] (iv) a monovalent heterocyclic group;

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

[0573] (vi)NR b R c -、NRb R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c The same or different, represents a hydrogen atom or a monovalent hydrocarbon group. );

[0574] (vii) nitro, sulfate, sulfonate, 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] A monovalent chain hydrocarbon group refers to a hydrocarbon group consisting solely of a chain structure, without a cyclic structure in the main chain. However, the chain structure may be straight-chain or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be straight-chain or branched.

[0578] The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the alkyl group having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0579] The alkenyl group is preferably an alkenyl group having 2 to 12 carbon atoms, more preferably an alkenyl group having 2 to 6 carbon atoms, and even more preferably an alkenyl group having 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the alkenyl group having 2 to 12 carbon atoms include ethenyl, propenyl, and n-butenyl.

[0580] Alkynyl groups are preferably those having 2 to 12 carbon atoms, more preferably those having 2 to 6 carbon atoms, and even more preferably those having 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of alkynyl groups having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

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

[0582] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group containing only an alicyclic hydrocarbon as a ring structure and not an aromatic ring. The alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons and may partially contain a chain structure. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl groups, cycloalkenyl groups, and cycloalkynyl groups, which may be either monocyclic or polycyclic.

[0583] The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 carbon atoms, more preferably a cycloalkyl group having 3 to 6 carbon atoms, and even more preferably a cycloalkyl group having 5 to 6 carbon atoms. The above carbon number does not include the carbon number of the substituent. Examples of the cycloalkyl group having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0584] The cycloalkenyl group is preferably a cycloalkenyl group having 3 to 12 carbon atoms, more preferably a cycloalkenyl group having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl group having 5 to 6 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the cycloalkenyl group having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0585] The cycloalkynyl group is preferably a cycloalkynyl group having 3 to 12 carbon atoms, more preferably a cycloalkynyl group having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl group having 5 to 6 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of the cycloalkynyl group having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

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

[0587] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to be composed solely of aromatic rings; a portion thereof may contain a chain structure or an alicyclic hydrocarbon, and the aromatic ring may be either monocyclic or polycyclic. Monovalent aromatic hydrocarbon groups are preferably aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in substituents. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

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

[0591] A monovalent heterocyclic group is a group formed by removing one hydrogen atom from the heterocyclic ring of a heterocyclic compound. A monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic group preferably contain one or more heteroatoms selected from oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contain one or more heteroatoms selected from oxygen, sulfur, and nitrogen atoms.

[0592] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and still more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furyl, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinoneyl, carbazolyl, fluorenyl, quinolyl, isoquinolyl, quinazolinyl, and phthalazinyl.

[0593] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and still more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. Examples of the monovalent non-aromatic heterocyclic group include an oxiranyl group, an aziridinyl group, an azetidinyl group, an oxetanyl group, a thietanyl group, a pyrrolidinyl group, a dihydrofuranyl group, a tetrahydrofuranyl group, a dioxolanyl group, a tetrahydrothiophenyl group, a pyrrolinyl group, an imidazolidinyl group, an oxazolidinyl group, a piperidinyl group, a dihydropyranyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group, a morpholinyl group, a thiomorpholinyl group, a pyrazinyl group, a dihydrooxazinyl group, a tetrahydrooxazinyl group, a dihydropyrimidinyl group, and a tetrahydropyrimidinyl group.

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

[0595] Preferably, the substituent may be the following group:

[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-membered or 6-membered heterocyclic ring;

[0600] (v')R a -O-、R a -C(=O)-, R a -OC(=O)- or Ra -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') The same groups as exemplified in the above (vii).

[0603] More preferably, the substituent may be the following group:

[0604] (i") halogen atoms;

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

[0606] (iii”)R a -O-、R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms. ); 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") The same groups as exemplified in the above (vii).

[0609] More preferably, the substituent may be the following group:

[0610] (i'') halogen atoms;

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

[0612] (iii”')R a -O-、R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ); 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 c are the same or different and represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. );

[0614] (v''") are the same groups as exemplified in the above-mentioned (vii).

[0615] Particularly preferably, the substituent may be the following group:

[0616] (i””) halogen atoms;

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

[0618] (iii””)R a -O-、R a -C(=O)-, R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ); 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 are the same or different and represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. );

[0620] (v") The same groups as exemplified in the above (vii).

[0621] More specifically, the leaving group may be any of the following (a) to (c).

[0622] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)];

[0623] (b) heteroarylene; or

[0624] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms).]

[0625] As the leaving group, (a) or (b) is preferred among (a), (b), and (c), and (a) is particularly preferred. Ring P in (a) is any one of an arylene group that may be substituted with an electron-withdrawing group, a heteroarylene group that may be substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine that may be substituted with a ring-contracting group, a 2,6-diketopiperidine that may be substituted with a ring-contracting group, a 2-ketopyrrolidine that may be substituted with a ring-contracting group, a 2-ketopiperidine that may be substituted with a ring-contracting group, and a 2-pyridone. Among them, an arylene group substituted with an electron-withdrawing group, a heteroarylene group substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine, and a 2,6-diketopiperidine are more preferred, and an arylene group substituted with an electron-withdrawing group, a 2,5-diketopyrrolidine, and a 2,6-diketopiperidine are even more 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 ring-condensable 2,5-diketopyrrolidine, a ring-condensable 2,6-diketopiperidine, a ring-condensable 2-ketopyrrolidine, a ring-condensable 2-ketopiperidine, and a 2-pyridone. The details of these groups are the same as those described as preferred examples of groups that enhance the ability to leave -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)-, -ON(R)- (here, 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 an imidazolediyl group, a triazolediyl group, a tetrazolediyl group, or a 2-pyridonediyl group (=2-hydroxypyridonediyl group), and more preferably an imidazolediyl group or a 2-pyridonediyl group.

[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 -ON(R)- (here, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), preferably -S-, -C≡C-CH2-O-, -N(OR)-, -N(R)- or -ON(R)-, and more preferably S, -N(OR) or -ON(R)-.

[0630] More specifically, a 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) indicates the bond with L1, and ● (black circle) indicates the bond with E1. ).

[0638] The 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 of 1 to 4, more preferably 1, 2 or 3. n is preferably an integer of 1 to 3, more preferably 1 or 2.

[0639] The divalent group containing a leaving group represented by L is a divalent group consisting of the above-mentioned leaving group, or a divalent group containing other divalent groups in addition to the above-mentioned leaving group. Examples of such other divalent groups include: a divalent hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -NR L -(R L represents a hydrogen atom or the above-mentioned substituent), -O-, -S-, -C(═S)-, and a group consisting of a combination of two or more of these (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4). The divalent hydrocarbon group and the divalent heterocyclic group may have, for example, 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 that such substituents are not present. On the other hand, when the above-mentioned groups have substituents, examples and preferred examples of such substituents are the same as those described above.

[0640] The divalent hydrocarbon group is a linear, branched, or cyclic divalent hydrocarbon group, preferably a linear or branched divalent hydrocarbon group. Examples of the divalent hydrocarbon group include an alkylene group, an alkenylene group, an alkynylene group, and an arylene group.

[0641] Alkylene groups are preferably those having 1 to 12 carbon atoms, more preferably those having 1 to 6 carbon atoms, and particularly preferably those having 1 to 4 carbon atoms. The carbon number above does not include the carbon number of substituents. Alkylene groups may be linear, branched, or cyclic, but linear alkylene groups are preferred. Examples of such alkylene groups include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0642] Alkenylene groups preferably have 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms, and particularly preferably 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Alkenylene groups may be straight-chain, branched, or cyclic, but straight-chain alkenylene groups are preferred. Examples of such alkenylene groups include ethenylene, propenylene, butenylene, pentenylene, and hexenylene.

[0643] Alkyneylene groups are preferably those having 2 to 12 carbon atoms, more preferably those having 2 to 6 carbon atoms, and particularly preferably those having 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Alkyneylene groups may be straight-chain, branched, or cyclic, but straight-chain alkynylene groups are preferred. Examples of such alkynylene groups include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

[0644] The arylene group is preferably an arylene group having 6 to 24 carbon atoms, more preferably an arylene group having 6 to 18 carbon atoms, further preferably an arylene group having 6 to 14 carbon atoms, and still further preferably an arylene group having 6 to 10 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. Examples of the arylene group include phenylene, naphthylene, and anthracene.

[0645] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic ring preferably contain at least one selected from oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably contain at least one selected from oxygen atoms, sulfur atoms, and nitrogen atoms.

[0646] The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 1 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 1 to 15 carbon atoms, further preferably a divalent aromatic heterocyclic group having 1 to 9 carbon atoms, and still further preferably a divalent aromatic heterocyclic group having 1 to 6 carbon atoms. The above carbon number does not include the carbon number of the substituent. More specifically, examples of the divalent aromatic heterocyclic group include a pyrrolediyl group, a furandiyl group, a thiophenediyl group, a pyridinediyl group, a pyridazinediyl group, a pyrimidinediyl group, a pyrazinediyl group, a triazinediyl group, a pyrazoldiyl group, an imidazolediyl group, a thiazolediyl group, an isothiazolediyl group, an oxazolediyl group, an isoxazolediyl group, a triazolediyl group, a tetrazolediyl group, an indolediyl group, a purinediyl group, an anthraquinonediyl group, a carbazolediyl group, a fluorenediyl group, a quinolinediyl group, an isoquinolinediyl group, a quinazolinediyl group, and a phthalazinediyl group.

[0647] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, further preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and still more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of the substituent. More specifically, examples of the divalent non-aromatic heterocyclic group include a pyrroledionediyl group, a pyrrolidinedionediyl group, a pyrrolinediyl group, an oxiranediyl group, an aziridinediyl group, an azetidinediyl group, an oxetanediyl group, a thietanediyl group, a pyrrolidinediyl group, a dihydrofurandiyl group, a tetrahydrofurandiyl group, a dioxolanediyl group, a tetrahydrothiophenediyl group, a pyrrolidinediyl group, an imidazolidinediyl group, an oxazolidinediyl group, a piperidinediyl group, a dihydropyrandiyl group, a tetrahydropyrandiyl group, a tetrahydrothiopyrandiyl group, a morpholinediyl group, a thiomorpholinediyl group, a pyrazinediyl group, a dihydrooxazinediyl group, a tetrahydrooxazinediyl group, a dihydropyrimidinediyl group, and a tetrahydropyrimidinediyl group.

[0648] In certain embodiments, L may be expressed 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 those of the other divalent groups when the divalent group represented by L including a leaving group is a divalent group including 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 those of the leaving group in L.

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

[0652] The length of the main chain of L (divalent group comprising a leaving group) or L1 (bond or divalent group)-L2 (leaving group) connecting A (affinity substance) and E (divalent group comprising an electrophilic group) can be appropriately designed according to various factors such as the relationship between the target site of the affinity substance in the antibody and the specific amino acid residue in the antibody to be position-selectively modified. The main chain of L or L1-L2 refers to a chain structure consisting of multiple atoms connected by covalent bonding connecting A and E, excluding hydrogen atoms, branched structure parts and substituents. If the compound represented by formula (I) is contacted with the antibody, then A first associates with the antibody. Then, the nucleophilic group in the side chain of the specific amino acid residue to be modified in the antibody near the antibody association site (for example, the amino group in the side chain of a lysine residue) reacts with the electrophilic group in E, so that the nucleophilic group is combined with the electrophilic group, and on the other hand, the leaving group contained in L or L1 can be detached from E. In this case, if no other amino acid residue of the same type as the specific amino acid residue is present in the vicinity between the antibody binding site and the specific amino acid residue to be modified, the electrophilic group in E can positionally selectively bind to the nucleophilic group in the side chain of the specific amino acid residue to be modified in the antibody, even if the length of the main chain is not strictly controlled. Of course, even if another amino acid residue of the same type as the specific amino acid residue is present in such a region, the electrophilic group in E can positionally selectively bind to the specific amino acid residue by controlling the length of the main chain.

[0653] The length of the main chain of L or L1-L2 connecting A and E is not particularly limited as long as it can position-selectively modify specific amino acid residues in the antibody. For example, when position-selectively modifying specific amino acid residues in human IgG Fc (e.g., Lys248 or Lys246, Lys288 or Lys290, Lys317, or other amino acid residues other than these residues based on Eu numbering), it is preferably composed of 20 atoms or less. The length of the main chain of L or L1-L2 may also be preferably 1 or more, more preferably 2 or more, even 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 may also be preferably 50 or less, more preferably 30 or less, even 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 may be preferably 1 to 50, more preferably 1 to 30, even 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 is preferably 2 to 30, more preferably 3 to 20, particularly preferably 4 to 15 or 5 to 10.

[0654] When the main chain has no ring structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure (excluding hydrogen atoms, branched structure portions, and atoms in substituents).

[0655] On the other hand, when the main chain includes a ring structure, the number of atoms in the main chain can be conveniently counted from the perspective of determining the main chain length. 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 structure that does not contain a bivalent ring structure in the main chain (excluding hydrogen atoms, branched structure parts, and atoms in substituents), and then counting the number of atoms in the shortest path connecting two bonds in the ring structure (for example, see the bold paths (a) to (d) below).

[0656] [Chemical Formula 10]

[0657]

[0658] ·It is a bonding key.

[0659] In the case of (a), the shortest path is the path in bold (boldface), and therefore the number of atoms in the bivalent 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 bivalent ring structure counted as the number of atoms in the main chain is 3.

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

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

[0663] Preferably, the main chain of L or L1-L2 may be a chain structure in which the "other divalent groups" in L or the "divalent groups" in L1 do not contain a divalent ring structure. Therefore, the "other divalent groups" in L or the "divalent groups" in L1 may be a divalent straight or branched hydrocarbon group, -C(=O)-, -NR L -(R L represents a hydrogen atom or the above-mentioned substituent), -O-, -S-, -C(═S)-, and a group consisting of a combination of two or more thereof (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4). A divalent straight-chain or branched hydrocarbon group may have, for example, 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 a substituent, examples and preferred examples of such substituents are the same as the substituents that may be possessed by the heteroarylene group as an example of a leaving group.

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

[0665] In formula (I), L is a divalent group comprising 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.

[0666] Examples of the nucleophilic group in the antibody 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. 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 linked to a leaving group and has the ability to react with the nucleophilic group in the antibody as described above can be used, but preferably a group selected from -C(=O)-, -SO2-, and -CH2-. As the electrophilic group, -CH2- can also be used depending on the electron balance with its adjacent groups (e.g., leaving group, L2, E2). For example, when a tosyl group is used as a leaving group, -CH2- can be appropriately used as an 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 further preferred.

[0668] The divalent group containing an electrophilic group represented by E is a divalent group consisting of the above-mentioned electrophilic group, or a divalent group containing other divalent groups in addition to the above-mentioned electrophilic group. Examples of such other divalent groups include: a divalent hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -NR E -(R E represents a hydrogen atom or the above-mentioned substituent), -O-, -S-, -C(=S)-, and a group consisting of a combination of two or more of these (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4). The divalent hydrocarbon group and the divalent heterocyclic group may have, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents, or may not have a substituent. From the viewpoint of synthesizing a compound with a simple chemical structure, it is preferred not to have such a substituent. On the other hand, in the case where the group as described above has a substituent, examples and preferred examples of such a substituent are the same as the substituents that the heteroarylene group, which is an example of a leaving group, may have. E (and E1-E2-E3 described later) can be designed so as not to include a peptide portion that has a potential immunogenicity and is easily hydrolyzed in the blood.

[0669] The divalent hydrocarbon group is a linear, branched, or cyclic divalent hydrocarbon group, preferably a linear or branched divalent hydrocarbon group. Examples of the divalent hydrocarbon group include an alkylene group, an alkenylene group, an alkynylene group, and an arylene group.

[0670] Alkylene groups are preferably those having 1 to 12 carbon atoms, more preferably those having 1 to 6 carbon atoms, and particularly preferably those having 1 to 4 carbon atoms. The carbon number above does not include the carbon number of substituents. Alkylene groups may be linear, branched, or cyclic, but linear alkylene groups are preferred. Examples of such alkylene groups include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0671] Alkenylene groups preferably have 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms, and particularly preferably 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Alkenylene groups may be straight-chain, branched, or cyclic, but straight-chain alkenylene groups are preferred. Examples of such alkenylene groups include ethenylene, propenylene, butenylene, pentenylene, and hexenylene.

[0672] Alkyneylene groups are preferably those having 2 to 12 carbon atoms, more preferably those having 2 to 6 carbon atoms, and particularly preferably those having 2 to 4 carbon atoms. The above carbon number does not include the carbon number of substituents. Alkyneylene groups may be straight-chain, branched, or cyclic, but straight-chain alkynylene groups are preferred. Examples of such alkynylene groups include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

[0673] The arylene group is preferably an arylene group having 6 to 24 carbon atoms, more preferably an arylene group having 6 to 18 carbon atoms, further preferably an arylene group having 6 to 14 carbon atoms, and still further preferably an arylene group having 6 to 10 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. Examples of the arylene group include phenylene, naphthylene, and anthracene.

[0674] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic ring preferably contain at least one selected from oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably contain at least one selected from oxygen atoms, sulfur atoms, and nitrogen atoms.

[0675] The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 1 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 1 to 15 carbon atoms, further preferably a divalent aromatic heterocyclic group having 1 to 9 carbon atoms, and still further preferably a divalent aromatic heterocyclic group having 1 to 6 carbon atoms. The above carbon number does not include the carbon number of substituents. More specifically, examples of the divalent aromatic heterocyclic group include a pyrrolediyl group, a furandiyl group, a thiophenediyl group, a pyridinediyl group, a pyridazinediyl group, a pyrimidinediyl group, a pyrazinediyl group, a triazinediyl group, a pyrazoldiyl group, an imidazolediyl group, a thiazolediyl group, an isothiazolediyl group, an oxazolediyl group, an isoxazolediyl group, a triazolediyl group, a tetrazolediyl group, an indolediyl group, a purinediyl group, an anthraquinonediyl group, a carbazolediyl group, a fluorenediyl group, a quinolinediyl group, an isoquinolinediyl group, a quinazolinediyl group, and a phthalazinediyl group.

[0676] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, further preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and still more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of the substituent. More specifically, examples of the divalent non-aromatic heterocyclic group include a pyrroledionediyl group, a pyrrolidinedionediyl group, a pyrrolinediyl group, an oxiranediyl group, an aziridinediyl group, an azetidinediyl group, an oxetanediyl group, a thietanediyl group, a pyrrolidinediyl group, a dihydrofurandiyl group, a tetrahydrofurandiyl group, a dioxolanediyl group, a tetrahydrothiophenediyl group, a pyrrolidinediyl group, an imidazolidinediyl group, an oxazolidinediyl group, a piperidinediyl group, a dihydropyrandiyl group, a tetrahydropyrandiyl group, a tetrahydrothiopyrandiyl group, a morpholinediyl group, a thiomorpholinediyl group, a pyrazinediyl group, a dihydrooxazinediyl group, a tetrahydrooxazinediyl group, a dihydropyrimidinediyl group, and a tetrahydropyrimidinediyl group.

[0677] In certain embodiments, E may be represented in the form of E1-E2-E3.

[0678] 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. The definition, examples and preferred embodiments of the electrophilic group of E1 are the same as those of the electrophilic group in E.

[0679] E2 is (a) or (b) below:

[0680] (a) -XY- [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

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

[0682] [Chemical Formula 11]

[0683]

[0684] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms on both sides thereof are carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms on both sides thereof are carbon atoms. · is a bonding bond.)

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

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

[0687] The X in E2 can also be specified in the relationship with a leaving group (e.g., with reference to L, L2). In the case where X is an atom or group such as N (R3), O, S or Se, not only is the leaving group but also X can be separated from the electrophilic group. However, in the case of a compound of a predetermined amount represented by formula (I) in a reaction with an antibody, the X in at least a portion of the compound represented by formula (I) can control the reaction so as not to separate from the electrophilic group, so an atom or group as described above 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) and the antibody and then improving the yield of the antibody with a bioorthogonal functional group, an atom or group (i.e., the pKa value of the atom or group is greater than the pKa value of the leaving group) that is more difficult to separate from the leaving group can be used as X. Therefore, from the viewpoint of more selectively allowing the leaving group to dissociate and suppressing the dissociation of X in E2 to improve the target reaction efficiency of the compound represented by formula (I) and the antibody, and thus to improve the yield of the antibody having a bioorthogonal functional group, X in E2 is preferably an X having a dissociation ability lower than that of the leaving group. Such X 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 of a group containing a group that improves the dissociation ability of the leaving group adjacent to the leaving group, and its type, as summarized below.

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

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

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

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

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

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

[0694] When the ring-constituting atom X' that is bonded to E1 is a carbon atom, ring Z is a divalent cyclic group in which the ring-constituting atom X' that is bonded to E1 and its two adjacent ring-constituting atoms are all carbon atoms. The divalent cyclic group may have, for example, 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 divalent cyclic group has a substituent, examples and preferred examples of such substituents are the same as the substituents that the heteroarylene group, which is an example of a leaving group, may have. Examples of such a divalent cyclic group include: cyclic divalent hydrocarbon groups (e.g., arylene groups, cyclic alkylene groups, cyclic alkenylene groups, cyclic alkynylene groups) and divalent heterocyclic groups (e.g., divalent aromatic heterocyclic groups, divalent non-aromatic heterocyclic groups).

[0695] The divalent hydrocarbon group is a cyclic divalent hydrocarbon group, and examples of the divalent hydrocarbon group include a cyclic alkylene group, a cyclic alkenylene group, a cyclic alkynylene group, and an arylene group.

[0696] The cyclic alkylene group is preferably an alkylene group having 3 to 12 carbon atoms, more preferably an alkylene group having 3 to 10 carbon atoms, and particularly preferably an alkylene group having 5 to 8 carbon atoms. The above carbon number does not include the carbon number of substituents. Examples of such alkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, and cyclodecylene.

[0697] The cyclic alkenylene group is preferably one having 3 to 12 carbon atoms, more preferably one having 3 to 10 carbon atoms, and particularly preferably one having 5 to 8 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms in the substituent. Examples of such alkenylene groups include cyclopropenylene, cyclobutenylene, cyclopentenylene, cyclohexenylene, cycloheptenylene, cyclooctenylene, cyclononenylene, and cyclodecenylene.

[0698] The cyclic alkynylene group is preferably an alkynylene group having 6 to 12 carbon atoms, more preferably an alkynylene group having 7 to 12 carbon atoms, and particularly preferably an alkynylene group having 8 to 12 carbon atoms. The above carbon number does not include the carbon number of substituents. The alkynylene group may be straight-chain, branched, or cyclic, with a straight-chain alkynylene group being preferred. Examples of such alkynylene groups include cyclohexynylene, cycloheptynylene, cyclooctynylene, cyclononynylene, cyclodecynylene, cycloundecynylene, and cyclododecynylene.

[0699] The arylene group is preferably an arylene group having 6 to 24 carbon atoms, more preferably an arylene group having 6 to 18 carbon atoms, further preferably an arylene group having 6 to 14 carbon atoms, and still further preferably an arylene group having 6 to 10 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. Examples of the arylene group include phenylene, naphthylene, and anthracene.

[0700] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic ring preferably contain at least one selected from oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably contain at least one selected from oxygen atoms, sulfur atoms, and nitrogen atoms.

[0701] The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, further preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and still further preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. The above carbon number does not include the carbon number of the substituent. More specifically, examples of the divalent aromatic heterocyclic group include a pyrrolediyl group, a furandiyl group, a thiophenediyl group, a pyridinediyl group, a pyridazinediyl group, a pyrimidinediyl group, a pyrazoldiyl group, an isothiazolediyl group, an isoxazolediyl group, an indolediyl group, an anthraquinonediyl group, a carbazolediyl group, a fluorenediyl group, a quinolinediyl group, an isoquinolinediyl group, a quinazolinediyl group, and a phthalazinediyl group.

[0702] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, further preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and still further preferably a non-aromatic heterocyclic group having 3 to 6 carbon atoms. The above carbon atom number does not include the carbon atom number of the substituent. More specifically, examples of the divalent non-aromatic heterocyclic group include a pyrrolidinedionediyl group, a pyrrolidinediyl group, an azetidinediyl group, an oxetanediyl group, a thietanediyl group, a pyrrolidinediyl group, a dihydrofurandiyl group, a tetrahydrofurandiyl group, a tetrahydrothiophenediyl group, a pyrrolidinediyl group, a piperidinediyl group, a dihydropyrandiyl group, a tetrahydropyrandiyl group, a tetrahydrothiopyrandiyl group, a pyrazinediyl group, a dihydrooxazinediyl group, a tetrahydrooxazinediyl group, a dihydropyrimidinediyl group, and a tetrahydropyrimidinediyl group.

[0703] When the ring-constituting atom X' bonding to E1 is a nitrogen atom, Ring Z is a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom, and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. Such a divalent heterocyclic group contains a nitrogen atom as a ring-constituting atom. Divalent heterocyclic groups containing a nitrogen atom as a ring-constituting atom are preferably those having 3 to 21 carbon atoms, more preferably those having 3 to 15 carbon atoms, even more preferably those having 3 to 9 carbon atoms, and even more preferably those having 3 to 6 carbon atoms. A divalent heterocyclic group may have, for example, 1 to 5 substituents, preferably 1 to 3, and more preferably 1 or 2 substituents, or may have no substituents. From the perspective of synthesizing a compound with a simple chemical structure, it is preferably free of such substituents. On the other hand, when the divalent heterocyclic group has substituents, examples and preferred examples of such substituents are the same as those that may be possessed by the heteroarylene group as an example of a leaving group. The above number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of divalent heterocyclic groups containing nitrogen atoms as ring-constituting atoms include divalent aromatic heterocyclic groups containing nitrogen atoms as ring-constituting atoms and divalent non-aromatic heterocyclic groups containing nitrogen atoms as ring-constituting atoms. Examples of divalent aromatic heterocyclic groups containing nitrogen atoms as ring-constituting atoms include pyrrolediyl, imidazolediyl, indolediyl, purinediyl, and carbazolediyl. Examples of divalent non-aromatic heterocyclic groups containing nitrogen atoms as ring-constituting atoms include pyrroledionediyl, pyrrolidinedionediyl, pyrrolinediyl, aziridinediyl, azetidinediyl, pyrrolidinediyl, pyrrolinediyl, imidazolidinediyl, piperidinediyl, morpholinediyl, thiomorpholinediyl, pyrazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0704] Preferably, (b) the group represented by the above formula (i) is (b') a 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 on both sides thereof are carbon atoms. · is a bonding bond.) The definition, examples, and preferred embodiments of the divalent cyclic group of ring Z in the group represented by the above formula (i') are the same as those of the divalent cyclic group of ring Z in the group represented by the above formula (i).

[0708] When E2 is -XY-, 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 when the divalent group represented by E containing 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 ((a) or (b) above)-E3 (bond or divalent group) connecting L (a divalent group containing a leaving group) and B (bioorthogonal functional group) is less important than the distance between the antibody and the bioorthogonal functional group in the antibody having the bioorthogonal functional group produced after the reaction, rather than the distance between the antibody and the bioorthogonal functional group, which can participate in the reaction, rather than the distance between the antibody and the bioorthogonal functional group. The main chain of E or E1-E2-E3 refers to a chain structure consisting of multiple atoms connected by covalent bonds, connecting L and B, excluding hydrogen atoms, branched structure parts, and substituents. Therefore, from the perspective of adjusting this distance, the length of the main chain of E or E1-E2-E3 can be appropriately designed.

[0710] The length of the main chain of E or E1-E2-E3 connecting L and B is not particularly limited, and may be composed of three or more atoms.

[0711] When the main chain has no ring structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure (excluding hydrogen atoms, branched structure portions, and atoms in substituents).

[0712] On the other hand, when the main chain includes a ring structure, the number of atoms in the main chain can be conveniently counted from the perspective of determining the length of 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 shortest path connecting two bonds in the ring structure, based on the number of atoms in the chain structure (excluding hydrogen atoms, branched structure portions, and substituents) that does not contain a bivalent ring structure in the main chain, as described above.

[0713] Preferably, the main chain of E or E1-E2-E3 can be a chain structure in which the "other divalent groups" in E or the "divalent groups" in E3 do not contain a divalent ring structure. Therefore, the "other divalent groups" in E or the "divalent groups" in E3 can be a divalent straight or branched hydrocarbon group, -C(=O)-, -NR E -(R Erepresents a hydrogen atom or the above-mentioned substituent), -O-, -S-, -C(═S)-, and a group consisting of a combination of two or more thereof (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4). A divalent straight-chain or branched hydrocarbon group may have, for example, 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 a substituent, examples and preferred examples of such substituents are the same as the substituents that may be possessed by the heteroarylene group as an example of a leaving group.

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

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

[0716] Bioorthogonal functional groups are groups that do not react with biological components (e.g., amino acids, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bioorthogonal functional groups are well known in the art (e.g., see Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

[0717] In the case where the target of the affinity substance is an antibody (protein), the bioorthogonal functional group is a bioorthogonal functional group for a protein. The bioorthogonal functional group for a protein refers to a group that does not react with the side chains of the 20 natural amino acid residues that constitute the protein, but reacts with a prescribed functional group. The 20 natural amino acids that constitute the protein are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H) and lysine (L). Among these 20 natural amino acids, glycine, which has no side chain (i.e., hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., their side chains do not contain heteroatoms selected from sulfur, nitrogen, and oxygen atoms), are inert to general reactions. Therefore, bioorthogonal functional groups for proteins are functional groups that do not react with "the side chains of these amino acids having side chains inert to general reactions" and also do 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, olefin residues (in other words, any residue having an ethenylene moiety as the smallest unit having a double bond between carbon atoms, the same below), alkyne residues (in other words, any residue having an ethynylene moiety as the smallest unit having a triple bond between carbon atoms, the same below), halogen residues, tetrazine 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, a chlorine atom, a bromine atom, or an iodine atom at the α-position, the same below), isonitrile residues, sydney ketone residues, and selenium residues. Antibodies can be proteins that do not contain free thiols. In proteins that do not contain free thiols, thiols function as bioorthogonal functional groups. Therefore, when the target of the affinity substance is an antibody, the bioorthogonal functional group includes a thiol. The thiol residue may be an unprotected thiol residue (i.e., -SH) or a protected thiol residue. Examples of protecting groups for thiol residues in protected thiol residues include hydrocarbon groups [e.g., alkyl, alkenyl, alkynyl, cycloalkyl, aryl (e.g., phenyl, naphthyl), arylalkyl (aralkyl)], acyl groups (e.g., acetyl, propoxy, tert-butoxycarbonyl and other butoxycarbonyl groups, benzoyl), arylalkyloxycarbonyl (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl, arylalkyl (aralkyl)oxycarbonyl (e.g., benzyloxycarbonyl), alkylthiol (tert-butylthio), and arylthiol (e.g., pyridyldisulfide). Alternatively, the protected thiol residue may be a disulfide residue. The arylalkyl group in the arylalkyl (aralkyl) and arylalkyl (aralkyl)oxycarbonyl groups is a group in which one or more (e.g., two, three, four, or five) aryl groups are bonded to an alkyl group. The number of carbon atoms in the protecting group of the thiol residue is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 15, further preferably 1 to 10, and particularly preferably 1 to 6. In the present invention, the compound represented by formula (I) may contain one or more (e.g., two, three, or four) bioorthogonal functional groups, and preferably, may contain one bioorthogonal functional group.

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

[0720] [Chemical Formula 13]

[0721]

[0722] [Where,

[0723] R 1f , one or more R 1gand one or more R 1h are the same or different and are atoms or groups selected from (a) to (g), or electron-withdrawing groups,

[0724] · is a bonding key. ].

[0725] The atom or group selected from (a) to (g) is selected from the following groups:

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

[0727] (b) a monovalent hydrocarbon group;

[0728] (c) aralkyl;

[0729] (d) a monovalent heterocyclic group;

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

[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 The same or different, represents a hydrogen atom or a monovalent hydrocarbon group. );

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

[0733] The halogen atoms, monovalent hydrocarbon groups, aralkyl groups, monovalent heterocyclic groups, and R a ~R c The definition, examples and preferred examples of the monovalent hydrocarbon group in are the same as those in (i) to (vii) above. It is particularly preferred that the atom or group selected from (a) to (g) is an atom or group of (a) or (b).

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

[0735] Examples of the electron-withdrawing group include the groups listed above, and a halogen atom, a boronic acid residue, a methanesulfonyl group, a toluenesulfonyl group, a trifluoromethanesulfonate, or an ester is preferred.

[0736] 1-6. Preferred structures of compounds 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] [Where,

[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 linked to a leaving group and (ii) has the ability to react with a nucleophilic group in an antibody,

[0744] E2 is (a) -XY- [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):

[0745] [Chemical Formula 14]

[0746]

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

[0748] When E2 is -XY-, 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 separated from E1 by the reaction between the above nucleophilic group and the above 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 the same as those described in (a) to (c).

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

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

[0753] [Where,

[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 PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)];

[0757] (b) heteroarylene; or

[0758] (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 -ON(R)- (wherein 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 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] [Where,

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

[0766] L2 is:

[0767] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)];

[0768] (b) heteroarylene; or

[0769] (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 -ON(R)- (wherein 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 formula (I-3) may be a compound represented by the following formula (I-4):

[0773] [Chemical Formula 16]

[0774]

[0775] [Where,

[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 PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, 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 -ON(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 the ring-constituting atoms on both sides thereof are carbon atoms.

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

[0784] In the compounds represented by formulae (I-1) to (I-4), the definitions, examples, and preferred embodiments of A, L1, L2, E1, E2, E3, and B, as well as -XY-, an alkyl group having 1 to 6 carbon atoms in R1 to R5, and the group represented by formula (i) (e.g., a bivalent cyclic group, a divalent heterocyclic group) are the same as those described above. Furthermore, the definitions, examples, and preferred embodiments of groups such as (a) to (c) representing L2, ring Z (e.g., a bivalent cyclic group in which the ring-constituting atom X' bonded to E1 and both adjacent ring-constituting atoms are carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and both adjacent ring-constituting atoms are carbon atoms), ring P, and ring Q (e.g., an alkyl group having 1 to 6 carbon atoms in R) are the same as those described above.

[0785] 1-7. Preparation Method

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

[0787] As an affinity substance (A) for an antibody, a substance having an arbitrary functional group can be appropriately selected. Therefore, by utilizing a reactive group that can react with the functional group, the affinity substance is reacted with the structural unit represented by LEB, and the structural unit represented by ALEB can be modulated. 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 (e.g., 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 further preferably 30 minutes to 8 hours.

[0788] In the reaction system, the molar ratio (Y / X) of the structural unit (Y) represented by LEB 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 is not particularly limited. For example, it is 0.1 to 50, preferably 0.5 to 40, more preferably 1 to 35, further preferably 2 to 25, and particularly preferably 3 to 15.

[0789] The formation of a compound or salt thereof having an affinity substance for an antibody and a bioorthogonal functional group can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, although this depends on the specific starting materials and the molecular weight of the product. The compound or salt thereof having an affinity substance for an antibody and a bioorthogonal functional group can be appropriately purified by any method, such as chromatography (e.g., the chromatography and affinity chromatography methods described above).

[0790] 1-8. Others

[0791] In the inventions described later [such as the invention represented by formula (II), (III) and their subordinate concepts and partial structural formulas], any symbols (such as A, L, E, B) and the details of the terms represented by the symbols (such as definitions, examples and preferred examples) are common to the invention of the compound represented by the formula (I) or its subordinate concepts or its salt. In addition, any technical elements (such as definitions, examples and preferred examples) that can specify specific groups (such as bioorthogonal functional groups, divalent groups, substituents) and specific numerical values ​​of the inventions described later may also be common to the contents described above. Therefore, unless otherwise specified, these matters may be appropriately cited in the inventions described later. Similarly, the specific invention technical elements described in the inventions described later may be appropriately cited as technical elements of the present invention and other inventions.

[0792] 2. Preparation of Antibodies with Bioorthogonal Functional Groups

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

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

[0795] ALEB (I)

[0796] [Where,

[0797] A is an affinity substance for antibodies,

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

[0799] E is a divalent group comprising an electrophilic group that is (i) linked to a leaving group and (ii) capable of reacting with a nucleophilic group in an antibody,

[0800] B is a bioorthogonal functional group,

[0801] The leaving group has the ability to be cleaved and separated from E by the reaction between the above nucleophilic group and the above electrophilic group. ];

[0802] Ab-EB (II)

[0803] [Where,

[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 preparation method of the antibody with bioorthogonal functional groups 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 (e.g., 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 (e.g., 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 (e.g., IgG1, IgG2, IgG3, IgG4) in the constant region.

[0807] Ab in formula (II) is the same as the above-mentioned antibody and is covalently bonded to the electrophilic group in E. Examples of the nucleophilic group in the antibody that is covalently bonded to 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.

[0808] E in formula (II) is the same as E in formula (I) above. E can be represented by E1-E2-E3. E1, E2, and E3 have the same meanings as the corresponding symbols above. As described above, E and E1-E2-E3 can be designed to exclude peptide moieties that pose potential immunogenicity and are easily hydrolyzed in blood. In this case, antibodies represented by formula (II) having bioorthogonal functional groups can be used to prepare antibodies containing functional substances that do not suffer from these issues.

[0809] The electrophilic group in E and the electrophilic group in E1 covalently bond to the nucleophilic group in the antibody. Examples of the electrophilic group that covalently bonds to the nucleophilic group in the antibody include NH-C(=O)-, NH-SO2-, and NH-CH2- (when the nucleophilic group in the antibody covalently bonded to E is NH2 in the side chain of a lysine residue), OC(=O)-, O-SO2-, and O-CH2- (when the nucleophilic group in the antibody covalently bonded to E is OH in the side chain of a tyrosine residue, a serine residue, or a threonine residue), and SC(=O)- and S-CH2- (when the nucleophilic group in the antibody covalently bonded to E is SH in the side chain of a cysteine ​​residue). As the electrophilic group that covalently bonds to the nucleophilic group in the antibody, preferred are NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody that covalently bonds to E is NH2 in the side chain of a lysine residue), OC(=O)- and O-SO2- (when the nucleophilic group in the antibody that covalently bonds to E is OH in the side chain of a tyrosine residue), more preferred are NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody that covalently bonds to E is NH2 in the side chain of a lysine residue), and even more preferred is NH-C(=O)- (when the nucleophilic group in the antibody that covalently bonds to E is NH2 in the side chain of a lysine residue).

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

[0811] Preferably, the antibodies having bioorthogonal functional groups produced by the production method of the present invention are antibodies having positionally selective bioorthogonal functional groups. When the compound having an affinity substance for the antibody and a bioorthogonal functional group or a salt thereof is a substance represented by formula (I), antibodies having positionally selective bioorthogonal functional groups represented by formula (II) can be produced. Antibodies having positionally selective bioorthogonal functional groups preferably have bioorthogonal functional groups only in the constant region, and more preferably have bioorthogonal functional groups only in the Fc region.

[0812] In this specification, "positionally selective" or "positionally selective" means that, although specific amino acid residues in an antibody are not unevenly present in a specific region, a specified structural unit that can bind to a specific amino acid residue in the antibody is unevenly present in a specific region in the antibody. Therefore, expressions related to positional selectivity, such as "positionally selectively possessing," "positionally selectively binding," and "positionally selectively binding," mean that the binding rate or retention rate of a specified structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention rate or binding rate of the structural unit in a non-target region containing multiple amino acid residues of the same type as the specific amino acid residue in the target region. Such positionally selective binding or retention can be achieved by the present invention, which does not allow a specified structural unit to react randomly with a specific amino acid residue in an antibody, but rather allows a specified structural unit to preferentially react with a specific amino acid residue in a target region in an antibody by using a compound containing an affinity substance for the antibody.

[0813] More specifically, when an antibody (Ab) contains one or more specific amino acid residues (e.g., lysine residues, tyrosine residues, threonine residues, serine residues, or cysteine ​​residues) in a target region consisting of 1 to 50 consecutive amino acid residues [e.g., a region consisting of amino acid residues 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residue 317 in the human IgG Fc region], and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to the one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, and 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 with bioorthogonal functional groups prepared by the preparation method of the present invention can have bioorthogonal functional groups (i.e., structural units represented by EB) according to the number of heavy chains. Antibodies with positionally selective bioorthogonal functional groups can be prepared as follows: First, the compound (ALEB) 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 the electrophilic group in E is reacted with the 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-mentioned antibody heavy chain). Therefore, by using an antibody with multiple (e.g., 1 to 8, preferably 1 to 4, more preferably 2) antibody heavy chains in the preparation method of the present invention, it is possible to prepare an antibody that selectively has multiple structural units represented by EB (or multiple structural units of its subordinate concepts) in the same target region of multiple antibody heavy chains. For example, by using an antibody having two heavy chains (e.g., IgG, IgD, IgE, F(ab')2 antibodies, Fc region proteins, or Fc fusion proteins) in the production methods of the present invention, it is possible to produce antibodies that positionally selectively possess two structural units represented by EB in the same target region of the two heavy chains. In other words, in antibodies having bioorthogonal functional groups, the modification pattern based on the bioorthogonal functional groups can be made the same across multiple (e.g., two) heavy chains.

[0815] Antibodies with bioorthogonal functional groups can also have homologous or heterologous bioorthogonal functional groups (e.g., structural units represented by EB) in multiple (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions of a single antibody heavy chain. In this case, in antibodies with bioorthogonal functional groups, the modification pattern based on the bioorthogonal functional groups can be made the same across multiple (e.g., two) heavy chains.

[0816] The preparation method of the present invention may further include subjecting the generated antibody to a specific treatment to generate a further modified antibody having a bioorthogonal functional group. Examples of such specific treatments include antibody fragmentation (e.g., treatment with a specific protease such as papain or pepsin).

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

[0818] 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, an antibody having a bioorthogonal functional group represented by the following formula (II-1) can be produced.

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

[0820] [Where,

[0821] Ab is antibody,

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

[0823] E2 is (a) -XY- [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 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] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms adjacent to it are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. · is a bonding bond.)

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

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

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

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

[0831] [Where,

[0832] Ab, X, Y and B have the same meanings as the corresponding symbols in 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 embodiment, when a compound represented by formula (I-3) is used as the compound represented by formula (I-1) in the production method of the present invention, an antibody having a bioorthogonal functional group represented by the following formula (II-3) can be produced.

[0836] [Chemical Formula 18]

[0837]

[0838] [Where,

[0839] Ab, the ring-constituting atoms X', the ring Z and B have the same meanings as the corresponding symbols in formula (II-1), E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0840] E3 is a bond or a divalent group. ].

[0841] 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 produced.

[0842] [Chemical Formula 19]

[0843]

[0844] [Where,

[0845] Ab, ring Z and B have the same meanings as the corresponding symbols in formula (II-1),

[0846] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0847] E3 is a bond or a divalent group. ].

[0848] The details (e.g., definitions, examples, and preferred embodiments) 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 represented by the symbols (e.g., antibodies, electrophilic groups, bioorthogonal functional groups) have the same meanings as those in the above-mentioned formula (I) or its subordinate formulas.

[0849] 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, at room temperature (e.g., about 15 to 30°C). The pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer 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 reactions, refer to, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0850] In the reaction system, the molar ratio (Y / X) of the compound having an affinity substance for an antibody and a bioorthogonal functional group or a salt thereof (Y) to the antibody (X) varies depending on the types of the compound having an affinity substance for an antibody and a bioorthogonal functional group or a salt thereof and the antibody, the number of sites in the antibody to be modified by the compound having an affinity substance for an antibody and a bioorthogonal functional group or a salt thereof (e.g., DAR), etc., and is therefore not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, further preferably 2 to 50, and particularly preferably 3 to 30.

[0851] The confirmation of the generation of antibodies with bioorthogonal functional groups, although also dependent on the molecular weight of its specific raw materials and products, 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, preferably by mass spectrometry. The confirmation of position selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, for example, it can be carried out by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. As protease, endoprotease is preferred. As such endoprotease, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N can be listed. The confirmation of the number of bioorthogonal functional groups possessed by antibodies with bioorthogonal functional groups can be carried out, for example, by electrophoresis, chromatography or mass spectrometry, preferably by mass spectrometry. Antibodies with bioorthogonal functional groups can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0852] 3. Method for preparing an antibody having a functional substance using a substance having affinity for an antibody and a compound having a bioorthogonal functional group or a salt thereof

[0853] The present invention provides a method for producing an antibody or a salt thereof having a functional substance, the method comprising the following steps.

[0854] (1) reacting a compound having an affinity for an antibody and a bioorthogonal functional group represented by the following formula (I) or a salt thereof with an antibody to produce an antibody having a bioorthogonal functional group represented by the following formula (II) or a salt thereof;

[0855] ALEB (I)

[0856] [Where,

[0857] A is an affinity substance for antibodies,

[0858] L is a divalent group containing a leaving group,

[0859] E is a divalent group comprising an electrophilic group that is (i) linked to a leaving group and (ii) capable of reacting with a nucleophilic group in an antibody,

[0860] B is a bioorthogonal functional group,

[0861] The leaving group has the ability to be cleaved and separated from E by the reaction between the above nucleophilic group and the above electrophilic group.

[0862] Ab-EB (II)

[0863] [Where,

[0864] E and B have the same meanings as the corresponding symbols in the above formula (I),

[0865] Ab is antibody.]; and

[0866] (2) The antibody or salt thereof having a bioorthogonal functional group represented by the above formula (II) is reacted with a functional substance via the bioorthogonal functional group to produce an antibody or salt thereof having a functional substance represented by the following formula (III):

[0867] Ab-E-B'-F (III)

[0868] [Where,

[0869] Ab has the same meaning as the corresponding symbols in formula (II),

[0870] E has the same meaning as the corresponding symbol in formula (I),

[0871] B' is a divalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0872] F is a functional substance. ].

[0873] Step (1) can be performed in the same manner as the method for producing an antibody having a bioorthogonal functional group.

[0874] The functional substance (F) used in step (2) is not particularly limited as long as it imparts any function to the antibody. Examples thereof include drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred. Furthermore, the functional substance may be a single functional substance or a combination of two or more functional substances.

[0875] As a drug, it can be a drug for any disease. As such diseases, for example, cancer (e.g., lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune disease-inflammatory disease (e.g., allergic disease, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve disease (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infection (e.g., bacterial infection, viral infection), hereditary-rare disease (e.g., hereditary spherocytosis, non-dystrophic myotonia syndrome), eye disease (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), orthopedic-plastic surgery field disease (e.g., osteoarthritis), blood disease (e.g., leukemia, purpura), other diseases (e.g., metabolic disorders such as diabetes, hyperlipidemia, liver disease, kidney disease, lung disease, circulatory system disease, digestive system disease). The drug may be a drug for treating or preventing a predetermined disease, or a drug for alleviating side effects associated with the use of an antibody against a target protein of a predetermined disease.

[0876] More specifically, the drug is an anticancer agent. Examples of anticancer agents include chemotherapeutic agents, toxins, radioactive isotopes, or substances containing the same. Examples of chemotherapeutic agents include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, microtubule binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin). Examples of radioactive isotopes include radioactive isotopes of hydrogen atoms (e.g., 3 H), radioactive isotopes of carbon atoms (such as 14 C), radioactive isotopes of phosphorus atoms (e.g. 32 P), radioactive isotopes of sulfur atoms (such as 35 S), radioactive isotopes of yttrium (e.g. 90 Y), radioactive isotopes of technetium (such as 99m Tc), radioactive isotopes of indium (such as 111 In), radioactive isotopes of iodine atoms (such as 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 (such as 212 Bi).

[0877] Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), radioactive isotopes (e.g., the radioactive isotopes mentioned above), or substances containing the same.

[0878] In addition, the functional substance is a high molecular weight compound, a medium molecular weight compound or a low molecular weight compound, preferably a low molecular weight compound. A low molecular weight compound refers to a compound with a molecular weight of 1500 or less. A low molecular weight compound is a natural compound or a synthetic compound. The molecular weight of the low molecular weight compound can 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 weight compound can be 30 or more, 40 or more, or 50 or more. The low molecular weight compound can be a drug or a marker as described above. Examples of low molecular weight compounds include amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids, and salts thereof.

[0879] Functional substances have various functional groups corresponding to their structures. In the case where a functional substance has a functional group that is easy to react 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 is easy to react with the bioorthogonal functional group can be different according to the specific type of the bioorthogonal functional group. If it is a person skilled in the art, a suitable functional group can be appropriately selected as a functional group that is easy to react with the bioorthogonal functional group (such as Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). As a functional group that is easy to react with a bioorthogonal functional group, for example, when the bioorthogonal functional group is an alkyne residue, an azide residue can be listed, when the bioorthogonal functional group is an aldehyde residue or a ketone residue, a hydrazine residue can be listed, and when the bioorthogonal functional group is a thiol residue, a maleimide residue and a disulfide residue can be listed, 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 the reverse thereof), the divalent group comprising a moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be a divalent group comprising 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 the reverse thereof), the divalent group comprising a moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be a divalent group comprising a triazole residue (which may or may not be fused with other rings); The divalent group containing the moiety 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 easily reacts with the bioorthogonal functional group is a maleimide residue or a disulfide residue (or the reverse thereof), the divalent group containing the moiety formed by the reaction between the functional substance and the bioorthogonal functional group can be a divalent group containing a sulfosuccinimide residue or a divalent group containing a disulfide residue (for example, Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Preferred examples of the divalent group containing the moiety formed by the reaction between the functional substance and the bioorthogonal functional group include a divalent group containing a triazole residue (which may or may not be fused with other rings), a divalent group containing a hydrazone residue, a divalent group containing a sulfosuccinimide residue, or a divalent group containing a disulfide residue.

[0880] On the other hand, when the functional substance does not have a functional group that readily reacts with the bioorthogonal functional group, a substance that has been derivatized to have the desired functional group can be used as the functional substance. For example, when the functional substance is a soluble protein, a substance that has been derivatized to have a functional group that the soluble protein does not naturally have can be used.

[0881] Derivatization is common technical knowledge in this field (e.g., International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, and U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization can be performed using a cross-linking agent as described above. Alternatively, derivatization can be performed using a specific linker having a 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 an appropriate environment (e.g., intracellular or extracellular). Examples of such linkers include peptidyl linkers that are degraded by specific proteases [e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secretory 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 local acidic sites present in the body (e.g., U.S. Patent No. 5,622,929, U.S. Patent No. 5,122,368, U.S. Patent No. 5,824,805). Linkers may be self-immolative (e.g., International Publication No. 02 / 083180, International Publication No. 04 / 043493, International Publication No. 05 / 112919). In the present invention, a derivatized functional substance may also be simply referred to as a "functional substance."

[0882] Ab in formula (III) is the same as the above-mentioned antibody and is covalently bonded to the electrophilic group in E. Examples of the nucleophilic group in the antibody that is covalently bonded to 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.

[0883] E in formula (III) is the same as E in formula (I) above. E can be represented by E1-E2-E3. E1, E2, and E3 have the same meanings as the corresponding symbols above. As described above, E and E1-E2-E3 can be designed to exclude peptide moieties that are potentially immunogenic and susceptible to hydrolysis in blood. In this case, the antibody having a functional substance represented by formula (III) can be suitable for use as a pharmaceutical.

[0884] The electrophilic group in E and the electrophilic group in E1 covalently bond to the nucleophilic group in the antibody. Examples of the electrophilic group that covalently bonds to the nucleophilic group in the antibody include NH-C(=O)-, NH-SO2-, and NH-CH2- (when the nucleophilic group in the antibody covalently bonded to E is NH2 in the side chain of a lysine residue), OC(=O)-, O-SO2-, and O-CH2- (when the nucleophilic group in the antibody covalently bonded to E is OH in the side chain of a tyrosine residue, a serine residue, or a threonine residue), and SC(=O)- and S-CH2- (when the nucleophilic group in the antibody covalently bonded to E is SH in the side chain of a cysteine ​​residue). As the electrophilic group that covalently bonds to the nucleophilic group in the antibody, preferred are NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody that covalently bonds to E is NH2 in the side chain of a lysine residue), OC(=O)- and O-SO2- (when the nucleophilic group in the antibody that covalently bonds to E is OH in the side chain of a tyrosine residue), more preferred are NH-C(=O)- and NH-SO2- (when the nucleophilic group in the antibody that covalently bonds to E is NH2 in the side chain of a lysine residue), and even more preferred is NH-C(=O)- (when the nucleophilic group in the antibody that covalently bonds to E is NH2 in the side chain of a lysine residue).

[0885] In a specific embodiment, when the functional substance has a functional group that readily reacts with a bioorthogonal functional group, or when it is derivatized to have a functional group that readily reacts with a bioorthogonal functional group, the functional group that readily reacts with the bioorthogonal functional group can be a group selected from the following: 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.

[0886] Moreover, in a specific embodiment, when the functional substance has a functional group that easily reacts with a bioorthogonal functional group, or when it is derivatized to have a functional group that easily reacts with a bioorthogonal functional group, the functional group that easily reacts with the above-mentioned bioorthogonal functional group can be a group selected from the groups represented by the following formula.

[0887] [Chemical Formula 20]

[0888]

[0889] [Where,

[0890] R 1f , one or more R 1g and one or more R 1h are the same or different and are atoms or groups selected from the above (i) to (vii), or electron-withdrawing groups, and are bonds to functional substances. ]

[0891] The divalent group represented by B' in formula (III) and comprising a portion generated by the reaction between the functional substance and the bioorthogonal functional group may be: (1) a divalent group comprising the residue mentioned in the preferred examples above, i.e., a triazole residue, a hydrazone residue, or a thiosuccinimide residue; or (2) not particularly limited, and may be, for example, a divalent group comprising a residue selected from the group consisting of a disulfide residue (the residue being the residue mentioned in the preferred examples above), 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 hydrazone-containing residue, an aminophosphoric acid ester residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, a residue containing an aromatic ring having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, a chain residue containing an unsaturated bond, and a glycosyl residue.

[0892] Furthermore, the divalent group comprising a moiety generated by the reaction between the functional substance and the bioorthogonal functional group is not particularly limited, and may be, for example, a divalent group comprising a residue corresponding to any one of the following chemical structures:

[0893] [Chemical Formula 21]

[0894]

[0895] [Here, the wavy lines perpendicular to the bonds represent the bonds formed by the reaction.

[0896] Multiple R 2a , multiple R 2b and multiple R 2c are the same or different, and are hydrogen atoms or the above-mentioned substituents,

[0897] J is -CH2-, -O- or -S-,

[0898] r is any integer from 1 to 4,

[0899] ○ (white circle) indicates the bond to the F side, ● (black circle) indicates the bond to the B' side,

[0900] When the chemical structure is asymmetric around the cleavable moiety, ● may represent a bond to the moiety on the B' side, and ○ may represent a bond to the moiety on the F side. ]

[0901] Preferably, the antibody having a functional substance prepared by step (2) of the preparation method of the present invention is an antibody having a functional substance selectively at a position. When the antibody having a bioorthogonal functional group selectively at a position is an antibody represented by formula (II), an antibody having a functional substance selectively at a position represented by formula (III) can be prepared. The antibody having a functional substance selectively at a position 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.

[0902] When an antibody (Ab) contains one or more specific amino acid residues (e.g., a lysine residue, a tyrosine residue, a threonine residue, a serine residue, or a cysteine ​​residue) in a target region consisting of 1 to 50 consecutive amino acid residues [e.g., a region consisting of amino acid residues 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residue 317 in the human IgG Fc region] and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to the one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, and 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%.

[0903] The antibody with functional substances prepared by the preparation method of the present invention can also have functional substances (i.e., structural units represented by EB'-F) according to the number of heavy chains. This is because: antibodies with functional substances can be prepared from antibodies having bioorthogonal functional groups that can have structural units represented by EB according to the number of heavy chains. Therefore, by using an antibody having multiple (e.g., 1 to 8, preferably 1 to 4, more preferably 2) antibody heavy chains in the preparation method of the present invention, it is possible to prepare antibodies that selectively have multiple structural units represented by EB'-F (or multiple structural units of its subordinate concepts) in the same target region of multiple antibody heavy chains. For example, by using an antibody having two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab')2 antibodies, Fc region proteins, Fc fusion proteins) in the preparation method of the present invention, it is possible to prepare antibodies that selectively have two structural units represented by EB'-F in the same target region of two antibody heavy chains. That is, in an antibody having a functional substance, the modification pattern based on the functional substance can be made the same among multiple (eg, two) heavy chains.

[0904] Antibodies carrying functional substances may also carry homologous or heterologous functional substances (e.g., structural units represented by E-B'-F) in multiple (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions of a single antibody heavy chain. In this case, in antibodies carrying functional substances, the modification pattern based on the functional substance can be made the same across multiple (e.g., two) heavy chains.

[0905] The preparation method of the present invention may further include subjecting the generated antibody to a specific treatment to generate a further modified antibody having a functional substance. Examples of such specific treatments include antibody fragmentation (e.g., treatment with a specific protease such as papain or pepsin).

[0906] When the 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-mentioned formula (II) can be prepared in step (1), and then an antibody having a functional substance represented by the above-mentioned formula (III) can be prepared in step (2).

[0907] 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 above-mentioned formula (II-1) can be prepared in step (1), and then, an antibody having a functional substance represented by the following formula (III-1) can be prepared in step (2).

[0908] Ab-E1-E2-E3-B'-F (III-1)

[0909] [Where,

[0910] Ab is antibody,

[0911] E1 is an electrophilic group connected to the nucleophilic group in the antibody,

[0912] E2 is (a) -XY- [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 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):

[0913] [Chemical Formula 22]

[0914]

[0915] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms adjacent to it are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. · is a bonding bond.)

[0916] E3 is a divalent group when E2 is -XY-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0917] B' is a divalent group including a portion generated by the reaction between the functional substance and the bioorthogonal functional group,

[0918] F is a functional substance. ].

[0919] In a preferred specific embodiment, when a compound represented by formula (I-2) is used as a compound represented by formula (I-1) in the preparation method of the present invention, an antibody having a bioorthogonal functional group represented by the above-mentioned formula (II-2) can be prepared in step (1), and then, an antibody having a functional substance represented by the following formula (III-2) can be prepared in step (2).

[0920] Ab-E1-XY-E3-B'-F (III-2)

[0921] [Where,

[0922] Ab, X, Y, B' and F have the same meanings as the corresponding symbols in the above formula (III-1),

[0923] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0924] E3 is a divalent group. ].

[0925] In another preferred specific embodiment, when a compound represented by formula (I-3) is used as a compound represented by formula (I-1) in the preparation method of the present invention, an antibody having a bioorthogonal functional group represented by the above-mentioned formula (II-3) can be prepared in step (1), and then, an antibody having a functional substance represented by the following formula (III-3) can be prepared in step (2).

[0926] [Chemical Formula 23]

[0927]

[0928] [Where,

[0929] Ab, ring-constituting atoms X', ring Z, B' and F have the same meanings as the corresponding symbols in formula (III-1),

[0930] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0931] E3 is a bond or a divalent group. ].

[0932] Preferably, when a compound represented by formula (I-4) is used as a compound represented by formula (I-3) in the preparation 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).

[0933] [Chemical Formula 24]

[0934]

[0935] [Where,

[0936] Ab, ring Z, B' and F have the same meanings as the corresponding symbols in formula (III-1),

[0937] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0938] E3 is a bond or a divalent group. ].

[0939] The details (e.g., definitions, examples, and preferred embodiments) of any symbol (e.g., E, E1, E2, E3, B) in formula (III), (III-1), (III-2), (III-3), or (III-4) and the terms represented by the symbols (e.g., antibodies, electrophilic groups, bioorthogonal functional groups) have the same meanings as those in the above-mentioned formula (I) or its subordinate formulas.

[0940] Antibodies with bioorthogonal functional groups can react with functional substances via the bioorthogonal functional groups. Such reactions can be suitably performed under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds), as described above.

[0941] 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 type of bioorthogonal functional group, functional substance, and antibody, and the number of sites in the antibody to be modified (e.g., DAR), and is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, further preferably 2 to 50, and particularly preferably 3 to 30.

[0942] The confirmation of the generation of antibodies with functional substances, although also dependent on the molecular weight of its specific raw materials and products, can be carried out by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC) or mass spectrometry, preferably by mass spectrometry. The confirmation of position selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, for example, it can be carried out by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. As protease, endoprotease is preferred. As such endoprotease, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N can be listed. The confirmation of the number of functional substances possessed by antibodies with functional substances can be carried out, for example, by electrophoresis, chromatography or mass spectrometry, preferably by mass spectrometry. The antibodies with functional substances can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0943] 4. Antibodies or salts thereof having positionally selective bioorthogonal functional groups or functional substances

[0944] The present invention provides an antibody or a salt thereof having a bioorthogonal functional group or a functional substance selectively at the position thereof.

[0945] The antibody or salt thereof having a bioorthogonal functional group selectively at a position is an antibody having a bioorthogonal functional group selectively at a position represented by the above formula (II-1). Preferably, as the antibody having a bioorthogonal functional group selectively at a position represented by the above formula (II-1), an antibody having a bioorthogonal functional group selectively at a position represented by the above formula (II-2) or (II-3) is provided. More preferably, as the antibody having a bioorthogonal functional group selectively at a position represented by the above formula (II-3), an antibody having a bioorthogonal functional group selectively at a position represented by the above formula (II-4) is provided. The details (e.g., definitions, examples, and preferred embodiments) 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 represented by the symbols (e.g., antibodies, electrophilic groups, bioorthogonal functional groups) have the same meanings as those in the above-mentioned formula (I) or its subordinate formulas.

[0946] The antibody or 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-mentioned formula (III-1). Preferably, as the antibody that selectively has a functional substance at a position represented by the above-mentioned formula (III-1), an antibody that selectively has a functional substance at a position represented by the above-mentioned 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-mentioned formula (III-3), an antibody that selectively has a functional substance at a position represented by the above-mentioned 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 terms (such as antibodies, electrophilic groups, bioorthogonal functional groups) represented by the symbols are the same as the corresponding meanings in the formulas of the above-mentioned formula (I) or its subordinate concepts.

[0947] Antibodies that selectively possess bioorthogonal functional groups or functional substances are the same as those described above. Preferably, such antibodies are monoclonal antibodies. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Monoclonal antibodies are full-length antibodies or antibody fragments (e.g., F(ab')2, Fab', Fab, Fv, single-chain antibodies), but full-length antibodies are preferred. Particularly preferably, such antibodies are human antibodies, humanized antibodies, or chimeric antibodies that possess human IgG (e.g., IgG1, IgG2, IgG3, IgG4) in the constant region.

[0948] The antibody having a bioorthogonal functional group or functional substance selectively located at a position preferably has the bioorthogonal functional group or functional substance only in the constant region of the antibody, and more preferably has the bioorthogonal functional group or functional substance only in the Fc region of the antibody.

[0949] When an antibody that positionally selectively possesses a bioorthogonal functional group or functional substance contains one or more specific amino acid residues (e.g., a lysine residue, a tyrosine residue, a threonine residue, a serine residue, or a cysteine ​​residue) in a target region consisting of 1 to 50 consecutive amino acid residues [e.g., (a) a region consisting of amino acid residues 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residue 317 in the human IgG Fc region] and contains five or more of the specific amino acid residues in a non-target region other than the target region, the bioorthogonal functional group or functional substance can be possessed with a positional selectivity of 30% or more in the one or more specific amino acid residues contained in the target region. The position selectivity may preferably be 40% or more, more preferably 50% or more, further 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%.

[0950] In addition, antibodies that selectively possess bioorthogonal functional groups or functional substances at certain positions can possess bioorthogonal functional groups (i.e., structural units represented by EB) or functional substances (i.e., structural units represented by E-B'-F) depending on the number of heavy chains. When an antibody that selectively possesses bioorthogonal functional groups or functional substances at certain positions has multiple (e.g., 1 to 8, preferably 1 to 4, and more preferably 2) antibody heavy chains, the bioorthogonal functional groups or functional substances can be selectively possessed at certain positions in the same target region of the multiple antibody heavy chains. That is, in antibodies that selectively possess bioorthogonal functional groups or functional substances at certain positions, the modification method based on the bioorthogonal functional groups or functional substances can be made the same between multiple (e.g., two) heavy chains.

[0951] Alternatively, antibodies that selectively harbor bioorthogonal functional groups or functional substances may harbor the same or different bioorthogonal functional groups (e.g., structural units represented by EB) or functional substances (e.g., structural units represented by E-B'-F) in multiple (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions within a single antibody heavy chain. In this case, in antibodies that selectively harbor bioorthogonal functional groups or functional substances, the modification pattern using the bioorthogonal functional groups or functional substances can be made the same across multiple (e.g., two) heavy chains.

[0952] 5. Compounds having affinity substances for antibodies and functional substances or their salts

[0953] The present invention provides a compound represented by formula (IV) having an affinity substance for an antibody and a functional substance, or a salt thereof.

[0954] ALEF (IV)

[0955] [Where,

[0956] A is an affinity substance for antibodies,

[0957] L is a divalent group containing a leaving group,

[0958] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[0959] F is a functional substance,

[0960] The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group and the electrophilic group. ]

[0961] The definitions, examples and preferred embodiments of the affinity substance (A) for antibodies, the divalent group (L) containing a leaving group, the divalent group (E) containing an electrophilic group and the functional substance (F) in the compound or its salt having an affinity substance and a functional substance for antibodies have the same meanings as those described above. Therefore, in the compound or its salt having an affinity substance and a functional substance for antibodies, A, L and E can be specified in the same manner as A, L and E in the compound or its salt having an affinity substance and a bioorthogonal functional group for antibodies (for example, with reference to formula (I)). In addition, in the compound or its salt having an affinity substance and a functional substance for antibodies, F can be specified in the same manner as F in the antibody or its salt having a functional substance (for example, with reference to formula (III)).

[0962] In a preferred embodiment, the compound represented by formula (IV) may be a compound represented by the following formula (IV-1):

[0963] A-L1-L2-E1-E2-E3-F (IV-1)

[0964] [Where,

[0965] A and F have the same meanings as the corresponding symbols in formula (IV),

[0966] L1 is a bond or a divalent group,

[0967] L2 is a leaving group,

[0968] 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,

[0969] E2 is (a) -XY- [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 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):

[0970] [Chemical Formula 25]

[0971]

[0972] (Here, ring Z is a divalent cyclic group in which the ring-constituting atom X' bonding to E1 and the ring-constituting atoms adjacent to it are all carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonding to E1 is a nitrogen atom and the ring-constituting atoms adjacent to the nitrogen atom are carbon atoms. · is a bonding bond.)

[0973] E3 is a divalent group when E2 is -XY-, and is a bond or a divalent group when E2 is a group represented by formula (i).

[0974] The leaving group has the ability to be cleaved and separated from E1 by the reaction between the above nucleophilic group and the above electrophilic group. ]

[0975] In formula (IV-1), the definition, examples, and preferred examples of the leaving group represented by L2 are the same as those described with respect to (a) to (c) in the above-mentioned "1-3. Divalent group (L) containing a leaving group".

[0976] In a preferred embodiment, the compound represented by formula (IV-1) may be a compound represented by the following formula (IV-2):

[0977] A-L1-L2-E1-XY-E3-F (IV-2)

[0978] [Where,

[0979] A, L1, X, Y and F have the same meanings as the corresponding symbols in formula (IV-1),

[0980] L2 is:

[0981] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)];

[0982] (b) heteroarylene; or

[0983] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.],

[0984] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0985] E3 is a divalent group. ].

[0986] In another preferred embodiment, the compound represented by formula (IV-1) may be a compound represented by the following formula (IV-3):

[0987] [Chemical Formula 26]

[0988]

[0989] [Where,

[0990] A, L1, ring Z, ring-constituting atoms X' and F have the same meanings as the corresponding symbols in the above formula (IV-1),

[0991] L2 is:

[0992] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)];

[0993] (b) heteroarylene; or

[0994] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.],

[0995] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[0996] E3 is a bond or a divalent group. ].

[0997] Preferably, the compound represented by formula (IV-3) may be a compound represented by the following formula (IV-4):

[0998] [Chemical Formula 27]

[0999]

[1000] [Where,

[1001] A, L1 and F have the same meanings as the corresponding symbols in the above formula (IV-1),

[1002] L2 is:

[1003] (a) Ring PQ- [herein, Ring P 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 ring-contracting 2,5-diketopyrrolidine, a ring-contracting 2,6-diketopiperidine, a ring-contracting 2-ketopyrrolidine, a ring-contracting 2-ketopiperidine, and a 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 -ON(R)- (herein, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.)];

[1004] (b) heteroarylene; or

[1005] (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 -ON(R)- (wherein R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.],

[1006] E1 is a group selected from -C(=O)-, -SO2-, and -CH2-,

[1007] Ring Z is a divalent cyclic group in which the ring-constituting atom bonded to E1 and the ring-constituting atoms on both sides thereof are carbon atoms.

[1008] E3 is a bond or a divalent group. ].

[1009] In the compounds represented by formulae (IV-1) to (IV-4), the definitions, examples, and preferred embodiments of A, L1, L2, E1, E2, E3, and F, as well as -XY-, an alkyl group having 1 to 6 carbon atoms in R1 to R5, and the group represented by formula (i) (e.g., a bivalent cyclic group, a divalent heterocyclic group) have the same meanings as those described above. Furthermore, the definitions, examples, and preferred embodiments of groups such as (a) to (c) representing L2, ring Z (e.g., a bivalent cyclic group in which the ring-constituting atom X' bonded to E1 and both adjacent ring-constituting atoms are carbon atoms, or a divalent heterocyclic group in which the ring-constituting atom X' bonded to E1 is a nitrogen atom and both adjacent ring-constituting atoms are carbon atoms), ring P, and Q (e.g., an alkyl group having 1 to 6 carbon atoms in R) have the same meanings as those described above.

[1010] A compound or a salt thereof having an affinity substance for an antibody and a functional substance can be appropriately modulated by reacting a compound or a salt thereof having an affinity substance for an antibody and a bioorthogonal functional group with a functional substance as described above via a 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 (e.g., about 15 to 200° C.). The reaction system may include 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.

[1011] In the reaction system, the molar ratio (Y / X) of the compound having an affinity substance for the antibody and a bioorthogonal functional group or its salt (Y) 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 is therefore not particularly limited. For example, it is 0.01 to 100, preferably 0.05 to 20, and more preferably 0.1 to 10.

[1012] The formation of a compound or salt thereof having an affinity for an antibody and a functional substance can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, although this depends on the specific starting materials and product molecular weights. The compound or salt thereof having an affinity for an antibody and a functional substance can be appropriately purified by any method such as chromatography (e.g., the chromatography and affinity chromatography methods described above).

[1013] 6. Method for producing an antibody having a functional substance using a compound or a salt thereof containing a substance having affinity for an antibody and a functional substance

[1014] The present invention provides a method for producing an antibody or a salt thereof having a functional substance, the method comprising the following steps.

[1015] A compound having an affinity for an antibody and a functional substance represented by the following formula (IV) or a salt thereof is reacted with an antibody to produce an antibody having a functional substance represented by the following formula (V) or a salt thereof:

[1016] ALEF (IV)

[1017] [Where,

[1018] A is an affinity substance for antibodies,

[1019] L is a divalent group containing a leaving group,

[1020] E is a divalent group comprising an electrophilic group, wherein the electrophilic group is (i) connected to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody.

[1021] F is a functional substance,

[1022] The leaving group has the ability to be cleaved and separated from E through the reaction between the nucleophilic group and the electrophilic group.];

[1023] Ab-EF (V)

[1024] [Where,

[1025] Ab is antibody,

[1026] E and F have the same meanings as the corresponding symbols in the above formula (IV).].

[1027] Preferably, the antibody containing a functional substance produced by the production method of the present invention is an antibody containing a functional substance selectively at a position. In this case, an antibody containing a functional substance selectively at a position represented by formula (V) can be produced. The antibody containing a functional substance selectively at a position preferably contains a functional substance only in the constant region, and more preferably contains a functional substance only in the Fc region.

[1028] When an antibody (Ab) contains one or more specific amino acid residues (e.g., a lysine residue, a tyrosine residue, a threonine residue, a serine residue, or a cysteine ​​residue) in a target region consisting of 1 to 50 consecutive amino acid residues [e.g., a region consisting of amino acid residues 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residue 317 in the human IgG Fc region] and contains five or more of the specific amino acid residues in a non-target region other than the target region, the partial structure other than the antibody can bind to the one or more specific amino acid residues contained in the target region with a positional selectivity of 30% or more. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, and 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%.

[1029] In addition, the antibody with a functional substance prepared by the preparation method of the present invention can have a functional substance (i.e., a structural unit represented by EF) according to the number of heavy chains. Therefore, by using an antibody having multiple (e.g., 1 to 8, preferably 1 to 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 represented by EF (or multiple structural units of its subordinate concepts) in the same target region of multiple antibody heavy chains. For example, by using an antibody having two antibody heavy chains (e.g., 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 two structural units represented by EF in the same target region of two antibody heavy chains. That is, in an antibody having a functional substance, the modification method based on the functional substance can be made the same between multiple (e.g., two) heavy chains.

[1030] Furthermore, an antibody containing a functional substance may contain a functional substance (e.g., a structural unit represented by EF) of the same or different species in multiple (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3) target regions of a single antibody heavy chain. In this case, the antibody containing a functional substance may have the same modification pattern based on the functional substance across multiple (e.g., two) heavy chains.

[1031] The preparation method of the present invention may further include subjecting the generated antibody to a specific treatment to generate a further modified antibody having a functional substance. Examples of such specific treatments include antibody fragmentation (e.g., treatment with a specific protease such as papain or pepsin).

[1032] When the compound represented by 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-mentioned formula (V) can be produced.

[1033] In a preferred embodiment, when a compound represented by formula (IV-1) is used as the compound represented by 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.

[1034] Ab-E1-E2-E3-F (V-1)

[1035] [Where,

[1036] Ab is antibody,

[1037] E1, E2, E3 and F have the same meanings as the corresponding symbols in the above formula (IV-1). ]

[1038] In a preferred specific embodiment, when a compound represented by formula (IV-2) is used as the compound represented by 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.

[1039] Ab-E1-XY-E3-F (V-2)

[1040] [Where,

[1041] Ab is antibody,

[1042] E1, X, Y, E3 and F have the same meanings as the corresponding symbols in the above formula (IV-2).

[1043] In another preferred embodiment, when a compound represented by formula (IV-3) is used as the compound represented by 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.

[1044] [Chemical Formula 28]

[1045]

[1046] [Where,

[1047] Ab is antibody,

[1048] E1, ring Z, ring-constituting atoms X', E3 and F have the same meanings as the corresponding symbols in the above formula (IV-3).

[1049] Preferably, when a compound represented by formula (IV-4) is used as the compound represented by formula (IV-3) in the production method of the present invention, an antibody having a functional substance represented by the following formula (V-4) can be produced.

[1050] [Chemical Formula 29]

[1051]

[1052] [Where,

[1053] Ab is antibody,

[1054] E1, ring Z, E3 and F have the same meanings as the corresponding symbols in the above formula (IV-4).

[1055] Compounds having affinity substances and functional substances for antibodies or their salts can react with antibodies because they have 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 (for example, cleavage of amide bonds). For example, such a reaction can be carried out in a suitable reaction system, for example, in a buffer, at room temperature (for example, about 15 to 30°C). The pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer 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 reactions, refer to, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[1056] In the reaction system, the molar ratio (Y / X) of the compound having an affinity substance and a functional substance for the antibody or its salt (Y) to the antibody (X) varies depending on the types of the compound having an affinity substance and a functional substance for the antibody or its salt and the antibody, the number of sites in the antibody to be modified by the compound having an affinity substance and a bioorthogonal functional group or its salt (e.g., DAR), etc., and is not particularly limited. For example, it is 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, further preferably 2 to 50, and particularly preferably 3 to 30.

[1057] The confirmation of the generation of antibodies with functional substances, although also dependent on the molecular weight of its specific raw materials and products, can be carried out by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC) or mass spectrometry, preferably by mass spectrometry. The confirmation of position selectivity can be carried out, for example, by peptide mapping. Regarding peptide mapping, for example, it can be carried out by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. As protease, endoprotease is preferred. As such endoprotease, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N can be listed. The confirmation of the number of functional substances possessed by antibodies with functional substances can be carried out, for example, by electrophoresis, chromatography or mass spectrometry, preferably by mass spectrometry. The antibodies with functional substances can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[1058] 7. Salt

[1059] 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 hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium and 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, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrochloric acid) or a salt with an organic acid (e.g., trifluoroacetic acid).

[1060] 8. Purpose

[1061] The compounds of the present invention or their salts having affinity substances for antibodies and bioorthogonal functional groups can be used, for example, for position-selective modification of antibodies based on bioorthogonal functional groups. Therefore, the present invention provides reagents for position-selective modification of antibodies based on bioorthogonal functional groups, comprising compounds or their salts having affinity substances for antibodies and bioorthogonal functional groups. In the reagents for position-selective modification of antibodies based on bioorthogonal functional groups, the details (e.g., definitions, examples, and preferred embodiments) of the compounds or their salts having affinity substances for antibodies and bioorthogonal functional groups have the same meanings as those described above.

[1062] The compound of the present invention or its salt having an affinity substance and a functional substance for an antibody can be used, for example, for position-selective modification of an antibody based on a functional substance. Therefore, the present invention provides a position-selective modification reagent for an antibody based on a functional substance, which comprises a compound or its salt having an affinity substance and a functional substance for an antibody. In the position-selective modification reagent for an antibody based on a functional substance, the details (e.g., definitions, examples, and preferred embodiments) of the compound or its salt having an affinity substance and a functional substance for an antibody are the same as those described above.

[1063] In the position-selective modifying agent of the present invention, the details of the position-selective modification of the modified antibody (such as definition, examples and preferred embodiments) have the same meanings as those described above.

[1064] The position selective modification reagent of the present invention can be provided in the form of a composition further comprising other components. Examples of such other components include: solutions, stabilizers (e.g., antioxidants, preservatives). As a solution, an aqueous solution is preferred. Examples of aqueous solutions include: water (e.g., distilled water, sterile distilled water, purified water, physiological saline), buffer solutions (e.g., aqueous phosphoric acid solution, Tris-hydrochloric acid buffer solution, carbonate-bicarbonate buffer solution, aqueous boric acid solution, glycine-sodium hydroxide buffer solution, citric acid 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 position selective modification reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[1065] An antibody or a salt thereof that selectively possesses a bioorthogonal functional group at a position selectively can be used as an intermediate in the preparation of an antibody or a salt thereof that selectively possesses a functional substance at a position selectively.

[1066] Antibodies or their salts that selectively possess functional substances at the position can be used as drugs or reagents (e.g., diagnostic drugs, research reagents), in particular as drugs. It has been reported that if the number and binding position of the drug of the antibody-drug complex (ADC) are changed, the in vivo kinetics or the release rate and effect of the drug will change. Based on these circumstances, for the next generation of ADCs, it is required to control the number and position of the conjugated drugs. It is generally believed that if the number and position are constant, the expected efficacy, the diversity of the conjugated agents, the batch difference, and the problem of standardization (regulation) are solved. The antibodies or their salts of the present invention that selectively possess functional substances at the position can solve such standardization problems. Therefore, the antibodies or their salts of the present invention that selectively possess functional substances at the position can be provided in the form of pharmaceutical compositions. Such pharmaceutical compositions may also include a pharmaceutically acceptable carrier in addition to the antibodies or their salts that selectively possess functional substances at the position. Examples of pharmaceutically acceptable carriers include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methyl cellulose, hydroxypropyl cellulose, polypropyl pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethyl cellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; and disintegrants such as magnesium stearate and silica aerogel. 1), lubricants such as talc and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersants such as surfactants; diluents such as water, physiological saline, and orange juice; base waxes such as cocoa butter, polyethylene glycol, and white kerosene, but are not limited to these examples. The antibody or salt thereof of the present invention that selectively possesses a functional substance may also have any modification (e.g., PEGylation) to achieve stability.

[1067] Preparations suitable for oral administration include: liquid preparations obtained by dissolving an effective amount of the ligand in a diluent such as water, physiological saline, orange juice, etc.; capsules, sachets or tablets containing an effective amount of the ligand in solid or granular form; suspensions in which an effective amount of the active ingredient is suspended in an appropriate dispersion medium; emulsions in which a solution containing an effective amount of the active ingredient is dispersed in an appropriate dispersion medium and emulsified.

[1068] Pharmaceutical compositions are suitable for parenteral administration (e.g., intravenous, subcutaneous, intramuscular, topical, or intraperitoneal administration). Examples of pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antimicrobial agents, and isotonic agents. Other examples include aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.

[1069] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, severity of the disease, animal species to be administered, drug tolerance, body weight, age of the subject, etc., and can be appropriately set.

[1070] Example

[1071] Next, the present invention will be described in further detail with reference to examples, but the present invention is not limited to the following examples.

[1072] [Example 1: Synthesis of IgG1 Fc Affinity Substance]

[1073] (1-1) Synthesis of affinity peptides for antibodies

[1074] The peptides shown below as affinity substances for antibodies were prepared in exactly the same way. Peptides with acetyl groups blocking the N-terminus (compounds 1, 2, 32-53) and peptides with 3-(triphenylmethylthio) propionic acid blocking the N-terminus (compounds 3, 31, 54) were prepared by peptide solid phase synthesis using Rink amide resin based on the Fmoc method, and stirred for 3 hours in a solution of trifluoroacetic acid: water: triisopropylsilane: ethanedithiol = 94: 2.5: 1.0: 2.5 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 crude crystals. It was purified by preparative HPLC to obtain the affinity peptide as the product.

[1075] Compounds 35-53, peptides with intramolecular S-S bonds (excluding those containing methionine), were synthesized using the following method after obtaining linear peptide precursors using the method described above. Several dozen mg of the resulting precursors were dissolved in 1 mL of DMSO, and 100 μL of NH₃ / MeOH and 10 μL of H₂O₂ were added, followed by stirring overnight. Completion of the reaction was confirmed by LCMS, followed by purification by preparative HPLC to yield the target affinity peptides.

[1076] Regarding compounds 33 and 34, peptides with SS bonds within the molecule (methionine-containing peptides), linear peptides were obtained as precursors using the method described above and then synthesized using the method described below. Several tens of mg of the resulting precursors were dissolved in 20 mL of 0.1 M Tris-HCl buffer (pH 8.00), 5.0 eq of oxidized glutathione was added, and the mixture was stirred overnight. Completion of the reaction was confirmed by LCMS, and purification was then performed by preparative HPLC to obtain the target affinity peptide.

[1077] [Chemical formula 30]

[1078]

[1079] (Amino acid sequence of the peptide portion SEQ ID NO: 5) MS (ESI) m / z: z = 3 1392 [M+3H] 3+ ,z=4 1044[M+4H] 4+ [Chemical Formula 31]

[1080]

[1081] (Amino acid sequence of the peptide portion SEQ ID NO: 6) MS (ESI) m / z: z = 3 1392 [M+3H] 3+ ,z=4 1044[M+4H] 4+ [Chemical Formula 32]

[1082]

[1083] (Amino acid sequence of the peptide portion SEQ ID NO: 7) MS (ESI) m / z: z = 3 1478 [M+3H] 3+ ,z=4 1108[M+4H] 4+ [Chemical Formula 33]

[1084]

[1085] (Amino acid sequence of the peptide portion is SEQ ID NO: 97)

[1086] 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+

[1087] [Chemical Formula 34]

[1088]

[1089] (Amino acid sequence of the peptide portion is SEQ ID NO: 98)

[1090] MS (ESI) m / z: z=3 1401 [M+3H] 3+ ,Z=4 1051[M+4H] 4+ ,z=5 841[M+5H] 5+

[1091] [Chemical Formula 35]

[1092]

[1093] (Amino acid sequence of the peptide portion is SEQ ID NO: 41)

[1094] MS (ESI) m / z: z=3 1426 [M+3H] 3+ ,Z=4 1070[M+4H] 4+ ,z=5 859[M+5H] 5+

[1095] [Chemical Formula 36]

[1096]

[1097] (Amino acid sequence of the peptide portion is SEQ ID NO: 42)

[1098] MS (ESI) m / z: z=3 1417 [M+3H] 3+ ,Z=4 1063[M+4H] 4+ ,z=5 851[M+5H] 5+

[1099] [Chemical Formula 37]

[1100]

[1101] (Amino acid sequence of the peptide portion is SEQ ID NO: 43)

[1102] MS (ESI) m / z: z=3 1425 [M+3H] 3+ ,Z=4 1069[M+4H] 4+ ,z=5 855[M+5H] 5+

[1103] [Chemical Formula 38]

[1104]

[1105] (Amino acid sequence of the peptide portion is SEQ ID NO: 46)

[1106] MS (ESI) m / z: z = 1 2090 [M + 1H] + ,Z=2 1045[M+2H] 2+ ,Z=3 697[M+3H] 3+

[1107] [Chemical Formula 39]

[1108]

[1109] (Amino acid sequence of the peptide portion is SEQ ID NO: 47)

[1110] MS (ESI) m / z: z = 1 2074 [M + 1H] + ,Z=2 1037[M+2H] 2+ ,Z=3 692[M+3H] 3+

[1111] [Chemical Formula 40]

[1112]

[1113] (Amino acid sequence of the peptide portion is SEQ ID NO: 48)

[1114] MS (ESI) m / z: z = 1 2061 [M + 1H] + ,Z=2 1031[M+2H] 2+ ,Z=3 687[M+3H] 3+

[1115] [Chemical Formula 41]

[1116]

[1117] (Amino acid sequence of the peptide portion is SEQ ID NO: 49)

[1118] MS (ESI) m / z: z = 1 2032 [M + 1H] + ,Z=2 1016[M+2H] 2+ ,Z=3 678[M+3H] 3+

[1119] [Chemical Formula 42]

[1120]

[1121] (Amino acid sequence of the peptide portion is SEQ ID NO: 50)

[1122] MS (ESI) m / z: z = 1 2089 [M + 1H] + ,Z=2 1044[M+2H] 2+ ,Z=3 696[M+3H] 3+

[1123] [Chemical Formula 43]

[1124]

[1125] (Amino acid sequence of the peptide portion is SEQ ID NO: 51)

[1126] MS (ESI) m / z: z = 1 2088 [M + 1H] + ,Z=2 1044[M+2H] 2+ ,Z=3 696[M+3H] 3+

[1127] [Chemical Formula 44]

[1128]

[1129] (Amino acid sequence of the peptide portion SEQ ID NO: 52) MS (ESI) m / z: z = 1 1561 [M+H] + ,Z=2 781[M+2H] 2+

[1130] [Chemical Formula 45]

[1131]

[1132] (Amino acid sequence of the peptide portion SEQ ID NO: 53) MS (ESI) m / z: z = 1 1548 [M+1H] + ,Z=2 774[M+2H] 2+ [Chemical Formula 46]

[1133]

[1134] (Amino acid sequence of the peptide portion SEQ ID NO: 55) MS (ESI) m / z: z = 2 1059 [M+2H] 2+ ,Z=3 706[M+3H] 3+ [Chemical Formula 47]

[1135]

[1136] (Amino acid sequence of the peptide portion SEQ ID NO: 57) MS (ESI) m / z: z = 2 1074 [M+2H]2+ ,Z=3 716[M+3H] 3+ [Chemical Formula 48]

[1137]

[1138] (Amino acid sequence of the peptide portion is SEQ ID NO: 59)

[1139] MS (ESI) m / z: z = 2 1081 [M + 2H] 2+ ,Z=3 721[M+3H] 3+ ,Z=4 541[M+4H] 4+

[1140] [Chemical Formula 49]

[1141]

[1142] (Amino acid sequence of the peptide portion is SEQ ID NO: 63)

[1143] MS (ESI) m / z: z = 2 1085 [M + 2H] 2+ ,Z=3 723[M+3H] 3+ ,Z=4 543[M+4H] 4+

[1144] [Chemical Formula 50]

[1145]

[1146] (Amino acid sequence of the peptide portion is SEQ ID NO: 71)

[1147] MS (ESI) m / z: z = 2 1045 [M + 2H] 2+ ,Z=3 697[M+3H] 3+

[1148] [Chemical Formula 51]

[1149]

[1150] (Amino acid sequence of the peptide portion SEQ ID NO: 72) MS (ESI) m / z: z = 1 1345 [M+1H] + ,Z=2 673[M+2H] 2+ [Chemical Formula 52]

[1151]

[1152] (Amino acid sequence of the peptide portion is SEQ ID NO: 77) MS (ESI) m / z: z = 2 1052 [M+2H] 2+ ,Z=3 702[M+3H] 3+ [Chemical Formula 53]

[1153]

[1154] (Amino acid sequence of the peptide portion SEQ ID NO: 81) MS (ESI) m / z: z = 2 1073 [M+2H] 2+ ,Z=3 715[M+3H] 3+ [Chemical Formula 54]

[1155]

[1156] (Amino acid sequence of the peptide portion SEQ ID NO: 82) MS (ESI) m / z: z = 2 1073 [M+2H] 2+ ,Z=3 716[M+3H] 3+ [Chemical Formula 55]

[1157]

[1158] (Amino acid sequence of the peptide portion is SEQ ID NO: 99)

[1159] MS (ESI) m / z: z = 2 966 [M + 2H] 2+ ,Z=3 644[M+3H] 3+

[1160] [Chemical Formula 56]

[1161]

[1162] (Amino acid sequence of the peptide portion is SEQ ID NO: 100)

[1163] MS (ESI) m / z: z=3 1381 [M+3H] 3+ ,Z=4 1036[M+4H] 4+ ,z=5 829[M+5H] 5+

[1164] (1-2) Synthesis of Antibody-Specific Affinity Peptide Azide Adducts

[1165] The peptide-azide adducts (Compounds 4 and 5) shown below were synthesized as follows. Subsequently, peptides capped with an acetyl group at the N-terminus were prepared by peptide solid-phase synthesis using Rink amide resin using the Fmoc method, using an amino acid in which only the functionalized Lys residue was protected with an mtt group. The mtt group was deprotected by stirring for one hour in a solution of dichloromethane:trifluoroacetic acid:triisopropylsilane (90:5:5). The resin was washed with DMF and then dissolved in 50 molar equivalents of triethylamine, 10 molar equivalents of azidoacetic acid NHS ester, and 4 mL of DMF. The mixture was then stirred for 16 hours. After removing the solution, the mixture was stirred for one hour in a solution of trifluoroacetic acid:water:triisopropylsilane (95:2.5:2.5) to cleave from the resin and deprotect. The resin was removed by filtration, and diethyl ether was added for precipitation. The diethyl ether was removed by decantation to obtain the peptide as crude crystals. This was purified by preparative HPLC to obtain the affinity peptide azide adduct as the product.

[1166] [Chemical Formula 57]

[1167]

[1168] (Amino acid sequence of the peptide portion SEQ ID NO: 8)

[1169] MS (ESI) m / z: z=3 1401 [M+3H] 3+ ,z=4 1051[M+4H] 4+

[1170] [Chemical Formula 58]

[1171]

[1172] MS (ESI) m / z: z=3 1420 [M+3H] 3+ ,z=4 1065[M+4H] 4+

[1173] [Example 2: Synthesis of Antibody-Modified Linkers and Linkage to IgG1 Fc Affinity Peptide Azide Adduct]

[1174] (2-1) Synthesis of Imidazolyl Carbonyl Compounds (2-1-1) Synthesis of Imidazolyl Carbonyl Compounds (Compound 6)

[1175] [Chemical Formula 59]

[1176]

[1177] 0.100 g (1.32 mmol) of 3-butyn-1-ol and 263 mg (1.62 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 and allowed to stand for 5 minutes. The 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 fractions containing the product were recovered and concentrated under reduced pressure to obtain 0.180 g (1.10 mmol) of 1H-imidazole-1-carboxylic acid-3-butynyl ester equivalent to compound 6.

[1178] 1 H 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] +

[1179] (2-1-2) Synthesis of Imidazolyl Carbonyl Compound (Compound 7)

[1180] [Chemical Formula 60]

[1181]

[1182] 0.100 g (0.745 mmol) of 9-decyn-1-ol and 148 mg (0.916 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 and allowed to stand for 5 minutes. The 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 0.152 g (0.612 mmol) of 1H-imidazole-1-carboxylic acid-9-butynyl ester equivalent to compound 7.

[1183] 1 H NMR (400MHz, chloroform-d) δ1.33-1.50 (m, 10H), 1.82 (m, 2H), 1.96 (t, J = 2.6Hz, 1H) 2.21 (td ,J=6.8,2.6Hz,2H),4.43(t,J=6.8Hz,2H)7.10(s,1H),7.45(brs,1H),8.16(s,1H).

[1184] MS (ESI) m / z: 271 [M+Na] +

[1185] (2-1-3) Synthesis of Imidazolyl Carbonyl Compound (Compound 9)

[1186] [Chemical Formula 61]

[1187]

[1188] 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 were dissolved in dichloromethane. After stirring at room temperature for 1 hour, 0.100 g (0.497 mmol) of 12-amino-dodecanol was added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with ethyl acetate, washed with water and brine, and then sodium sulfate was added. The mixture was allowed to stand for 5 minutes. The sodium sulfate was removed by filtration, and the mixture was concentrated 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.

[1189] MS (ESI) m / z: 281 [M+H] +

[1190] [Chemical Formula 62]

[1191]

[1192] 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 and allowed to stand for 5 minutes. The 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 fractions containing the product were 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-oxo)aminododecyl ester, corresponding to compound 9.

[1193] 1 H NMR (400MHz, chloroform-d) δ1.23-1.50(m,20H),1.81(t,J=2.6Hz,1H),2.36(m,2H)2.53(td,J=6.8,2.6Hz,2H),3.23(t,J= 6.8Hz,2H)4.41(t,J=6.8Hz,2H),5.64(brs,1H),7.07(s,1H),7.43(brs,1H),8.13(s,1H).MS(ESI)m / z: 398[M+Na] +

[1194] (2-2) Synthesis of Imidazole Carbonyl Compounds and Their Conjugation to Affinity Peptide Azides

[1195] The affinity peptide azide (compound 4) synthesized in Example 1 and the imidazolyl carbonyl compounds (compounds 6, 7, and 9) synthesized in Example 2 were linked using 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 100 mg / mL solution of the imidazolyl carbonyl compound in dimethyl sulfoxide were added. To this reaction mixture, a separately prepared aqueous solution of 4 molar equivalents of copper sulfate monohydrate and 20 molar equivalents of tris(3-hydroxypropyltriazolylmethyl)amine was added and stirred. The reaction was monitored by LC-MS. After confirming the disappearance of the starting material, the reaction mixture was concentrated and diluted with water four times by ultrafiltration (Amicon Ultra, 3K MWCO). The resulting aqueous solution was freeze-dried to obtain the peptide-imidazolyl carbonyl compounds (compounds 10, 11, and 12) shown below.

[1196] [Chemical Formula 63]

[1197]

[1198] (Amino acid sequence of the peptide portion SEQ ID NO: 8)

[1199] MS (ESI) m / z: z=3 1455 [M+3H] 3+ ,z=4 1092[M+4H] 4+

[1200] [Chemical Formula 64]

[1201]

[1202] (Amino acid sequence of the peptide portion SEQ ID NO: 8)

[1203] MS (ESI) m / z: z=3 1483 [M+3H] 3+ ,z=4 1113[M+4H] 4+

[1204] [Chemical Formula 65]

[1205]

[1206] (Amino acid sequence of the peptide portion SEQ ID NO: 8)

[1207] MS (ESI) m / z: z=3 1525 [M+3H]3+ ,z=4 1144[M+4H] 4+

[1208] [Reference Example 1: Verification of the difference in reactivity due to the difference in the number of atoms of the affinity peptide-antibody modifying group (electrophilic group) through model experiment]

[1209] (1-1) Synthesis of IgG Antibody Trastuzumab-Peptide Complex

[1210] 20 μg of anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 2.0 μL of 100 mM HEPES buffer (pH 7.2). 20 molar equivalents of the peptide-imidazolyl carbonyl compounds (Compounds 10, 11, and 12) synthesized in (2-2) of Example 2 were added to the antibody, and the mixture was stirred at 37 degrees for 4 hours. The reaction solution was replaced with water by ultrafiltration (Amicon Ultra, 3K MWCO), and the peptide reagent was removed to stop the reaction, thereby obtaining an IgG antibody trastuzumab-peptide complex. (1-2) Analysis of IgG antibody trastuzumab-peptide complex based on SDS-PAGE

[1211] The three IgG antibody trastuzumab-peptide complexes obtained in (1-1) were analyzed by SDS-PAGE (Mini-PROTEANT GX gel, 4-20%, Bio-RAD; under reducing conditions; stained with Coomassie Brilliant Blue G-250 dye) using three peptide-imidazolyl carbonyl compounds (compounds 10, 11, and 12). The results are shown in FIG. Figure 2 From these results, it was found that the antibody modification reaction proceeded when using compounds 10 and 11, but did not proceed when using compound 12. [Example 3: Synthesis of Maleimide-Carrying Antibody Affinity Reagent]

[1212] (3-1) Synthesis of Protected Thiol Compounds (3-1-1) Synthesis of Protected Thiol Compounds (Compound 14)

[1213] [Chemical Formula 66]

[1214]

[1215] 100 mg (0.287 mmol) of 3-(triphenylmethylthio)propionic acid was dissolved in THF, and 42.7 μL (0.316 mmol) of isobutyl chlorocarbonate and 69.4 μL (0.631 mmol) of N-methylmorpholine were added at 0°C. The mixture was stirred for 30 minutes to prepare the corresponding mixed anhydride. 33.6 mg (0.287 mmol) of 5-aminovaleric acid was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, and the THF solution of the mixed anhydride was added dropwise at room temperature. After stirring at room temperature for 16 hours, the reaction mixture was washed with water and ethyl acetate, and the aqueous phase was recovered. 6 M aqueous hydrochloric acid was added to the aqueous phase, and the pH of the system was adjusted to 3.0. The reaction mixture was then separated and extracted with ethyl acetate. The organic phase was then washed with brine, and anhydrous magnesium sulfate was added, and the mixture was allowed to stand for 5 minutes. The magnesium sulfate was removed by filtration, and the mixture was concentrated under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography. The fractions containing the product were recovered and concentrated under reduced pressure to obtain 110 mg (0.246 mmol) of 5-[3-(triphenylmethyl)sulfanyl-propyl-1-oxo]amino-hexanoic acid corresponding to Compound 13.

[1216] 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).

[1217] MS (ESI) m / z: 470 [M+Na] +

[1218] [Chemical Formula 67]

[1219]

[1220] 20.2 mg (0.045 mmol) of 5-(3-triphenylmethyl-propyl-1-oxo)aminocaproic acid was dissolved in THF. 6.70 μL (0.050 mmol) of isobutyl chlorocarbonate and 10.9 μL (0.0991 mmol) of N-methylmorpholine were added at 0°C, and the mixture was stirred for 30 minutes to prepare the corresponding mixed anhydride. 16.6 μL (0.226 mmol) of propargylamine was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, followed by the dropwise addition of the mixed anhydride solution in THF at room temperature. The mixture was stirred at room temperature for 16 hours, after which the reaction mixture was washed with water and ethyl acetate, and the aqueous phase was recovered. 6 M aqueous hydrochloric acid was added to the aqueous phase, and the pH was adjusted to 3.0. Extraction was then performed with ethyl acetate, and the organic phase was washed with brine. Anhydrous magnesium sulfate was then added, and the mixture was allowed to stand for 5 minutes. The magnesium sulfate was removed by filtration, and the mixture was concentrated under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography. The fractions containing the product were recovered and concentrated under reduced pressure to obtain 20.0 mg (0.041 mmol) of N-propynyl-5-(3-triphenylmethylsulfanyl-propyl-1-oxo)amino-hexanamide corresponding to Compound 14.

[1221] 1 H NMR(400MHz, chloroform-d)δ1.52(dt,J=8.2,6.5Hz,2H),1.59-1.72(m,2H),2.02-2.09(m,2H),2.17-2.28(m,2H)2.52( t,J=7.2Hz,2H),3.21(brs,2H),4.01(dd,J=5.3,2.6Hz,2H),6.04(s,1H),7.19-7.35(m,9H),7.40-7.49(m,6H).

[1222] MS (ESI) m / z: 507 [M+H] +

[1223] (3-1-2) Synthesis of protected thiol compound (Compound 15)

[1224] [Chemical Formula 68]

[1225]

[1226] 100 mg (0.287 mmol) of 3-(triphenylmethylthio)propionic acid was dissolved in THF, and 42.7 μL (0.316 mmol) of isobutyl chlorocarbonate and 69.4 μL (0.631 mmol) of N-methylmorpholine were added at 0°C. The mixture was stirred 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 mixed anhydride was added dropwise at room temperature. After stirring at room temperature for 16 hours, the reaction mixture was washed with water and ethyl acetate, and the aqueous phase was recovered. 6 M aqueous hydrochloric acid was added to the aqueous phase, and the pH of the system was adjusted to 3.0. Extraction was then carried out with ethyl acetate, and the organic phase was washed with brine. Anhydrous magnesium sulfate was then added, and the mixture was allowed to stand for 5 minutes. The magnesium sulfate was removed by filtration, and the mixture was concentrated under reduced pressure to obtain 110 mg (0.286 mmol) of N-propynyl-3-triphenylmethylsulfanyl-propionamide, corresponding to compound 15.

[1227] 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).

[1228] MS (ESI) m / z: 408 [M+Na] +

[1229] (3-2) Linking of Affinity Peptides to Protected Thiol Compounds (3-2-1) Synthesis of Peptide-Protected Thiol Linkers (Compound 16)

[1230] [Chemical Formula 69]

[1231]

[1232] (Amino acid sequence of the peptide portion SEQ ID NO: 6)

[1233] Compound 5 synthesized in Example 1 was dissolved in N,N'-dimethylformamide. Five molar equivalents of 3-(triphenylmethylthio)propionic 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, the mixture was purified by preparative HPLC. The fraction containing the target compound was confirmed by ESI-MS and freeze-dried to obtain the peptide-protected thiol linker (Compound 16).

[1234] MS (ESI) m / z: z=3 1502 [M+3H] 3+ ,z=4 1125[M+4H] 4+

[1235] (3-2-2) Synthesis of Peptide-Protected Thiol Linkers (Compounds 17 and 18)

[1236] The affinity peptide azide (Compound 5) synthesized in Example 1 and the protected thiol compounds (Compounds 17 and 18) synthesized in (4-1-1) and (4-1-2) were linked using 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 100 mg / mL solution of the protected thiol compound in dimethyl sulfoxide were added. A separately prepared aqueous solution of 4 molar equivalents of copper sulfate monohydrate and 20 molar equivalents of tris(3-hydroxypropyltriazolylmethyl)amine was added to the reaction mixture and stirred. The reaction was monitored by LC-MS. After confirming the disappearance of the starting material, the solution was concentrated by ultrafiltration (Amicon Ultra-4, 3K MWCO) and diluted with water four times. The resulting aqueous solution was freeze-dried to obtain the peptide-protected thiol linker (Compounds 17 and 18).

[1237] [Chemical Formula 70]

[1238]

[1239]

[1240] (Amino acid sequence of the peptide portion SEQ...

Claims

1. A compound having an affinity peptide and a bioorthogonal functional group represented by the following formula (I) or a salt thereof: ALEB(I) Where, A is an affinity peptide having 10 to 40 amino acid residues that has the ability to bind to the Fc region of an antibody, L is a divalent group containing a leaving group, wherein the leaving group is -S- or -O-, E is a divalent group consisting of (a) an electrophilic group linked to the leaving group and (b) a linear divalent group, wherein the electrophilic group is -C(=O)-, and the linear divalent group is a linear divalent hydrocarbon group, -NH-, -O-, -S-, or a group consisting of a combination of 2 to 4 of these groups, and E does not contain a peptide portion. B is a bioorthogonal functional group, wherein the bioorthogonal functional group comprises an azide residue or an olefin residue, The leaving group has the ability to be cleaved and separated from E by the reaction between the nucleophilic group in the antibody and the electrophilic group.

2. The compound or salt thereof according to claim 1, wherein The above-mentioned straight-chain divalent group is a straight-chain divalent hydrocarbon group.

3. The compound or salt thereof according to claim 1, wherein The linear divalent group is a group composed of a combination of (i) a linear divalent hydrocarbon group and (ii) one heteroatom selected from -O- and -S-.

4. The compound or salt thereof according to claim 1, wherein The linear divalent hydrocarbon group is a linear alkylene group, an arylene group, or a group consisting of a combination thereof.

5. The compound or salt thereof according to claim 4, wherein The above-mentioned straight-chain alkylene group is a straight-chain alkylene group having 1 to 12 carbon atoms.

6. The compound or salt thereof according to claim 4, wherein The above-mentioned straight-chain alkylene group is a straight-chain alkylene group having 1 to 6 carbon atoms.

7. The compound or salt thereof according to claim 4, wherein The above-mentioned straight-chain alkylene group is a straight-chain alkylene group having 1 to 4 carbon atoms.

8. The compound or salt thereof according to claim 1, wherein The above-mentioned azide residue is The above olefin residue is Here, R 1f and R 1g is a hydrogen atom, and is a bonding bond. A reagent for position-selective modification of an antibody based on a bioorthogonal functional group, comprising the compound or a salt thereof according to any one of claims 1 to 8.

10. A method for preparing an antibody or a salt thereof having a bioorthogonal functional group, the method comprising: The compound or salt thereof according to any one of claims 1 to 8 is reacted with an antibody to produce an antibody or salt thereof having a bioorthogonal functional group represented by the following formula (II), Ab-EB(II) Where, Ab is antibody, E is a divalent group composed of (a) an electrophilic group linked to the amino group in the side chain of a lysine residue present in the constant region of the antibody and (b) a linear divalent group, wherein the electrophilic group is -C(=O)-, and the linear divalent group is a linear divalent hydrocarbon group, -NH-, -O-, -S-, or a group composed of a combination of two to four of these, and E does not contain a peptide portion. B has the same meaning as the corresponding symbol in the above formula (I).

11. An antibody having a bioorthogonal functional group represented by the following formula (II) or a salt thereof: Ab-EB(II) Where, Ab is antibody, E is a divalent group composed of (a) an electrophilic group linked to the amino group in the side chain of a lysine residue present in the constant region of the antibody and (b) a linear divalent group, wherein the electrophilic group is -C(=O)-, and E does not contain a peptide portion. B is a bioorthogonal functional group, and the bioorthogonal functional group includes an azide residue or an olefin residue.

12. The antibody or salt thereof according to claim 11, wherein The above-mentioned straight-chain divalent group is a straight-chain divalent hydrocarbon group.

13. The antibody or salt thereof according to claim 11, wherein The linear divalent group is a group composed of a combination of (i) a linear divalent hydrocarbon group and (ii) one heteroatom selected from -O- and -S-.

14. The antibody or salt thereof according to claim 11, wherein The linear divalent hydrocarbon group is a linear alkylene group, an arylene group, or a group consisting of a combination thereof.

15. The antibody or salt thereof according to claim 14, wherein The above-mentioned straight-chain alkylene group is a straight-chain alkylene group having 1 to 12 carbon atoms.

16. The antibody or salt thereof according to claim 14, wherein The above-mentioned straight-chain alkylene group is a straight-chain alkylene group having 1 to 6 carbon atoms.

17. The antibody or salt thereof according to claim 14, wherein The above-mentioned straight-chain alkylene group is a straight-chain alkylene group having 1 to 4 carbon atoms.

18. The antibody or salt thereof according to any one of claims 11 to 17, wherein The above-mentioned azide residue is The above olefin residue is Here, R 1f and R 1g is a hydrogen atom, and is a bonding bond.

19. The antibody or salt thereof according to any one of claims 11 to 17, wherein The above-mentioned antibody is IgG.

20. The antibody or salt thereof according to claim 19, wherein The above-mentioned IgG is human IgG.

21. The antibody or salt thereof according to any one of claims 11 to 17, wherein The lysine residue present in the constant region of the above-mentioned antibody is a lysine residue present in the CH2 region of the antibody.

22. The antibody or salt thereof according to claim 20, wherein The lysine residues present in the constant region of the above-mentioned antibody are lysine residues at positions 246 to 248 or 288 to 290 based on Eu numbering in human IgG Fc.

23. The antibody or salt thereof according to any one of claims 11 to 17, wherein The above-mentioned antibody has a plurality of structural units represented by EB.

24. The antibody or salt thereof according to any one of claims 11 to 17, wherein The above-mentioned antibody has 2 to 4 structural units represented by EB.

25. An antibody or a salt thereof having a functional substance represented by the following formula (III): Ab-E-B'-F(III) Where, Ab is antibody, E is a divalent group composed of (a) an electrophilic group linked to the amino group in the side chain of a lysine residue present in the constant region of the antibody and (b) a linear divalent group, wherein the electrophilic group is -C(=O)-, and the linear divalent group is a linear divalent hydrocarbon group, -NH-, -O-, -S-, or a group composed of a combination of two to four of these, and E does not contain a peptide portion. B' is a divalent group comprising a portion generated by a reaction between a functional substance and a bioorthogonal functional group, wherein the bioorthogonal functional group comprises an azide residue or an olefin residue, F is a functional substance, which is a drug, a marker or a stabilizer.

26. The antibody or salt thereof according to claim 25, wherein The above-mentioned antibody has a plurality of structural units represented by E-B'-F.

27. The antibody or salt thereof according to claim 25, wherein The above-mentioned antibody has 2 to 4 structural units represented by EB'-F.

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