Affinity substances for antibodies, and compounds having bioorthogonal functional groups or their salts.

Abstract

To provide a technique enabling the modification of antibodies, particularly regioselective modification of antibodies.SOLUTION: The present invention provides a substance having affinity for an antibody, and a compound or salt thereof having a bioorthogonal functional group, and the like, which are represented by the following formula (I): A-L-E-B (I) [where, A is a substance having affinity for an antibody, L is a divalent group containing a leaving group, E is a divalent group containing an electrophilic group that (i) is linked to the leaving group and (ii) has a capacity for reacting with a nucleophilic group in the antibody, B is a bioorthogonal functional group, and the leaving group has a capacity for separating from and leaving E due to a reaction between the nucleophilic group and the electrophilic group].SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to substances that have affinity for antibodies, and compounds having bioorthothic functional groups or salts thereof. [Background technology]

[0002] In recent years, research and development of antibody-drug conjugates (ADCs) has been actively pursued. As the name suggests, ADCs are drugs in which an antibody is conjugated with a drug (e.g., an anticancer drug), and they have direct cytotoxic activity against cancer cells and other cells. A representative ADC is T-DM1 (trade name: Kadcyla®) (Non-patent documents 1-3).

[0003] ADCs, including T-DM1, have been plagued by heterogeneity since their initial development. Specifically, because small-molecule drugs are randomly reacted with approximately 70-80 lys residues in the antibody, the drug-antibody ratio (DAR) and conjugation site are not consistent. Typically, such random conjugation methods result in a DAR ranging from 0 to 8, producing multiple drugs with varying numbers of conjugated molecules. Recently, it has been reported that altering the number and location of drug binding in ADCs changes pharmacokinetics, drug release rates, and efficacy. Therefore, next-generation ADCs require control over the number and location of conjugated drugs. It is believed that consistent number and location will resolve issues such as expected efficacy, variations in conjugated drugs, and lot-to-lot variability (i.e., regulation problems) (Non-Patent Literature 4).

[0004] While site-selective modification of antibodies is being studied worldwide, most of these methods involve genetic engineering or enzymatic modification. Regarding genetic engineering, although site-selectivity and number-selectivity can be controlled, problems such as decreased antibody expression efficiency (reduced overall yield when preparing ADCs) have been pointed out. Furthermore, the long time required to construct antibody expression systems is a significant issue (Non-Patent Documents 5-7).

[0005] Furthermore, methods for chemically modifying proteins in complex environments such as cells using small molecule probes have been reported in recent years. This technique is used for receptor identification in imaging and repositioning of small molecule drugs. In addition, organic chemical protein modification methods using synthetic small molecule probes are attracting attention in the field of chemical biology (Non-patent documents 8-11).

[0006] Recently, the CCAP (Chemical Conjugation by Affinity Peptide) method has been developed. This method successfully achieves regioselective modification of antibodies by reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide (i.e., a method for producing ADCs via a linker containing the peptide portion). This method is the first in the world to successfully achieve regioselective modification of the antibody Fc region with a drug using a chemical synthesis method, and has also shown good practical results [reaction time 30 minutes, yield 70% (for DAR 1), regioselectivity 100%]. It has been demonstrated that the DAR can be controlled to 2 by adding about 5 equivalents of the peptide reagent, and is groundbreaking in that the modification site can also be controlled (Patent Document 1). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2016 / 186206 [Non-patent literature]

[0008] [Non-Patent Document 1] Reichert JM et al., Nat Biotechnol 2005;23:1073-8 [Non-Patent Document 2] Kubota T et al., Cancer Sci 2009;100:1566-72 [Non-Patent Document 3] Wu AM et al., Nat Biotechnol 2005;23:1137-46 [Non-Patent Document 4] Junutula JR et al., Nat Biotechnol 2008;26:925-32 [Non-Patent Document 5] Shen BQ et al., Nat Biotechnol 2012;30:184-9 [Non-Patent Document 6] Hofer T et al., Biochemistry 2009;48:12047-57 [Non-Patent Document 7] Liu W et al., Nat Methods 2007;4:239-44 [Non-Patent Document 8] S.T. Laughlin et al., Science 2008;320,664 [Non-Patent Document 9] A.E. Speers et al., ChemBioChem 2004;5,41 [Non-Patent Document 1] 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] [Problems to be Solved by the Invention]

[0009] The present invention aims to develop a technology that enables antibody modification, particularly regioselective modification of antibodies. [Means for solving the problem]

[0010] As a result of diligent research, the inventors have found that compounds or salts thereof having a structural unit called ALE (where A is an antibody affinity substance, L is a divalent group containing a predetermined leaving group, and E is a divalent group containing an electrophile that (i) is linked to the leaving group and (ii) has the ability to react with a nucleophile in the antibody) are useful for regio-directed modification of antibodies. For example, a predetermined compound represented by formula (I), which has an antibody affinity substance and a bioorthogonal functional group, was found to be useful for regio-directed modification of antibodies (see Figure 1, various examples). Furthermore, a compound represented by formula (IV), which has an antibody affinity substance and a functional substance, or a salt thereof, was found to be useful for regio-directed modification of antibodies (see Examples 13 and 14). The inventors have further found that by using such compounds, it is possible to prepare antibodies (antibody-drug conjugates (ADCs)) that regioselectively contain a functional substance (e.g., a drug) and do not contain a peptide moiety as a linker. Avoiding the use of linkers containing peptide portions that have potential immunogenicity and are easily hydrolyzed in the blood is desirable in the clinical application of ADCs. In other words, according to the method developed by the present inventors, the antibody Fc region can be regioselectively modified with a drug by chemical synthesis and without using a linker containing a peptide portion.

[0011] In other words, the present invention is as follows: [1] The following formula (I): ALEB (I) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, B is a bioorthogonal functional group, The leaving group has the ability to be cleaved and eliminated from E by a reaction between the nucleophile and the electrophile. A substance that has affinity for antibodies, and a compound having a bioorthothic functional group or a salt thereof, represented as ]. [2] The compound of [1] or a salt thereof, wherein the affinity substance is a peptide. [3] The compound or salt thereof of [2], wherein the peptide is a peptide having the ability to bind to the constant region of a monoclonal antibody. [4] A compound or salt thereof of [2] or [3], wherein the peptide is a peptide having the ability to bind to the Fc region of a monoclonal antibody. [5] The compound of [4] or a salt thereof, wherein the peptide is a peptide having the ability to bind to the Fc region of IgG. [6] The peptide is any of the compounds [2] to [5] or a salt thereof, having 10 to 40 amino acid residues. [7] The peptide is (a)(a-1-1)FNMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (Sequence ID 11) amino acid sequence, or (a-1-2)FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (Sequence ID 12) amino acid sequence, Any one to three amino acid residues in the sequence are substituted by one amino acid residue each selected from the group consisting of lysine residues, aspartic acid residues, and glutamic acid residues, which may be the same or different. The amino acid sequence of (a-2-1)β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (Sequence ID 13), or In the amino acid sequence of (a-2-2)β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 14), Any one to three amino acid residues in the sequence are replaced by one amino acid residue each selected from the group consisting of lysine residues, aspartic acid residues, and glutamic acid residues, and (b) A compound according to any one of [2] to [6] or a salt thereof, comprising an amino acid sequence having 85% or more identity to each of the amino acid sequences of SEQ ID NOs: 11 to 14. [8] The peptide is Formula 1-1: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-I-W-C-(X 0-3 ) b (SEQ ID NO: 15) 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) Formula 1-3: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-V-V-W-C-(X 0-3 ) 1] b (SEQ ID NO: 17) Formula 1-4: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-A-V-W-C-(X 0-3 ) b (SEQ ID NO: 18) Formula 1-5: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-L-W-C-(X 0-3 ) b (SEQ ID NO: 19) Formula ;1-6: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-I-W-C-(X 0-3 ) b (SEQ ID NO: 20) Formula 1-7: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-F-C-(X 0-3 ) b (SEQ ID NO: 21) Formula 1-8: (X0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-QVWC-(X 0-3 ) b (Sequence ID 22) Formula 1-9:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-EVWC-(X 0-3 ) b (Sequence ID 23) [During the ceremony, (X 0-3 ) a is none, arginine residue-glycine residue-asparagine residue, glycine residue-asparagine residue, aspartic acid residue, or asparagine residue. (X 0-3 ) b It is none, a threonine residue-tyrosine residue-histidine residue, or a threonine residue. Xaa1 is an alanine residue, Xaa2 is a tyrosine residue, a tryptophan residue, or a histidine residue. Xaa3 is a histidine residue, phenylalanine residue, tyrosine residue, tryptophan residue, arginine residue, or glycine residue. Xaa4 is a lysine residue, an aspartic acid residue, or a glutamic acid residue. Xaa5 is a glycine residue, serine residue, asparagine residue, glutamine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, tyrosine residue, tryptophan residue, histidine residue, threonine residue, leucine residue, alanine residue, valine residue, isoleucine residue, or arginine residue. Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. Formula 2-1:(X 0-3 ') a -C-(Xaa1')-(Xaa2')-(Xaa3')-(Xaa4')-(Xaa5')-(Xaa6')-LVWC-(X 0-3 ')b (Sequence ID 24) [During the ceremony, (X 0-3 ') a and (X 0-3 ') b Each of the above (X 0-3 ) a and (X 0-3 ) b It is the same as, Xaa1', Xaa2', Xaa3', Xaa4', Xaa5', and Xaa6' are the same as Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6, respectively. A compound or salt thereof from any of [2] to [6] containing any of the amino acid sequences of ]. [9] Any compound from [1] to [8] or a salt thereof, wherein the leaving group is (1) a group selected from the group consisting of -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.

[10] Any compound from [1] to [9] or a salt thereof, wherein the nucleophile is a group 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.

[11] Any compound from [1] to

[10] or a salt thereof, wherein the electrophile is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-.

[12] Any compound from [1] to

[11] or a salt thereof, wherein the bioorthogonal functional group is a group selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene residues, halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isonitrile residues, cydonone residues, and selenium residues.

[13] Any compound from [1] to

[12] or a salt thereof, wherein the bioorthogonal functional group is a group selected from the group consisting of azide residues, thiol residues, alkyne residues, maleimide residues, and disulfide residues.

[14] The compound represented by formula (I) is the following formula (I-1): A-L1-L2-E1-E2-E3-B (I-1) [During the ceremony, A and B are the same as those in formula (I) above, L1 is a bond or a divalent group. L2 is a leaving group, E1 is an electrophile that (i) is linked to the leaving group and (ii) has the ability to react with the nucleophile in the antibody, E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). The leaving group has the ability to be cleaved and eliminated from E1 by a reaction between the nucleophile and the electrophile. A compound represented by [1] to

[13] or a salt thereof.

[15] The above L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [wherein Q is a group selected from the group consisting of -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)],

[14] compounds or salts thereof.

[16] A compound or salt of

[14] or

[15] wherein L2 is a group selected from the group consisting of the following structural formulas: [ka] (Here, EWG is an electron-withdrawing group, m is an integer between 0 and 4. n is an integer between 0 and 3. R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ○ (white circle) represents a connection to L1, and ● (black circle) represents a connection to E1.

[17] Any compound or salt thereof from [1] to

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

[18] The compound represented by formula (I-1) is the following: A-L1-L2-E1-XY-E3-B (I-2) [During the ceremony, A, L1, X, Y, and B are the same as those in formula (I-1) above. L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a divalent group. A compound represented by

[14] to

[17] , or a salt thereof.

[19] The compound represented by formula (I-1) is the following formula (I-3): [ka] [During the ceremony, A, L1, ring Z, and B are the same as those in formula (I-1) above. L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group. A compound represented by

[14] to

[17] or a salt thereof.

[20] The following formula (I): ALEB (I) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, B is a bioorthogonal functional group, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A regioselective modification reagent for antibodies by bioorthogonal functional groups, comprising an affinity substance for antibodies represented by [ ], and a compound having a bioorthogonal functional group or a salt thereof.

[21] A method for producing an antibody having a bioorthogonal functional group or a salt thereof, Formula (I): ALEB (I) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, B is a bioorthogonal functional group, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A substance that has affinity for antibodies, represented by ], and a compound having a bioorthothic functional group or a salt thereof are reacted with an antibody. Formula (II) below: Ab-EB (II) [During the ceremony, E and B are the same as those in formula (I) above, Ab is an antibody. A method comprising producing an antibody or a salt thereof having a bioorthogonal functional group represented by [ ].

[22] A method for producing an antibody or a salt thereof having a functional substance, (1) The following formula (I): ALEB (I) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, B is a bioorthogonal functional group, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A substance that has affinity for antibodies, represented by ], and a compound having a bioorthothic functional group or a salt thereof are reacted with an antibody. Formula (II) below: Ab-EB (II) [During the ceremony, E and B are the same as those in formula (I) above, Ab is an antibody. To produce an antibody or salt thereof having a bioorthogonal functional group represented by ]; and (2) An antibody or salt thereof having a bioorthogonal functional group represented by formula (II) above is reacted with a functional substance via the bioorthogonal functional group, Formula (III) below: Ab-E-B'-F (III) [During the ceremony, Ab is the same as that in formula (II) above, E is the same as that in equation (I) above, B' is a divalent group that includes a moiety generated by a reaction between a functional substance and a bioorthogonal functional group. A method comprising producing an antibody or a salt thereof having a functional substance represented by [ ].

[23] The following formula (II-1): Ab-E1-E2-E3-B (II-1) [During the ceremony, Ab is an antibody, E1 is an electrophile linked to a nucleophile in the antibody. E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). B is a bioorthogonal functional group. An antibody or salt thereof having a bioorthogonal functional group regioselectively represented by [ ].

[24] The antibody or salt thereof according to

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

[25] The antibody or salt thereof of

[23] or

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

[26] Any antibody or salt thereof from

[23] to

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

[27] The antibody represented by formula (II-1) is given by the following formula (II-2): Ab-E1-XY-E3-B (II-2) [During the ceremony, Ab, X, Y, and B are the same as those in formula (II-1) above. E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a divalent group. An antibody having a regioselective bioorthogonal functional group represented by ], any antibody or salt thereof from

[23] to

[26] .

[28] The antibody represented by formula (II-1) is given by the following formula (II-3): [ka] [During the ceremony, Ab, ring constituent atom X', ring Z, and B are the same as those in formula (II-1) above. E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a conjugated or divalent group. An antibody having a regioselective bioorthogonal functional group represented by ], any antibody or salt thereof from

[23] to

[26] .

[29] The following formula (III-1): Ab-E1-E2-E3-B'-F (III-1) [During the ceremony, Ab is an antibody, E1 is an electrophile linked to a nucleophile in the antibody. E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). B' is a divalent group that includes a moiety generated by a reaction between a functional substance and a bioorthogonal functional group. F is a functional substance. An antibody or a salt thereof that regioselectively contains a functional substance represented by [ ].

[30] The antibody according to

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

[31] The antibody or salt thereof of

[29] or

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

[32] Any antibody or salt thereof from

[29] to

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

[33] The antibody represented by formula (III-1) is given by the following formula (III-2): Ab-E1-XY-E3-B'-F (III-2) [During the ceremony, Ab, X, Y, B', and F are the same as those in formula (III-1) above. E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. An antibody having a functional substance regioselectively represented as E3 is a divalent group, any antibody or salt thereof from

[29] to

[32] .

[34] The antibody represented by formula (III-1) is given by the following formula (III-3): [ka] [During the ceremony, Ab, ring constituent atom X', ring Z, B', and F are the same as those in formula (III-1) above. E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a conjugated or bivalent group. An antibody having a functional substance regioselectively represented by

[29] to

[32] , any antibody or salt thereof.

[35] The following formula (IV): ALEF (IV) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, F is a functional substance, The leaving group has the ability to be cleaved and eliminated from E by a reaction between the nucleophile and the electrophile. A compound or salt thereof having an affinity substance for an antibody and a functional substance, represented as ].

[36] The following formula (IV): ALEF (IV) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, F is a functional substance, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A regioselective modification reagent for antibodies by a functional substance, comprising a compound or salt thereof having an affinity substance for an antibody and a functional substance, represented by [ ].

[37] A method for producing an antibody or a salt thereof having a functional substance, Formula (IV): ALEF (IV) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, F is a functional substance, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A compound or salt thereof having an affinity substance for an antibody and a functional substance, represented by ], is reacted with an antibody. Formula (III) below: Ab-EF (III) [During the ceremony, Ab is an antibody, A method comprising producing an antibody or a salt thereof having a functional substance represented by formula (IV) above. [Effects of the Invention]

[0012] The compounds of the present invention, or salts thereof, having an affinity substance for antibodies and a bioorthogonal functional group or functional substance, are useful, for example, for regioselective modification of antibodies. The antibody or salt thereof of the present invention, which has a regioselectively located bioorthogonal functional group, is useful, for example, as an intermediate in the preparation of an antibody or salt thereof that has a regioselectively located functional substance. The antibodies or salts thereof of the present invention, which possess functional substances in a regioselective manner, are useful, for example, as pharmaceuticals or reagents (e.g., diagnostic agents, research reagents). [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a schematic diagram illustrating the outline of the present invention. [Figure 2] Figure 2 shows the results of SDS-PAGE analysis in the synthesis of the IgG antibody trastuzumab-peptide complex. Lanes 1, 3, 6, 8: Molecular weight markers; Lane 2: Unreacted IgG antibody trastuzumab (control, the band around 50,000 molecular weight indicates the heavy chain and the band around 25,000 molecular weight indicates the light chain); Lane 4: Complex of IgG antibody trastuzumab and compound 10 (the upper band around 50,000 molecular weight indicates the conjugation of compound 10 to the trastuzumab heavy chain. The lower band around 50,000 molecular weight indicates the unreacted heavy chain, and the band around 25,000 molecular weight indicates the unreacted light chain); Lane 5: IgG antibody trastuzumab Mab and compound 11 complex (the upper band around molecular weight 50,000 indicates conjugation of compound 11 to the heavy chain of trastuzumab. The lower band around molecular weight 50,000 indicates the unreacted heavy chain, and the band around molecular weight 25,000 indicates the unreacted light chain); Lane 7: Reaction mixture after conjugation of IgG antibody trastuzumab and compound 12 (the band around molecular weight 50,000 indicates the unreacted heavy chain, and the band around molecular weight 25,000 indicates the unreacted light chain. No bands indicating conjugation of compound 12 are visible). [Figure 3]Figure 3 shows the results of SDS-PAGE analysis of trastuzumab-peptide complexes synthesized by regioselectively introducing maleimide into the IgG antibody trastuzumab and then conjugating it 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), regioselectively introducing maleimide, and then conjugating it with compound 25. The upper band around molecular weight 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band around molecular weight 50,000 indicates the unreacted heavy chain, and the band around molecular weight 25,000 indicates the unreacted light chain. Lane 3: Complex obtained by treating the IgG antibody trastuzumab with compound 22 (20 molar equivalents relative to the antibody), regioselectively introducing maleimide, and then conjugating it with compound 25. The upper band around a molecular weight of 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band around 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 4: A complex obtained by treating the IgG antibody trastuzumab with compound 22 (40 molar equivalents relative to the antibody), regioselectively introducing maleimide, and then conjugating with compound 25. The upper band around a molecular weight of 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band around 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: A complex obtained by treating the IgG antibody trastuzumab with compound 23 (10 molar equivalents relative to the antibody), regioselectively introducing maleimide, and then conjugating with compound 25. The upper band around a molecular weight of 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band around 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 6: A complex obtained by treating the IgG antibody trastuzumab with compound 23 (20 molar equivalents relative to the antibody), regioselectively introducing maleimide, and then conjugating it with compound 25.The upper band around a molecular weight of 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band around 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 7: A complex obtained by treating the IgG antibody trastuzumab with compound 23 (40 molar equivalents relative to the antibody), regioselectively introducing maleimide, and then conjugating it with compound 25. The upper band around a molecular weight of 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band around 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 8, 10: Unreacted IgG antibody trastuzumab (control, bands around molecular weight 50,000 indicate the heavy chain and bands around molecular weight 25,000 indicate the light chain). Lane 11: Reaction mixture after treating IgG antibody trastuzumab with compound 24 (10 molar equivalents relative to the antibody) and adding compound 25. Bands around molecular weight 50,000 indicate the unreacted heavy chain, and bands around molecular weight 25,000 indicate the unreacted light chain. Lane 12: Reaction mixture after treating IgG antibody trastuzumab with compound 24 (20 molar equivalents relative to the antibody) and adding compound 25. Bands around molecular weight 50,000 indicate the unreacted heavy chain, and bands around molecular weight 25,000 indicate the unreacted light chain. Lane 13: Reaction mixture after treating IgG antibody trastuzumab with compound 24 (40 molar equivalents relative to the antibody) and adding compound 25. Bands around molecular weight 50,000 indicate the unreacted heavy chain, and bands around molecular weight 25,000 indicate the unreacted light chain. [Figure 4] Figure 4 shows the ESI-TOFMS results of the trastuzumab-maleimide modified product synthesized in (4-5-1) under reduced conditions. The upper panel shows the results of unreacted trastuzumab, and the lower panel shows the modified product. [Figure 5] Figure 5 shows the ESI-TOFMS of the trastuzumab-azide modified antibody (azide-modified antibody 1) synthesized in (6-1-1) under reduced conditions. The lower panel shows the measurement of unreacted trastuzumab, and the upper panel shows the modified antibody. [Figure 6]Figure 6 shows the ESI-TOFMS results of the trastuzumab-azide modified antibody (azide-modified antibody 3) synthesized in (6-1-2) under reduced conditions. The lower panel shows the results of measuring unreacted trastuzumab, and the upper panel shows the modified antibody. [Figure 7] Figure 7 shows the ESI-TOFMS of the trastuzumab-azide modified antibody (azide-modified antibody 6) synthesized in (6-1-3) under reduced conditions. The upper panel shows the measurement of unreacted trastuzumab, and the lower panel shows the modified antibody. [Figure 8] Figure 8 shows the ESI-TOFMS of the trastuzumab-azide modified antibody (azide-modified antibody 8) synthesized in (6-1-3) under reduced conditions. The upper panel shows the measurement of unreacted trastuzumab, and the lower panel shows the modified antibody. [Figure 9] Figure 9 shows the ESI-TOFMS results of the trastuzumab-azide modified antibody (azide-modified antibody 10) synthesized in (6-1-3) under reduced conditions. The upper panel shows the results of measuring unreacted trastuzumab, and the lower panel shows the modified antibody. [Figure 10] Figure 10 shows the ESI-TOFMS of the adalimumab-azide modified antibody (azide-modified antibody 28) synthesized in (6-1-4) under reduced conditions. The upper panel shows the measurement of unreacted adalimumab, and the lower panel shows the modified antibody. [Figure 11] Figure 11 shows the ESI-TOFMS of the denosumab-azide-modified antibody (azide-modified antibody 29) synthesized in (6-1-4) under reduced conditions. The upper panel shows the measurement of unreacted denosumab, and the lower panel shows the modified product. [Figure 12] Figure 12 shows the ESI-TOFMS of the dupilumab-azide-modified antibody (azide-modified antibody 30) synthesized in (6-1-4) under reduced conditions. The upper panel shows the measurement of unreacted dupilumab, and the lower panel shows the modified product. [Figure 13] Figure 13 shows the ESI-TOFMS of the rituximab-azide modified antibody (azide modified antibody 31) synthesized in (6-1-4) under reduced conditions. The lower panel shows the measurement of unreacted rituximab, and the upper panel shows the modified antibody. [Figure 14]Figure 14 shows the ESI-TOFMS results of the trastuzumab-protective thiol modified product synthesized in (8-1-1) under reduced conditions. The upper panel shows the unreacted trastuzumab, and the lower panel shows the modified product. [Figure 15] Figure 15 shows the ESI-TOFMS of the trastuzumab-thiol modified product deprotected in (8-3-1) under reduced conditions. The upper panel shows the measurement of unreacted trastuzumab, and the lower panel shows the modified product. [Figure 16] Figure 16 shows the ESI-TOFMS results of the trastuzumab-azide modified product synthesized in (9-1-1) under reduced conditions. The upper panel shows the results of unreacted trastuzumab, and the lower panel shows the modified product. [Figure 17] Figure 17 shows the ESI-TOFMS results of the trastuzumab-azide modified product synthesized in (9-1-5) under reduced conditions. The upper panel shows the results of unreacted trastuzumab, and the lower panel shows the modified product. [Figure 18] Figure 18 shows the ESI-TOFMS results of the trastuzumab-azide modified product synthesized in (9-2-2) under reduced conditions. The upper panel shows the results of unreacted trastuzumab, and the lower panel shows the modified product. [Figure 19] Figure 19 shows the ESI-TOFMS results of the trastuzumab-Cy3 complex synthesized in (10-1-1). The bottom panel shows the results of measuring unreacted trastuzumab, the middle panel shows the results of measuring the trastuzumab-azide modified compound synthesized in (6-1-1), and the top panel shows the trastuzumab-Cy3 complex. [Figure 20] Figure 20 shows ESI-TOFMS of the trastuzumab-Cy3 complex treated with (10-1-2) under reducing conditions. The bottom panel shows measurements of unreacted trastuzumab, the middle panel shows measurements of the trastuzumab-azide modified compound synthesized with (6-1-1), and the top panel shows the trastuzumab-Cy3 complex. [Figure 21]Figure 21 shows the ESI-TOFMS results of the trastuzumab-peptide complex synthesized in (10-2-2). The bottom panel shows the measurement of unreacted trastuzumab, the middle panel shows the measurement of the trastuzumab-azide modified compound synthesized in (6-1-1), and the top panel shows the trastuzumab-Cy3 complex. [Figure 22] Figure 22 shows ESI-TOFMS of the trastuzumab-Cy3 complex treated with (10-2-3) under reducing conditions. The bottom panel shows measurements of unreacted trastuzumab, the middle panel shows measurements of the trastuzumab-azide modified compound synthesized with (6-1-1), and the top panel shows the trastuzumab-peptide complex. [Figure 23] Figure 23 shows ESI-TOFMS results under reducing conditions for the trastuzumab-maleimide compound complex treated with (10-3-1). The upper panel shows the thiolated trastuzumab, and the lower panel shows the trastuzumab-maleimide compound complex. [Figure 24] Figure 24 shows the ESI-TOFMS of the reaction products treated in (10-3-2) under reducing conditions. The upper panel shows the thiol-transformed trastuzumab, and the lower panel shows the trastuzumab-maleimide compound complex. [Figure 25] Figure 25 shows (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). [Figure 26] Figure 26 shows (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). [Figure 27] Figure 27 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). [Figure 28] Figure 28 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 29] Figure 29 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 30] Figure 30 shows the MS spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 31] Figure 31 shows the CID spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification site to the lysine residue (amine benzoate derivative (+119.037Da)) obtained by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 32] Figure 32 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of mercaptopropionate-added maleimide-modified trastuzumab. [Figure 33] Figure 33 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of maleimide MPA-modified trastuzumab. [Figure 34] Figure 34 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of alkyl azide-modified trastuzumab. [Figure 35] Figure 35 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of alkyl azide-modified trastuzumab. [Figure 36] Figure 36 shows the MS spectrum of the peptide fragment VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 101), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 37] Figure 37 shows the CID spectrum of the peptide fragment VVSVLTVLHQDWLNGKEYK (SEQ ID NO: 101), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 38] Figure 38 shows the results of an analysis by BioPharma Finder, which indicates that the lysine residue at position 317 of azidobenzoic acid-modified trastuzumab is highly selectively modified. [Figure 39] Figure 39 shows the MS spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 40] Figure 40 shows the CID spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 41] Figure 41 shows the results of an analysis by BioPharma Finder, which indicates that the lysine residue at position 288 or 290 of azidobenzoate-modified trastuzumab is highly selectively modified. [Figure 42] Figure 42 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 43] Figure 43 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 44] Figure 44 shows the results of an analysis by BioPharma Finder, which indicates that the lysine residue at position 246 or 248 of azidobenzoic acid-modified trastuzumab is highly selectively modified. [Figure 45] Figure 45 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of acetylthiol-modified trastuzumab. [Figure 46] Figure 46 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of acetylthiol-modified trastuzumab. [Figure 47] Figure 47 shows the results of an analysis by BioPharma Finder, which indicates that the lysine residue at position 246 or 248 of acetylthiol-modified trastuzumab is highly selectively modified. [Figure 48] Figure 48 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 49] Figure 49 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 50] Figure 50 shows the MS spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 51]Figure 51 shows the CID spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification site to the lysine residue by trypsin digestion of azidobenzoic acid-modified trastuzumab. [Figure 52] Figure 52 shows the MS spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification sites to the lysine residues by trypsin digestion of acetylthiol and azidobenzoic acid-modified trastuzumab. [Figure 53] Figure 53 shows the CID spectrum of the peptide fragment THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 11), including the modification sites to the lysine residues by trypsin digestion of acetylthiol and azidobenzoic acid-modified trastuzumab. [Figure 54] Figure 54 shows the MS spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification sites to lysine residues by trypsin digestion of acetylthiol and azidobenzoic acid-modified trastuzumab. [Figure 55] Figure 55 shows the CID spectrum of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12), including the modification sites to lysine residues by trypsin digestion of acetylthiol and azidobenzoic acid-modified trastuzumab. [Figure 56] Figure 56 shows the results of an analysis by BioPharma Finder, which indicates that lysine residues at positions 246 or 248 and 288 or 290 of acetylthiol and azidobenzoic acid-modified trastuzumab are highly selectively modified. [Figure 57] Figure 57 shows the MS spectrum of the peptide fragment CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR (SEQ ID NO: 102), including the modification site to the lysine residue by trypsin digestion of benzoic acid-modified denosumab. [Figure 58]Figure 58 shows the CID spectrum of the peptide fragment CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR (SEQ ID NO: 102), including the modification site to the lysine residue by trypsin digestion of benzoic acid-modified denosumab. [Figure 59] Figure 59 shows the results of an analysis by BioPharma Finder, which indicates that the lysine residue at position 247 or 249 of benzoic acid-modified denosumab is highly selectively modified. [Figure 60] Figure 60 shows the MS spectrum of the peptide fragment YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 103), including the modification site to the lysine residue by trypsin digestion of benzoic acid-modified dupilumab. [Figure 61] Figure 61 shows the CID spectrum of the peptide fragment YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 103), including the modification site to the lysine residue by trypsin digestion of benzoic acid-modified dupilumab. [Figure 62] Figure 62 shows the results of an analysis by BioPharma Finder, which indicates that the lysine residue at position 251 or 253 of benzoic acid-modified dupilumab is highly selectively modified. [Figure 63] Figure 63 shows the results of the analysis of the trastuzumab-DM1 conjugate synthesized in (12-1-1) by ESI-TOFMS (under non-reducing conditions). [Figure 64] Figure 64 shows the results of the analysis of the trastuzumab-DM1 conjugate synthesized in (12-1-1) by ESI-TOFMS (under reducing conditions). [Figure 65] Figure 65 shows the results of the analysis of the trastuzumab-MMAE conjugate synthesized in (12-2-1) by ESI-TOFMS (under non-reducing conditions). [Figure 66] Figure 66 shows the results of the analysis of the trastuzumab-MMAE conjugate synthesized in (12-2-1) by ESI-TOFMS (under reducing conditions). [Figure 67] Figure 67 is a diagram showing the analysis result of the rituximab-DM1 conjugate synthesized in (12-3-1) by ESI-TOFMS (under non-reducing conditions). [Figure 68] Figure 68 is a diagram showing the analysis result of the rituximab-DM1 conjugate synthesized in (12-3-1) by ESI-TOFMS (under reducing conditions). [Figure 69] Figure 69 is a diagram showing the analysis result of the rituximab-DM1 conjugate synthesized in (12-4-1) by ESI-TOFMS (under non-reducing conditions). [Figure 70] Figure 70 is a diagram showing the analysis result of the rituximab-DM1 conjugate synthesized in (12-4-1) by ESI-TOFMS (under reducing conditions). [Figure 71] Figure 71 shows (1) the consensus amino acid sequence (SEQ ID NO: 1) of the Fc region in the heavy chain of trastuzumab and the IgG1 Fc region, and (2) the amino acid sequence (SEQ ID NO: 3) of the IgG1 Fc region.

Mode for Carrying Out the Invention

[0014] 1. An affinity substance for an antibody, and a compound having a bioorthogonal functional group or a salt thereof 1-1. Overview The present invention provides a compound having an affinity substance for an antibody and a bioorthogonal functional group, or a salt thereof, represented by formula (I). A-L-E-B (I) 〔In the formula, A is an affinity substance for an antibody, L is a divalent group containing a leaving group, E is a divalent group containing an electrophilic group that (i) is linked to the leaving group and (ii) has the ability to react with a nucleophilic group in the antibody, B is a bioorthogonal functional group, The leaving group has the ability to be cleaved from E and eliminated by the reaction between the nucleophilic group and the electrophilic group.〕

[0015] In the notation of formula (I) and other formulas presented in connection with the present invention, a hyphen (-) indicates that two units on either side of it 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 antibody affinity substance represented by formula (I), and compounds having bioorthothic functional groups or salts thereof, indicate that the antibody affinity substance (A) contains a structural unit having an LEB via a covalent bond between A and L. Therefore, in formula (I), the antibody affinity substance (A) may have one structural unit having an LEB, or a plurality of structural units (homogeneous or heterogeneous) having LEBs (e.g., 2 to 5, preferably 2 to 4, more preferably 2 or 3). In other formulas as well, the affinity substance (A) or antibody (Ab) for the antibody indicates that it contains specific structural units (structural units other than A or Ab) in the formula via covalent bonds. Therefore, in other formulas as well, the affinity substance (A) or antibody (Ab) for the antibody 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 types).

[0016] 1-2. Affinity substances for antibodies (A) In formula (I), A is an antibody affinity substance. An antibody affinity substance is a substance that has the ability to bind to an antibody via a non-covalent bond.

[0017] The affinity substance used in the present invention targets an antibody. The antibody may be modified with a biomolecule (e.g., sugar) or may be an unmodified antibody. Any antibody can be used against any component, such as biological components, viral components, and components found in the environment, but antibodies against biological components or viral components are preferred. Examples of biological components include components (e.g., proteins) derived from animals such as mammals and birds (e.g., chickens), insects, microorganisms, plants, fungi, and fish. Preferably, the biological component is derived from a mammal. Examples of mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep). The biological components are more preferably components derived from primates or rodents (e.g., proteins), and even more preferably, from the viewpoint of the clinical application of the present invention, components derived from humans (e.g., proteins). Examples of virus-derived components include components derived from influenza viruses (e.g., avian influenza viruses, swine influenza viruses), HIV, Ebola viruses, and phage viruses (e.g., proteins).

[0018] Antibodies may also be polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which specific glycans have been added (e.g., antibodies modified to have a glycan-binding consensus sequence such as an N-linked glycan-binding consensus sequence), bispecific antibodies, scFv antibodies, Fab antibodies, F(ab')2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins. Antibodies may also be bivalent antibodies (e.g., IgG, IgD, IgE) or tetravalent or higher antibodies (e.g., IgA antibodies, IgM antibodies).

[0019] The antibody, which is the target of the affinity substance, may also be composed of any amino acid residue, but preferably of the 20 natural L-α-amino acid residues that normally make up proteins. Examples of such amino acid residues include L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (the notation L will be omitted below). The antibody may consist 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. The antibody may also consist of, 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 consist 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. If the antibody is an antibody (e.g., a monoclonal antibody as described above), the above number of amino acid residues may correspond to the amino acid residues of the antibody's heavy chain.

[0020] The antibody, which is the target of the affinity substance, is a protein that contains, preferably at one or more positions (preferably at multiple positions) a specific amino acid residue having a side chain or terminal (N-terminus and / or C-terminus), preferably with a side chain, to which a bioorthogonal functional group, as described later, can react. Examples of such specific amino acid residues include those described later, but preferably they are amino acid residues selected from the group consisting of lysine residues, tyrosine residues, serine residues, threonine residues, and cysteine ​​residues. In view of the fact that the compound of the present invention can regioselectively modify antibodies, antibodies containing such specific amino acid residues at multiple positions are preferred. The multiple positions are not particularly limited as long as there are two or more positions, but for example, there may be three or more positions, preferably five or more positions, more preferably ten or more positions, even more preferably twenty or more positions, and especially preferably thirty or more positions. The multiple positions may also be, for example, positions 200 or less, preferably 180 or less, more preferably 150 or less, even more preferably 120 or less positions, and especially preferably 100 or less positions. More specifically, the multiple positions may be, for example, positions 3 to 200, preferably positions 5 to 180, more preferably positions 10 to 150, even more preferably positions 20 to 120, and particularly preferably positions 30 to 100. Even in antibodies containing such specific amino acid residues at multiple positions, the compounds of the present invention can regioselectively modify one or two specific amino acid residues located in a specific region. For example, the number of lysine residues in human IgG1 is generally said to be around 70 to 90, although this depends on the amino acid composition in the variable region. In the present invention, we have succeeded in regioselectively modifying one or two lysine residues located in a specific region of such human IgG1.

[0021] More specifically, in the present invention, from the viewpoint of modifying amino acid residues located at specific target sites in the antibody while maintaining antibody function (i.e., maintaining native folding without denaturing the antibody), regioselective modification of amino acid residues exposed on the surface of the antibody is preferred. For example, in human IgG such as human IgG1, exposed lysine and tyrosine residues are located at the following positions (according to EU numbering; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). (1) Exposed lysine residues CH2 domains (246th, 248th, 274th, 288th, 290th, 317th, 320th, 322nd, 338th) CH3 domains (360th, 414th, 439th) (2) Exposed tyrosine residues CH2 domains (ranked 278th, 296th, and 300th) CH3 domain (ranked 436th) (3) Exposed serine residues CH2 domains (254th, 267th, 298th, 324th) CH3 domains (375th, 400th, 415th, 440th, 442nd) (4) Exposed threonine residues CH2 domains (256th, 289th, 307th) CH3 domains (335th, 359th, 393rd, 437th) Therefore, when human IgG such as human IgG1 is modified with a lysine residue or a tyrosine residue, modification at the above-mentioned position is preferred.

[0022] Preferably, when human IgG such as human IgG1 is modified with a lysine residue, tyrosine residue, serine residue, or threonine residue, the lysine residue, tyrosine residue, serine residue, or threonine residue located at the following positions among the positions (1) to (4) above, which have high surface exposure, may be modified. (1') Exposed lysine residue CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322) CH3 domain (positions 360, 414, 439) (2’) exposed tyrosine residue CH2 domain (positions 278, 296, 300) CH3 domain (position 436) (3’) exposed serine residue CH2 domain (positions 254, 267, 298) CH3 domain (positions 400, 415, 440) (4’) exposed threonine residue CH2 domain (positions 256, 289) CH3 domain (positions 335, 359) Therefore, when human IgG such as human IgG1 is modified with a lysine residue, tyrosine residue, serine residue or threonine residue, modification at the above positions is more preferable.

[0023] More preferably, when human IgG such as human IgG1 is modified with a lysine residue, among the positions in (1) above, lysine residues present at predetermined positions (e.g., positions 246, 248, 288, 290, 317) in the CH2 domain that can be efficiently modified in the present invention may be modified.

[0024] In certain embodiments, if the antibody that is the target of the affinity substance contains specific amino acid residues at multiple positions as described above, it may contain one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and five or more specific amino acid residues in a non-target region other than the target region. The target region may preferably consist of 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, or 1 to 3 (i.e., 1, 2, or 3) amino acid residues. Particularly preferably, the target region may be a region consisting of specific amino acid residues located at specific positions. Such specific positions vary depending on the target protein and the type of affinity substance, but may, for example, be specific positions in specific regions within the constant region of the antibody (e.g., CH1, CH2, CH3), and preferably in CH2 of the antibody. More specifically, the target region may be the following residues according to the Eu numbering in human IgG Fc: (1) Lys248 residue (hereinafter also referred to simply as "Lys248" in this specification, and corresponding to the 18th residue of the human IgG CH2 region (SEQ ID NO: 1)) or Lys246 residue (hereinafter also referred to simply as "Lys246" in this specification, and corresponding to the 16th residue of the human IgG CH2 region (SEQ ID NO: 1): (2) Lys288 residue (hereinafter also referred to simply as "Lys288" in this specification, and corresponding to the 58th residue of the human IgG CH2 region (SEQ ID NO: 1)) or Lys290 residue (hereinafter also referred to simply as "Lys290" in this specification, and corresponding to the 60th residue of the human IgG CH2 region (SEQ ID NO: 1)); (3) Lys317 residue (hereinafter referred to simply as "Lys317" in this specification, and corresponding to the 87th residue of the human IgG CH2 region (SEQ ID NO: 1)).

[0025] According to the present invention, specific amino acid residues in the target region can be modified with high regioselectivity. Such regioselectivity may be, for example, 30% or more, preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0026] The target region may also be such that a specific amino acid residue located at a particular position does not contain any amino acid residues of the same type as the specific amino acid residue located at that particular position, in the region extending to a number of amino acid residues (where a is any integer from 1 to 10) at the N-terminal and C-terminal ends of that particular position, respectively. a is preferably an integer from 1 to 5, more preferably an integer from 1 to 3, even more preferably 1 or 2, and particularly preferably 1.

[0027] In preferred embodiments, the antibody is a monoclonal antibody. Examples of antibody isotypes, such as monoclonal antibodies, include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. The monoclonal antibody may be 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 preferred is a human antibody, a humanized antibody, or a chimeric antibody having human IgG (e.g., IgG1, IgG2, IgG3, IgG4) in its constant region.

[0028] An antibody is an antibody against any antigen. For example, such an antigen may be a component found in the organisms or viruses mentioned above. Other examples of such antigens include proteins (including oligopeptides and polypeptides; proteins modified with biomolecules such as sugars (e.g., glycoproteins)), glycans, nucleic acids, and small molecule compounds.

[0029] Preferably, the antibody may be an antibody that uses a protein as an antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0030] More specifically, the antigen protein of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0031] (1) Oncology PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1(CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue Factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfeline Receiver, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lysil B Antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 Integrin, TAG-72 (CA72-4), CD70,

[0032] (2) Autoimmune diseases and inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP(Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT

[0033] (3) Neurological diseases CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0034] (4) Infectious disease Clostridium Difficile toxin B, cytomegalovirus, RSV, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0035] (5) Hereditary and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0036] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0037] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0038] (8) Blood disorders vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0039] (9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

[0040] 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 IgG (e.g., IgG1, IgG2, IgG3, IgG4) is preferred. Preferably, the monoclonal antibody is a full-length monoclonal antibody.

[0041] In a more preferred embodiment, the antibody affinity substance is a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, or an antibody affinity substance for human antibodies (e.g., IgG1, IgG2, IgG3, IgG4, etc.).

[0042] In a particularly preferred embodiment, the affinity substance for the antibody is an affinity substance for an antibody that contains one Fc region protein selected from the group consisting of (A) to (C) below and has antigen-binding ability: (A) Fc region protein containing the amino acid sequence of Sequence ID No. 1; (B) Fc region proteins containing an amino acid sequence in which one or more amino acid residues are inserted, added, deleted, or substituted in the amino acid sequence of Sequence ID No. 1; or (C) Fc region protein containing an amino acid sequence that shows more than 90% identity with the amino acid sequence of Sequence ID No. 1.

[0043] The amino acid sequence of SEQ ID NO: 1 is an Fc region protein. Such Fc region proteins are known to have secretory ability. Therefore, the Fc region proteins described in (A) to (C) above can have secretory ability. Furthermore, antibodies containing such Fc region proteins can 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 later, and even more preferably leucine, isoleucine, or alanine, and is 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, and even more 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, and even more 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, and 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 nonpolar side chain, and even more preferably methionine, leucine, or isoleucine, and particularly preferably methionine or leucine. The amino acid residue at position 177 in SEQ ID NO: 1 is any amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a non-charged polar side chain as described later, or an amino acid residue having a nonpolar side chain, and even more preferably threonine, alanine, or glycine, and particularly preferably threonine or alanine.

[0044] In a preferred embodiment, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence consisting of amino acid residues at positions 220 to 449 of the amino acid sequence of SEQ ID NO: 2.

[0045] 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 of the amino acid sequence of SEQ ID NO: 3.

[0046] In certain embodiments, an antibody comprising an Fc region protein containing the amino acid sequence described above may also be an antibody comprising an Fc region protein containing the amino acid sequence described above, and a constant region of the antibody. Such a constant region of the antibody may be a chimeric antibody, a humanized antibody, or the constant region of a human antibody (e.g., IgG1, IgG2, IgG3, IgG4, etc.).

[0047] In Fc region proteins (B), one or more amino acid residues may be modified by one, two, three, or four types of mutations selected from the group consisting of deletion, substitution, addition, and insertion of amino acid residues. Amino acid residue mutations may be introduced into one region of the amino acid sequence or into multiple different regions. The term "one or several" indicates a number that does not significantly impair the activity of the protein. The number indicated by the term "one or several" 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 (e.g., 1, 2, 3, 4, or 5).

[0048] For Fc region proteins (C), the identity percentage with the amino acid sequence of SEQ ID NO: 1 may be 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. In this invention, the identity percentage of peptides or polypeptides (proteins) can be calculated using the algorithm blastp. More specifically, the identity percentage of polypeptides can be calculated using the algorithm blastp provided by the National Center for Biotechnology Information (NCBI) with default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11 Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment). Furthermore, the identity percentage of polynucleotides (genes) can be calculated using the algorithm blastn. More specifically, the percentage of polynucleotide identity can be calculated using the blastn algorithm provided by NCBI, with default Scoring Parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear).

[0049] In the context of secretory ability, "secretion" refers to the secretion of secretory proteins (so-called solubility). Therefore, "having secretory ability" means that it functions as an antibody, just like a normal antibody.

[0050] Antibodies containing the above-mentioned Fc region protein may have mutations introduced at specific sites, as long as they retain the desired properties (e.g., secretory ability, antigen-binding ability). The locations of amino acid residues in which mutations may be introduced while retaining the desired properties are obvious to those skilled in the art. Specifically, those skilled in the art can 1) compare the amino acid sequences of several proteins having similar properties, 2) identify relatively conserved and relatively unconserved regions, and then 3) predict from the relatively conserved and relatively unconserved regions which regions may play an important role in function and which may not play an important role in function, respectively, thus recognizing the correlation between structure and function. Therefore, those skilled in the art can identify the locations of amino acid residues in the amino acid sequence of antibodies containing the above-mentioned Fc region protein in which mutations may be introduced.

[0051] When an amino acid residue is mutated by substitution, the substitution may be a conservative substitution. As used herein, the term "conservative substitution" means substituting a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Preferably, the conservative amino acid substitutions may be substitutions between aspartic acid and glutamic acid, between arginine, lysine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine, between leucine, isoleucine and alanine, and between glycine and alanine.

[0052] Examples of antibodies used in the present invention, or antibodies containing any one Fc region selected from the group consisting of (A) to (C) above, include chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab), humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, allentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, ecrizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), Examples include mepolizumab, elotuzumab, daratumumab, ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab, and human antibodies (e.g., adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, laxibakumab, ramucirumab, nivolumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesitumumab, brodalumab (IgG2), olaratumab, and dupilumab (IgG4)) (whereas the IgG subtype is not mentioned, it is assumed to be IgG1).

[0053] Examples of substances with affinity for antibodies as described above include peptides (including oligopeptides, polypeptides, and proteins), small molecule compounds, nucleic acids, nucleic acid-peptide complexes, peptide-small molecule compound complexes, and nucleic acid-small molecule complexes.

[0054] In certain embodiments, the antibody affinity substance described above may be a peptide (including oligopeptides, polypeptides, and proteins; it may also be a glycoprotein). Examples of such peptides include: (1) IgG-binding peptides that have affinity for specific regions (CH2 regions) of human IgG in general (i.e., human IgG1, IgG2, IgG3, and IgG4; the same applies hereafter) (see, for example, International Publication No. 2016 / 186206, International Publication No. 2013 / 027796, and International Publication No. 2008 / 054030); (2) Protein A mimetic (PAM) peptides that have affinity for a specific region (CH2 region) of human IgG in general (see, e.g., Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, VOL.6, 564-569); (3) EPIHRSTLTALL (SEQ ID NO: 25) which has affinity for a specific region (CH2 region) of human IgG in general (see, for example, Ehrlich GK et al., J. Biochem. Biophys. Methods, 2001, VOL. 49, 443-454); (4) (NH2-Cys1-X1-X2-X3-X4)2-Lys-Gly-OH that has affinity for a specific region (Fc region) of human IgG in general (see, for example, Ruvo M et al., ChemBioChem, 2005, VOL.6, 1242-1253); (5) FARLVSSIRY (SEQ ID NO: 26), FGRLVSSIRY (SEQ ID NO: 27), and TWKTSRISIF (SEQ ID NO: 28) which have affinity for specific regions (Fc regions) of human IgG in general (see, for example, Krook M et al., Journal of Immunological Methods, 1998, VOL.221, 151-157); (6) QSYP (Sequence ID 29) that has affinity for specific regions of human IgG in general (see, e.g., Jacobs JM et al., Bio.Techniques, 2003, VOL.34, 132-141); (7) HWRGWV (SEQ ID NO: 30), HYFKFD (SEQ ID NO: 31), and HFRRHL (SEQ ID NO: 32) which have affinity for specific regions (Fc regions) of human IgG in general (see, e.g., Carbonell RG et al., Journal of Chromatography A, 2009, VOL.1216, 910-918); (8) DAAGs (SEQ ID NO: 33) that have affinity for specific regions (Fc regions) of human IgG in general (see, e.g., Lund LN et al., Journal of Chromatography A, 2012, VOL.1225, 158-167); (9) Fc-I, Fc-II, and Fc-III which have affinity for specific regions (Fc regions) of human IgG in general (e.g., Warren L. Delano et al., Science, 2000, VOL.287, 1279-1283; see International Publication 2001 / 045746); and (10) NARKFYKG (SEQ ID NO: 34) and NKFRGKYK (SEQ ID NO: 35) which have affinity for specific regions (Fc regions) of human IgG in general (see, e.g., Biochemical Engineering Journal, 2013, VOL.79, 33-40).

[0055] In another specific embodiment, the antibody affinity substance described above may be a substance other than a peptide. Examples of such substances include aptamers that have affinity for specific regions of human IgG (e.g., human IgG1-4) (CH2 region, particularly the side chain of Lys340) [e.g., GGUG(C / A)(U / T) motif-containing aptamers such as GGUGCU and GGUGAU] (see, e.g., 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).

[0056] Affinity substances for antibodies as described above can be obtained by any known method in the field. For example, they can be obtained by producing antibodies using the whole antibody or a partial peptide within the antibody (e.g., a partial peptide present in a known surface region of the antibody) (e.g., hybridoma method), or by screening affinity substances from libraries where affinity substances are available (e.g., peptide libraries, antibody libraries, antibody-producing cell libraries, aptamer libraries, phage libraries, mRNA libraries, cDNA libraries) (e.g., phage display method, SELEX method, mRNA display method, ribosome display method, cDNA display method, yeast display method). Furthermore, if the affinity substance for an antibody is an affinity substance for the Fc region (soluble region) of the antibody, affinity substances (e.g., antibodies, aptamers) that can selectively bind to any part of the Fc region of an antibody can be efficiently obtained by using a partial peptide present in a specific region (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 is a mixture of those with relatively strong affinity binding ability and those with weak affinity binding ability. However, even affinity substances with weak affinity binding ability can have their affinity binding ability reinforced by using them in excess.

[0057] In preferred embodiments, the affinity substance for the antibody is a peptide. Such peptides are preferably those having binding ability to the constant region of a monoclonal antibody, more preferably those having binding ability to the Fc region of a monoclonal antibody, and even more preferably those having binding ability to the Fc region of IgG. The length of the peptide is not particularly limited, but for example, it is a peptide consisting of 10 to 40 amino acid residues (e.g., 10 to 20, 20 to 30, and 30 to 40). Examples of amino acid residues constituting the peptide include the 20 L-α-amino acid residues that constitute natural proteins and their stereoisomers (e.g., D-amino acids), and their isomers (e.g., β-amino acids).

[0058] In certain embodiments, the affinity substance for the antibody described above may be a peptide containing any of the following amino acid sequences. (a-1-1) The amino acid sequence of (SEQ ID NO: 11) FNMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (see, for example, compounds 22-24, 29), or (a-1-2) In the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (Sequence ID 12) (an amino acid sequence in which two Ks are replaced with Rs in the known sequence Z34C), any one to three amino acid residues in the sequence are replaced by one amino acid residue each selected from the group consisting of lysine residues, aspartic acid residues, and glutamic acid residues, which may be the same or different. (a-2-1) The amino acid sequence of (a-2-1)β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (see, for example, compounds 26-28), or, In the amino acid sequence of (a-2-2)β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (Sequence ID 14) (an amino acid sequence in which two Ks are replaced with Rs in the known sequence Z34C, and the N-terminal F is replaced with β-Ala), any one to three amino acid residues in the sequence are replaced by one amino acid residue each selected from the group consisting of lysine residues, aspartic acid residues, and glutamic acid residues, and (b) A peptide comprising an amino acid sequence having 85% or more identity with each of the aforementioned amino acid sequences of Sequence IDs 11 to 14. Such peptides have the ability to bind to the Fc region of monoclonal antibodies.

[0059] The peptide consisting of the amino acid sequence of SEQ ID NO: 12 is obtained by changing the two K (lysine) atoms at positions 26 and 28 from the N-terminus to R (arginine) in the affinity peptide known as Z34C, for convenience in peptide reagent synthesis. Compounds of the present invention containing the peptide with the above amino acid sequence are useful for regioselective modification of specific amino acid residues in human IgG Fc (e.g., Lys248 or Lys246, Lys288 or Lys290, Lys317, or other amino acid residues following Eu numbering). The amino acid sequence of Z34C is FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (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)).

[0060] Affinity peptides can have affinity for human IgG (e.g., human IgG as described above; preferably human IgG1). If the affinity peptide contains two cysteine ​​residues (e.g., at positions 5 and 34), the two cysteine ​​residues may be disulfide-bonded to form a cyclic peptide.

[0061] Any position can be used for introducing lysine residues, aspartic acid residues, or glutamic acid residues (amino acid residues that are easily modified by a crosslinking agent), as long as they have affinity for human IgG such as human IgG1. Such positions can be easily identified by those skilled in the art. The positions for introducing lysine residues, aspartic acid residues, or glutamic acid residues may also be amino acid residues other than cysteine ​​residues. More preferably, examples of amino acid residues that can be easily modified by a crosslinking agent for introduction include positions 1, 3, 6, 7, 13, 20, 24, 31, and 32.

[0062] Preferably, the amino acid sequence having the characteristics of (a) and (b) has one specific amino acid residue (preferably a lysine residue) selected from the group consisting of lysine residues, aspartic acid residues, and glutamic acid residues (amino acid residues that can be easily modified by a crosslinking agent) at a predetermined position, and it is also preferable that it has mutations in the 20 ordinary amino acid residues that constitute natural proteins (preferably 17 amino acid residues other than lysine residues, aspartic acid residues, and glutamic acid residues, and more preferably 19 amino acid residues other than lysine residues) at positions other than the predetermined position. Such predetermined positions are not particularly limited and include, for example, positions 1, 3, 6, 7, 13, 20, 24, 31, and 32. The amino acid sequence having the characteristics of (a) and (b) maintains two cysteine ​​residues, and these two cysteine ​​residues may be linked by a disulfide bond. An amino acid sequence having 85% or more identity with the aforementioned amino acid sequences of SEQ ID NOs. 11-14 may be a sequence in which 1-3 amino acid residues (preferably 1 or 2, more preferably 1) have been modified by 1, 2, 3, or 4 types of mutations (preferably substitutions) selected from the group consisting of deletion, substitution, addition, and insertion of amino acid residues. The amino acid residue mutation may be introduced into one region of the amino acid sequence, or it may be introduced into multiple different regions.

[0063] More preferably, the above amino acid sequence having the characteristics of (a) and (b) may be (c) or (d) below. (c) An amino acid sequence selected from the group consisting of the following amino acid sequences (1) to (16): (1) FNMQQQRRFYEALHDPNLNEEQRNARIRSIKDD(Sequence ID 5); (2) FNMQQQRRFYEALHDPNLNEEQRNARIKSIRDD(Sequence ID 6); (3) β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD(Sequence ID 7); (4) FNMQQQRRFYEALHDPNLNEEQRNAKIKSIKDD(Sequence ID 8); (5) KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (Sequence ID 37); (6)FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC(Sequence ID 38); (7)FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC(Sequence ID 39); (8)FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC(Sequence ID 40); (9)FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC(Sequence ID 41); (10)FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC(Sequence ID 42); (11)FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC(Sequence ID 43); (12)FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC(Sequence ID 44); (13)FNMQCQRRFYEALHDPNLNEEQRNARIRSIRKDC(Sequence ID 45); (14)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSI(Sequence ID 97); (15)FNMQQQRRFYEALHDPNLNKEQRNARIRSIRDD(Sequence ID 98); and (16)β-Ala-NMQQQRRFYEALHDPNLEEQRNARIRSIKDD(Sequence ID 100); or (d) An amino acid sequence having 90% or more identity to any of the amino acid sequences (1) to (16) above, with mutations in 19 amino acid residues other than lysine residues at positions other than one lysine residue and two cysteine ​​residues (e.g., positions 1, 3, 6, 7, 13, 20, 24, 31, and 32) (this may also involve modifications of the number of amino acid residues as described above). Peptides containing such amino acid sequences have been confirmed to have the ability to bind to the Fc region of IgG. The affinity peptide having the above amino acid sequence in (d) is preferably a peptide that has the ability to bind to the constant region of a monoclonal antibody, more preferably a peptide that has the ability to bind to the Fc region of a monoclonal antibody, and even more preferably a peptide that has the ability to bind to the Fc region of IgG.

[0064] The affinity peptides described above may have further amino acid residue mutations in addition to the introduction of a single amino acid residue that can be easily modified by a crosslinking agent, as long as they have 85% or more identity with the amino acid sequences of SEQ ID NOs. Positions where further amino acid mutations can be introduced can be easily identified by those skilled in the art. For example, 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 asparagine residue at position 24, or the arginine residue at position 31 (excluding positions where an amino acid residue that can be easily modified by a crosslinking agent has already been introduced) can also be used. Examples of amino acids that can be introduced through further amino acid mutations include 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, any of these 19 amino acids other than lysine may be used. The amino acids may be either L-forms or D-forms, but L-forms are preferred (in the examples, all amino acid residues constituting the peptide are L-forms).

[0065] The degree of identity % with respect to the amino acid sequences of SEQ ID NOs. 11-14 or (1)-(16) can be determined as described above. The degree of identity % may preferably be 90% or more, 92% or more, more preferably 94% or more, even more preferably 95% or more, and particularly preferably 97% or more (i.e., having only one amino acid residue mutation).

[0066] In another specific embodiment, the affinity substance for the antibody described above is a peptide containing one of the following amino acid sequences. Formula 1-1:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IIWC-(X 0-3 ) b (Sequence ID 15) Formula 1-2:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IVWC-(X 0-3 ) b (Sequence ID 16) Formula 1-3:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-VVWC-(X 0-3 ) b (Sequence ID 17) Formula 1-4:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-AVWC-(X 0-3 ) b (Sequence No. 18) Formula 1-5:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-LLWC-(X 0-3 ) b (Sequence ID 19) Formula 1-6:(X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-LIWC-(X 0-3 )b (SEQ ID NO: 20) Formula 1-7: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-V-F-C-(X 0-3 ) b (SEQ ID NO: 21) 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) Formula 1-9: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-E-V-W-C-(X 0-3 )<​​​​​​​​​​​​​​​​​​​​​​​​​​Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue.], or Formula 2-1: (X 0-3 ’) a -C-(Xaa1’)-(Xaa2’)-(Xaa3’)-(Xaa4’)-(Xaa5’)-(Xaa6’)-L-V-W-C-(X 0-3 ’) b (SEQ ID NO: 24) 〔In the formula, (X 0-3 ’) a and (X 0-3 ’) b are each the same as the aforementioned (X 0-3 ) a and (X 0-3 ) b , and Xaa1’, Xaa2’, Xaa3’, Xaa4’, Xaa5’, Xaa6’ are each the same as the aforementioned Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6. 〕. Such a peptide has the ability to bind to the Fc region of a monoclonal antibody.

[0067] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, (X 0-3 ) a is none, an arginine residue-glycine residue-asparagine residue, a glycine residue-asparagine residue, an aspartic acid residue, or an asparagine residue, preferably none, an arginine residue-glycine residue-asparagine residue, an aspartic acid residue, or an asparagine residue.

[0068] In the amino acid sequences represented by the above formulas 1-1 to 1-9 and formula 2-1, Xaa2 is a tyrosine residue, a tryptophan residue, or a histidine residue, preferably a tyrosine residue, or a tryptophan residue.

[0069] In the amino acid sequences represented by formulas 1-1 to 1-9 and formula 2-1 above, Xaa3 is a histidine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, an arginine residue, or a glycine residue, and is preferably a histidine residue.

[0070] In the amino acid sequences represented by formulas 1-1 to 1-9 and formula 2-1 above, Xaa4 is a lysine residue, an aspartic acid residue, or a glutamic acid residue, and is preferably a lysine residue.

[0071] In the amino acid sequences represented by formulas 1-1 to 1-9 and formula 2-1 above, Xaa5 is a glycine residue, serine residue, asparagine residue, glutamine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, tyrosine residue, tryptophan residue, histidine residue, threonine residue, leucine residue, alanine residue, valine residue, isoleucine residue, or arginine residue, and is preferably a glycine residue, threonine residue, or leucine residue.

[0072] In the amino acid sequences represented by formulas 1-1 to 1-9 and formula 2-1 above, Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue, and more preferably a glutamine residue.

[0073] Preferably, a peptide containing any of the amino acid sequences represented by formulas 1-1 to 1-9 and formula 2-1 is a peptide containing an amino acid sequence selected from the group consisting of the following: (1') RGNCAYHKGQIIWCTYH(Sequence ID 46); (2') RGNCAYHKGQIVWCTYH(Sequence ID 47); (3') RGNCAYHKGQVVWCTYH(Sequence ID 48); (4') RGNCAYHKGQAVWCTYH(Sequence ID 49); (5') RGNCAYHKGQLLWCTYH(Sequence ID 50); (6') RGNCAYHKGQLIWCTYH(Sequence ID 51); (7') DCAYHKGQIVWCT (Sequence ID 52); (8') DCAYHKGQVVWCT (Sequence ID 53); (9') DCAYHKGQAVWCT (Sequence ID 54); (10')RGNCAYHKSQIIWCTYH(Sequence ID 55); (11')RGNCAYHKNQIIWCTYH(Sequence ID 56); (12')RGNCAYHKDQIIWCTYH(Sequence ID 57); (13')RGNCAYHKQQIIWCTYH(Sequence ID 58); (14')RGNCAYHKEQIIWCTYH(Sequence ID 59); (15')RGNCAYHKFQIIWCTYH(Sequence ID 60); (16')RGNCAYHKYQIIWCTYH(Sequence ID 61); (17')RGNCAYHKWQIIWCTYH(Sequence ID 62); (18')RGNCAYHKHQIIWCTYH(Sequence ID 63); (19')RGNCAYHKTQIIWCTYH(Sequence ID 64); (20')RGNCAYHKLQIIWCTYH(Sequence ID 65); (21') CAYHKLQIVWC (Sequence ID 66); (22') CAYHKLQLIWC (Sequence ID 67); (23') CAYHKSQIVWC (Sequence ID 68); (24')RGNCAYHKGQLVFCTYH(Sequence ID 69); (25')RGNCAYHKGQQVWCTYH(Sequence ID 70); (26')RGNCAYHKGQEVWCTYH(Sequence ID 71); (27') CAYHKGQLVWC (Sequence ID 72); (28')RGNCAYHKAQLVWCTYH(Sequence ID 73); (29')RGNCAYHKVQLVWCTYH(Sequence ID 74); (30')RGNCAYHKLQLVWCTYH(Sequence ID 75); (31')RGNCAYHKIQLVWCTYH(Sequence ID 76); (32')RGNCAYHKSQLVWCTYH(array_77); (33')RGNCAYHKTQLVWCTYH(Sequence ID 78); (34')RGNCAYHKNQLVWCTYH(Sequence ID 79); (35')RGNCAYHKDQLVWCTYH(Sequence ID 80); (36')RGNCAYHKQQLVWCTYH(Sequence ID 81); (37')RGNCAYHKEQLVWCTYH(Sequence ID 82); (38')RGNCAYHKFQLVWCTYH(Sequence ID 83); (39')RGNCAYHKRQLVWCTYH(Sequence ID 84); (40')RGNCAYHKHQLVWCTYH(Sequence ID 85); (41')RGNCAYHKWQLVWCTYH(Sequence ID 86); (42')RGNCAYHKYQLVWCTYH(Sequence ID 87); (43')RGNCAYFKGQLVWCTYH(Sequence ID 88); (44')RGNCAYYKGQLVWCTYH(Sequence ID 89); (45')RGNCAYWKGQLVWCTYH(Sequence ID 90); (46')RGNCAYRKGQLVWCTYH(Sequence ID 91); (47')RGNCAYGKGQLVWCTYH(Sequence ID 92); (48') DCAYHKGQLVWC (Sequence ID 93); (49') NCAYHKGQLVWC (Sequence ID 94); (50') CAYHKGQLVWCT (Sequence ID 95); (51') CAYHKSQLVWC (array code 96); (52') GNCAYHKGQIIWCTYH(sequence number 99); and (53')RGNCAYHEGQIIWCTYH(Sequence ID 108). Peptides containing such amino acid sequences have been confirmed to have the ability to bind to the Fc region of IgG.

[0074] At least two spaced cysteine ​​residues in each amino acid sequence of the above peptide can form a cyclic peptide via disulfide bonds. Alternatively, in the above peptide, the sulfide groups in the two cysteine ​​residues may be linked by a carbonyl group-containing linker represented below.

[0075] [ka]

[0076] The dashed lines in the carbonyl group-containing linker shown above represent the bonding portion with the sulfide group. This linker is more stable against reduction reactions than ordinary disulfide bonds. Such peptides can be prepared, for example, by the method described in International Publication No. 2016 / 186206.

[0077] Compounds of the present invention comprising a peptide containing the above amino acid sequence are useful for regioselective modification of specific amino acid residues in human IgG Fc (e.g., Lys248 or Lys246, Lys288 or Lys290, Lys317, or other amino acid residues following Eu numbering). The amino acids constituting the peptide may be either L-forms or D-forms, but L-forms are preferred (in the examples, all amino acid residues constituting the peptide are L-forms).

[0078] The above peptide may be modified by a crosslinking agent to modify specific amino acid residues. Examples of such specific amino acid residues include lysine residues, aspartic acid residues, and glutamic acid residues, but lysine residues are preferred. Examples of crosslinking agents include crosslinking agents that preferably contain two or more succinimidyl groups, such as DSG (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate); crosslinking agents that preferably contain two or more imido acid portions, such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2HCl), and DMS (dimethyl suberimidate·2HCl); and crosslinking agents having SS bonds, such as DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., International Publication No. 2016 / 186206).

[0079] When the affinity substance for an antibody is a peptide, the amino and carboxyl groups at the ends of the peptide may be protected. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., acetyl groups, propoxy groups, tert-butoxycarbonyl groups, etc.), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl group), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl group). The acetyl group is preferred as the protecting group for the N-terminal amino group. Examples of protecting groups for the C-terminal carboxyl group include groups capable of forming esters or amides. Examples of groups capable of forming esters or amides include alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups. An amino group is preferred as the protecting group for the C-terminal carboxyl group.

[0080] 1-3. Divalent groups (L) containing leaving groups In formula (I), L is a divalent group containing a leaving group.

[0081] A leaving group is a group in an antibody that has the ability to be cleaved and eliminated from a divalent group (E) containing an electrophile through a reaction between the nucleophile in the antibody and the electrophile contained in the electrophile. Such leaving groups are common technical knowledge in this field (e.g., Fujishima, S. et al J.Am.Chem.Soc, 2012, 134, 3961-3964 (mentioned above); Chem.Sci. 2015 3217-3224; Nature Chemistry volume 8, pages 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 described above, but examples include (1) a group selected from the group consisting of -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), and (2) heteroarylenes.

[0082] Examples of alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, and hexyl. Alkyl groups having 1 to 4 carbon atoms are preferred over alkyl groups having 1 to 6 carbon atoms.

[0083] The group selected from the group consisting of -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, and -ON(R)- is either (1) or (2) below: (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 (2) Groups containing -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- and groups that enhance the ability to leave the group.

[0084] A group that enhances 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 is a group that is adjacent to -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)-, and the group is -O -, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- are groups that enhance 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 the electrophile, compared to when they are not present adjacent to -, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)-. In other words, groups that enhance 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 are groups that have the ability to attract electrons from oxygen, sulfur, selenium, or nitrogen atoms.

[0085] Preferred examples of groups that enhance the ability to eliminate -O-, -S-, -Se-, -SO2-O-, -SO2-N(R)-, -SO2-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- include arylenes which may be substituted with electron-withdrawing groups, heteroarylenes which may be substituted with electron-withdrawing groups, 2,5-diketopyrrolidines which may be fused, 2,6-diketopiperidines which may be fused, 2-ketopyrrolidines which may be fused, 2-ketopiperidines which may be fused, and 2-pyridones. The number of electron-withdrawing groups that arylenes and heteroarylenes may have is one or more (e.g., 1 to 3, preferably 1 or 2). Examples of electron-withdrawing groups include halogen atoms, alkyl groups substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl, and keto groups (e.g., acyl).

[0086] In "arylene which may be substituted with an electron-withdrawing group," the "arylene" is preferably an arylene having 6 to 24 carbon atoms, more preferably an arylene having 6 to 18 carbon atoms, even more preferably an arylene having 6 to 14 carbon atoms, and most preferably an arylene having 6 to 10 carbon atoms. The arylene which may be substituted with an electron-withdrawing group may also be substituted with substituents other than the electron-withdrawing group, or it may not be substituted. The number of carbon atoms mentioned above does not include the number of carbon atoms of the electron-withdrawing group and other substituents. Examples of arylenes include phenylene, naphthylene, and anthracenylene.

[0087] In "heteroarylenes which may be substituted with electron-withdrawing groups," the "heteroarylenes" are preferably heteroarylenes having 1 to 21 carbon atoms, more preferably heteroarylenes having 1 to 15 carbon atoms, even more preferably heteroarylenes having 1 to 9 carbon atoms, and most preferably heteroarylenes having 1 to 6 carbon atoms. Heteroarylenes which may be substituted with electron-withdrawing groups may also be substituted with substituents other than electron-withdrawing groups, or they may not be substituted. The number of carbon atoms mentioned above does not include the number of carbon atoms of electron-withdrawing groups and other substituents. Heteroarylenes contain one or more heteroatoms (for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3) selected from the group consisting of nitrogen atoms, oxygen atoms, and sulfur atoms as ring constituent atoms. Examples of heteroarylenes include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolindiyl, and phthalazinediyl.

[0088] 2,5-diketopyrrolidine, 2,6-diketopyrrolidine, 2-ketopyrrolidine, 2-ketopyrrolidine, and 2-pyridone may be fused or unsubstituted with substituents such as electron-withdrawing groups.

[0089] An example of a leaving group heteroarylene is a heteroarylene with a low π electron density (i.e., less than 1). Preferred leaving group heteroarylenes include those containing a nitrogen atom as a ring element. Specifically, heteroarylenes with 1 to 21 carbon atoms containing a nitrogen atom are preferred, those with 1 to 15 carbon atoms containing a nitrogen atom are more preferred, and those with 1 to 9 carbon atoms containing a nitrogen atom are even more preferred. The leaving group heteroarylene may or may not be substituted with substituents such as electron-withdrawing groups. The carbon atom count does not include the carbon atoms of substituents. Examples of leaving group heteroarylenes containing a nitrogen atom as a ring element include imidazole diyl, triazole diyl, tetrazole diyl, and 2-pyridone diyl (i.e., 2-hydroxypyridine diyl).

[0090] Groups such as "arylenes that may be substituted with electron-withdrawing groups," which are 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)-, as well as "heteroarylenes," which are examples of leaving groups, may have, for example, 1 to 5 substituents, preferably 1 to 3, more preferably 1 or 2 substituents, or they may not have substituents. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that they do not have such substituents. On the other hand, if such groups have substituents, examples of such substituents include the following: (i) halogen atom; (ii) Monovalent hydrocarbon group; (iii) Aralkir; (iv) Monovalent heterocyclic group; (v)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or a monovalent hydrocarbon group. ); or (vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents a hydrogen atom or a monovalent hydrocarbon group, either identical or distinct. (vii) Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

[0091] Examples of halogen atoms include fluorine, chlorine, bromine, and iodine.

[0092] Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0093] A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure, and whose main chain does not contain a cyclic structure. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched.

[0094] As alkyl groups, alkyl groups having 1 to 12 carbon atoms are preferred, alkyl groups having 1 to 6 carbon atoms are more preferred, and alkyl groups having 1 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkyl groups having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0095] As the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0096] As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0097] Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0098] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.

[0099] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0100] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0101] As for cycloalkynyls, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0102] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0103] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are 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 number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0104] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0105] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups, with alkyl being more preferred.

[0106] An aralkyl refers to an arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyls are as described above. Preferred aralkyls have 3 to 15 carbon atoms. Examples of such aralkyls include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0107] A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. A monovalent heterocyclic group is either a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heterocyclic group preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0108] As monovalent aromatic heterocyclic groups, those having 1 to 15 carbon atoms are preferred, those having 1 to 9 carbon atoms are more preferred, and those having 1 to 6 carbon atoms are even more preferred. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazonal, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0109] As monovalent non-aromatic heterocyclic groups, non-aromatic heterocyclic groups having 2 to 15 carbon atoms are preferred, non-aromatic heterocyclic groups having 2 to 9 carbon atoms are more preferred, and non-aromatic heterocyclic groups having 2 to 6 carbon atoms are even more preferred. The number of carbon atoms in the above carbon atoms does not include the number of carbon atoms of substituents. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinil, azetidinil, oxetanil, thietanil, pyrrolidinil, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinil, oxazolidinil, piperidinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinil, dihydrooxazinil, tetrahydrooxazinil, dihydropyrimidinil, and tetrahydropyrimidinil.

[0110] Among these, a 5-membered or 6-membered heterocyclic group is preferred as the monovalent heterocyclic group.

[0111] Preferably, the substituents may be: (i') Halogen atom; (ii') Alkyl, phenyl, or naphthyl atoms having 1 to 12 carbon atoms; (iii') Aralkyl groups with 3 to 15 carbon atoms; (iv') A complex ring with 5 or 6 members; (v')R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 12 carbon atoms. (vi')NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R cThis represents an identical or distinct hydrogen atom, or an alkyl group having 1 to 12 carbon atoms. (vii') The same base as those listed in (vii) above.

[0112] More preferably, the substituents may be: (i'') halogen atom; (ii'') Alkyl atoms with 1 to 12 carbon atoms; (iii'')R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 12 carbon atoms. (iv'')NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents an identical or distinct hydrogen atom, or an alkyl group having 1 to 12 carbon atoms. (v'') The same base as those listed in (vii) above.

[0113] More preferably, the substituents may be: (i''') halogen atom; (ii''') Alkyl atoms with 1 to 6 carbon atoms; (iii''')R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. (iv''')NR b R c -, NR b Rc -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. (v''') The same base as those listed in (vii) above.

[0114] Particularly preferred, the substituents may be: (i'''') halogen atom; (ii'''') Alkyl atoms with 1 to 4 carbon atoms; (iii'''')R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 4 carbon atoms. (iv'''')NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. (v'''') The same base as those listed in (vii) above.

[0115] More specifically, the leaving group can be any of the following (a) to (c). (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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).]

[0116] Of (a), (b), and (c), (a) or (b) is preferred as the leaving group, and (a) is particularly preferred. The ring P in (a) is any of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopiperidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopiperidine which may be fused, or 2-pyridone. Of these, arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine, and 2,6-diketopiperidine are more preferred, and arylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine, and 2,6-diketopiperidine are even more preferred.

[0117] The ring P in (a) is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, fusible 2,5-diketopyrrolidine, fusible 2,6-diketopiperidine, fusible 2-ketopyrrolidine, fusible 2-ketopiperidine, and 2-pyridone. Details of these groups are the same as those described as preferred examples of groups that enhance the ability to eliminate -N(R)-, -N(OR)-, -O-, -S-, or -Se-.

[0118] In (a), Q is a group selected from the group consisting of -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). Of these, -O-, -S-, -SO2-O-, or -SO2-N(R)- are preferred, -O- or -S- are more preferred, and -O- is even more preferred.

[0119] (b) is a heteroarylene, but imidazole diyl, triazole diyl, tetrazole diyl, or 2-pyridone diyl (=2-hydroxypyridine diyl) is preferred, and imidazole diyl or 2-pyridone diyl is more preferred.

[0120] In (C), Q is a group selected from the group consisting of -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), but -S-, -C≡C-CH2-O-, -N(OR)-, -N(R)-, or -ON(R)- are preferred, and S, -N(OR), or -ON(R)- are more preferred.

[0121] More specifically, the preferred leaving group may be a group selected from the group consisting of the following structural formulas. [ka] (Here, EWG is an electron-withdrawing group, m is an integer between 0 and 4. n is an integer between 0 and 3. R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ○ (white circle) represents a connection to L1, and ● (black circle) represents a connection to E1. Details of the electron-withdrawing group and the alkyl group having 1 to 6 carbon atoms are as described above. m is preferably an integer from 1 to 4, more preferably 1, 2, or 3. n is preferably an integer from 1 to 3, more preferably 1 or 2.

[0122] A divalent group containing a leaving group, represented by L, is a divalent group consisting of the above leaving group, or a divalent group containing another divalent group in addition to the above leaving group. Examples of such other divalent groups include a divalent hydrocarbon group, a divalent heterocyclic group, -C(=O)-, and -NR. L -(R L Examples include groups consisting of a hydrogen atom (wherein represents the substituents described above), -O-, -S-, -C(=S)-, and combinations of two or more of these (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4). Divalent hydrocarbon groups and divalent heterocyclic groups may have, for example, 1 to 5 substituents, preferably 1 to 3, more preferably 1 or 2 substituents, or they may not. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that such substituents are absent. On the other hand, when such groups have substituents, examples and preferred examples of such substituents are the same as described above.

[0123] The divalent hydrocarbon group can be a linear, branched, or cyclic divalent hydrocarbon group, preferably a linear or branched divalent hydrocarbon group. Examples of divalent hydrocarbon groups include alkylene, alkenylene, alkylene, and arylene.

[0124] The alkylene is preferably one having 1 to 12 carbon atoms, more preferably one having 1 to 6 carbon atoms, and particularly preferably one having 1 to 4 carbon atoms. The number of carbon atoms does not include the number of substituent carbon atoms. The alkylene may be linear, branched, or cyclic, but linear alkylene is preferred. Examples of such alkylenes include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0125] The alkenylene is preferably one having 2 to 12 carbon atoms, more preferably one having 2 to 6 carbon atoms, and particularly preferably one having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of substituent carbon atoms. The alkenylene may be linear, branched, or cyclic, but linear alkenylene is preferred. Examples of such alkenylenes include ethyleneylene, propynylene, butenylene, pentenylene, and hexenylene.

[0126] The alkynylene is preferably one having 2 to 12 carbon atoms, more preferably one having 2 to 6 carbon atoms, and particularly preferably one having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of substituent carbon atoms. The alkynylene may be linear, branched, or cyclic, but linear alkynylene is preferred. Examples of such alkynylenes include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

[0127] As for the arylene, arylenes having 6 to 24 carbon atoms are preferred, arylenes having 6 to 18 carbon atoms are more preferred, arylenes having 6 to 14 carbon atoms are even more preferred, and arylenes having 6 to 10 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of arylenes include phenylene, naphthylene, and anthracenylene.

[0128] A divalent heterocyclic group is either a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0129] 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, even more preferably a divalent aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably a divalent aromatic heterocyclic group having 1 to 6 carbon atoms. The number of carbon atoms in the above figures does not include the number of carbon atoms of substituents. More specifically, examples of divalent aromatic heterocyclic groups include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl.

[0130] As for the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 21 carbon atoms is preferred, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is more preferred, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is even more preferred, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is even more preferred. The number of carbon atoms in the above figures does not include the number of carbon atoms of substituents. More specifically, examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, pyrrolinediyl, oxylandiyl, aziridindiyl, azetidinediyl, oxetanediyl, thietandiyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0131] In certain embodiments, L can be represented as L1-L2.

[0132] L1 is a bond or a divalent group. The definition, examples, and preferred examples of a divalent group represented by L1 are the same as those of other divalent groups, where a divalent group containing a leaving group represented by L is a divalent group containing another divalent group in addition to the leaving group.

[0133] L2 is a leaving group. The definition, examples, and preferred examples of a leaving group represented by L2 are the same as those for a leaving group in L.

[0134] Preferably, the leaving group represented by L2 is one of (a) to (c) above. Examples and preferred examples of the leaving group represented by L2 are also the same as those described in (a) to (c) above.

[0135] The length of the main chain of L (a divalent group containing a leaving group) or L1 (a binding or divalent group)-L2 (a leaving group) linking A (an affinity substance) and E (a divalent group containing an electrophile) can be appropriately designed depending on various factors such as the type of antibody and affinity substance, and the relationship between the target site of the affinity substance in the antibody and the specific amino acid residue in the antibody that should be regioselectively modified. The L or L1-L2 main chain refers to a chain-like structure consisting of multiple atoms linked by covalent bonds that connect A and E, excluding hydrogen atoms, branched structures, and substituents. When the compound represented by formula (I) is brought into contact with an antibody, A first associates with the antibody. Then, a nucleophile in the side chain of the specific amino acid residue to be modified in the antibody, located near the antibody association site (e.g., an amino group in the side chain of a lysine residue), reacts with the electrophile in E, causing the nucleophile to bind to the electrophile, while the leaving group contained in L or L1 can leave E. In this case, if there are no other amino acid residues of the same type as the specific amino acid residue to be modified in the vicinity between the antibody association site and the specific amino acid residue to be modified, the electrophile at E can regioselectively bind to the nucleophile in the side chain of the specific amino acid residue to be modified in the antibody, even without strictly controlling the length of the main chain. Of course, even if other amino acid residues of the same type as the specific amino acid residue exist in such a region, the electrophile at E can regioselectively bind to the specific amino acid residue by controlling the length of the main chain.

[0136] The length of the L or L1-L2 main chain linking A and E is not particularly limited as long as it can regioselectively modify specific amino acid residues in the antibody. However, when regioselectively modifying specific amino acid residues in human IgG Fc (e.g., Lys248 or Lys246, Lys288 or Lys290, Lys317, or other amino acid residues according to Eu numbering), it is preferable that the length consists of 20 atoms or less. The length of the L or L1-L2 main chain is also 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 L or L1-L2 main chain is also 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 L or L1-L2 is 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 L or L1-L2 is preferably 2 to 30, even more preferably 3 to 20, and particularly preferably 4 to 15 or 5 to 10.

[0137] If the main chain does not have a 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 structural portions, and atoms in substituents).

[0138] On the other hand, if the main chain has a structure that includes a ring structure, the number of atoms in the main chain can be conveniently counted from the viewpoint of defining the length of the main chain. Specifically, in such cases, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure that does not include a divalent ring structure (excluding hydrogen atoms, branched structural portions, and atoms in substituents), in addition to the number of atoms in the shortest path connecting two bonds in the ring structure (see, for example, the bolded paths in (a) to (d) below). [ka] • is a coupling. In case (a), the shortest path is the bold path, so the number of atoms in the divalent ring structure that can be counted as the number of atoms in the main chain is 2. In case (b), the shortest path is the bolded path, so the number of atoms in the divalent ring structure that can be counted as the number of atoms in the main chain is 3. In case (c), since both paths are the shortest paths (equidistances), the number of atoms in the divalent ring structure that can be counted as atoms in the main chain is 4. In case (d), since the condensation site is the shortest path, the number of atoms in the divalent ring structure that can be counted as the number of atoms in the main chain is 4.

[0139] Preferably, the main chain of L or L1-L2 may be a chain-like structure in which the "other divalent group" in L or the "divalent group" in L1 does not contain a divalent ring structure. Therefore, the "other divalent group" in L or the "divalent group" in L1 may be a divalent straight-chain or branched-chain hydrocarbon group, -C(=O)-, -NR L -(R L The group may consist of a hydrogen atom (wherein represents the substituents described above), -O-, -S-, -C(=S)-, and combinations of two or more of these (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4). The divalent linear or branched hydrocarbon group may have, for example, 1 to 5 substituents, preferably 1 to 3, more preferably 1 or 2 substituents, or it may not have any substituents. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that such substituents are absent. On the other hand, if such a group has substituents, examples and preferred examples of such substituents are the same as the substituents that heteroarylene, which is an example of a leaving group, may have.

[0140] 1-4. Divalent groups containing electrophiles (E) In formula (I), L is a divalent group that (i) is linked to a leaving group and (ii) contains an electrophile capable of reacting with a nucleophile in the antibody.

[0141] Examples of nucleophiles in antibodies include NH2 in the side chain of lysine residues, OH in the side chain of tyrosine residues, OH in the side chain of serine residues, OH in the side chain of threonine residues, and SH in the side chain of cysteine ​​residues. Preferably, the nucleophile in the antibody is NH2 in the side chain of lysine residues or OH in the side chain of tyrosine residues, with NH2 in the side chain of lysine residues being more preferred.

[0142] Any electrophile that can be linked to a leaving group and has the ability to react with the nucleophile in the antibody as described above can be used as the electrophile in E, but a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2- is preferred. As an electrophile, -CH2- can also be used depending on the electronic balance with its adjacent groups (e.g., leaving group, L2, E2). For example, when a tosyl group is used as the leaving group, -CH2- can be suitably used as the electrophile (Tsukiji et al., Nature Chemical Biology, Vol.5, No.5, May 2009). As an electrophile, -C(=O)- or -SO2- is more preferred, and -C(=O)- is even more preferred.

[0143] A divalent group containing an electrophile, represented by E, is a divalent group consisting of the above-mentioned electrophile, or a divalent group containing the above-mentioned electrophile plus other divalent groups. Examples of such other divalent groups include divalent hydrocarbon groups, divalent heterocyclic groups, -C(=O)-, and -NR. E -(R EExamples of groups include those consisting of a hydrogen atom (wherein represents the substituents described above), -O-, -S-, -C(=S)-, and combinations of two or more of these (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4). Divalent hydrocarbon groups and divalent heterocyclic groups may have, for example, 1 to 5 substituents, preferably 1 to 3, more preferably 1 or 2 substituents, or they may not. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that they do not have such substituents. On the other hand, if such groups have substituents, examples and preferred examples of such substituents are the same as the substituents that heteroarylene, which is an example of a leaving group, may have. E (and E1-E2-E3 described later) can be designed so as not to contain peptide moieties that have potential immunogenicity and are easily hydrolyzed in the blood.

[0144] The divalent hydrocarbon group can be a linear, branched, or cyclic divalent hydrocarbon group, preferably a linear or branched divalent hydrocarbon group. Examples of divalent hydrocarbon groups include alkylene, alkenylene, alkylene, and arylene.

[0145] The alkylene is preferably one having 1 to 12 carbon atoms, more preferably one having 1 to 6 carbon atoms, and particularly preferably one having 1 to 4 carbon atoms. The number of carbon atoms does not include the number of substituent carbon atoms. The alkylene may be linear, branched, or cyclic, but linear alkylene is preferred. Examples of such alkylenes include methylene, ethylene, propylene, butylene, pentylene, and hexylene.

[0146] The alkenylene is preferably one having 2 to 12 carbon atoms, more preferably one having 2 to 6 carbon atoms, and particularly preferably one having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of substituent carbon atoms. The alkenylene may be linear, branched, or cyclic, but linear alkenylene is preferred. Examples of such alkenylenes include ethyleneylene, propynylene, butenylene, pentenylene, and hexenylene.

[0147] The alkynylene is preferably one having 2 to 12 carbon atoms, more preferably one having 2 to 6 carbon atoms, and particularly preferably one having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of substituent carbon atoms. The alkynylene may be linear, branched, or cyclic, but linear alkynylene is preferred. Examples of such alkynylenes include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

[0148] As for the arylene, arylenes having 6 to 24 carbon atoms are preferred, arylenes having 6 to 18 carbon atoms are more preferred, arylenes having 6 to 14 carbon atoms are even more preferred, and arylenes having 6 to 10 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of arylenes include phenylene, naphthylene, and anthracenylene.

[0149] A divalent heterocyclic group is either a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0150] 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, even more preferably a divalent aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably a divalent aromatic heterocyclic group having 1 to 6 carbon atoms. The number of carbon atoms in the above figures does not include the number of carbon atoms of substituents. More specifically, examples of divalent aromatic heterocyclic groups include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl.

[0151] As for the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 21 carbon atoms is preferred, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is more preferred, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is even more preferred, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is even more preferred. The number of carbon atoms in the above figures does not include the number of carbon atoms of substituents. More specifically, examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, pyrrolinediyl, oxylandiyl, aziridindiyl, azetidinediyl, oxetanediyl, thietandiyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0152] In certain embodiments, E can be represented as E1-E2-E3.

[0153] E1 is an electrophile that (i) is linked to the leaving group and (ii) has the ability to react with the nucleophile in the antibody. The definition, examples, and preferred examples of the electrophile of E1 are the same as those of the electrophile in E.

[0154] E2 is either (a) or (b): (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)]; or (b) The following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which the ring constituent atom X' bonded to E1 and all of the ring constituent atoms on either side of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on either side of the nitrogen atom are carbon atoms. · represents a bond.)

[0155] If E2 is as described in (a) above, then the alkyl groups with 1 to 6 carbon atoms in R1 to R5 are the same as those described above.

[0156] When E2 is as described in (a) above, the X that bonds with E1 is preferably C(R1)(R2), N(R3), O, S, or Se, more preferably C(R1)(R2), N(R3), O, or S, even more preferably C(R1)(R2), N(R3), or O, still even more preferably C(R1)(R2), or N(R3), and most preferably C(R1)(R2).

[0157] In E2, X can also be defined in relation to the leaving group (e.g., see L, L2). When X is an atom or group such as N(R3), O, S, or Se, not only the leaving group but also X can leave the electrophile. However, when a predetermined amount of the compound represented by formula (I) is used in a reaction with an antibody, the reaction can be controlled so that X in at least some of the compounds represented by formula (I) does not leave the electrophile. Therefore, in the present invention, the atoms or groups described above can be used as X. Preferably, from the viewpoint of improving the efficiency of the target reaction between the compound represented by formula (I) and the antibody, and consequently improving the yield of antibodies having bioorthothic functional groups, an atom or group that is less likely to leave than the leaving group (i.e., an atom or group whose pKa value is greater than the pKa value of the leaving group) can be used as X. Therefore, from the viewpoint of improving the efficiency of the target reaction between the compound represented by formula (I) and the antibody, and consequently improving the yield of antibodies having bioorthogonal functional groups, it is preferable that X in E2 has a leaving ability less than or equal to that of the leaving group, by more selectively removing the leaving group and suppressing the removal of X in E2. Such X may vary depending on the type of leaving group (e.g., -N(R)-, -N(OR)-, -O-, -S-, or -Se-, or heteroarylene), as well as the presence and type of a group containing a group that enhances the leaving ability of the leaving group adjacent to the leaving group, but in general terms, it is as follows. (1) When the leaving group is -N(R)- or -N(OR)-, X is preferably C(R1)(R2) or N(R3), and more preferably C(R1)(R2). (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 even more preferably C(R1)(R2). (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, even more preferably C(R1)(R2), or N(R3), and still even more preferably C(R1)(R2). (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, even more preferably C(R1)(R2), N(R3), or O, still even more preferably C(R1)(R2), or N(R3), and most preferably C(R1)(R2). (5) When the leaving group is a heteroarylene, X is preferably C(R1)(R2), N(R3), O, S, or Se, more preferably C(R1)(R2), N(R3), O, or S, even more preferably C(R1)(R2), N(R3), or O, still even more preferably C(R1)(R2), or N(R3), and most preferably C(R1)(R2).

[0158] If E2 is as described in (b) above, the ring-forming atom X' that is bonded to E1 in ring Z is either a carbon atom or a nitrogen atom.

[0159] If the ring-forming atom X' bonded to E1 is a carbon atom, then ring Z is a divalent ring group in which the ring-forming atom X' bonded to E1 and all of the ring-forming atoms on either side of it are carbon atoms. The divalent ring group may have, for example, 1 to 5 substituents, preferably 1 to 3, and more preferably 1 or 2 substituents, or it may not have any substituents. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that it does not have such substituents. On the other hand, if the divalent ring group has substituents, examples and preferred examples of such substituents are the same as the substituents that heteroarylenes, which are examples of leaving groups, may have. Examples of such divalent ring groups include cyclic divalent hydrocarbon groups (e.g., arylenes, cyclic alkylenes, cyclic alkenylenes, cyclic alkynylenes) and divalent heterocyclic groups (e.g., divalent aromatic heterocyclic groups, divalent non-aromatic heterocyclic groups).

[0160] Examples of divalent hydrocarbon groups include cyclic divalent hydrocarbon groups, such as cyclic alkylenes, cyclic alkenylenes, cyclic alkynylenes, and arylenes.

[0161] As for the cyclic alkylene, alkylenes having 3 to 12 carbon atoms are preferred, alkylenes having 3 to 10 carbon atoms are more preferred, and alkylenes having 5 to 8 carbon atoms are particularly preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of such alkylenes include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, and cyclodextrine.

[0162] As for the cyclic alkenylene, alkenylenes having 3 to 12 carbon atoms are preferred, alkenylenes having 3 to 10 carbon atoms are more preferred, and alkenylenes having 5 to 8 carbon atoms are particularly preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of such alkenylenes include cyclopropenylene, cyclobutenylene, cyclopentenylene, cyclohexenylene, cycloheptenylene, cyclooctenylene, cyclononenylene, and cyclodekenylene.

[0163] As for cyclic alkynylenes, those having 6 to 12 carbon atoms are preferred, those having 7 to 12 carbon atoms are more preferred, and those having 8 to 12 carbon atoms are particularly preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. The alkynylene may be linear, branched, or cyclic, but linear alkynylenes are preferred. Examples of such alkynylenes include cyclohexynylene, cycloheptynylene, cyclooctynylene, cyclononynylene, cyclodexynylene, cycloundexynylene, and cyclododexynylene.

[0164] As for the arylene, arylenes having 6 to 24 carbon atoms are preferred, arylenes having 6 to 18 carbon atoms are more preferred, arylenes having 6 to 14 carbon atoms are even more preferred, and arylenes having 6 to 10 carbon atoms are even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. Examples of arylenes include phenylene, naphthylene, and anthracenylene.

[0165] A divalent heterocyclic group is either a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0166] The divalent aromatic heterocyclic groups 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. The carbon atoms of substituents are not included in the above carbon atom count. More specifically, examples of divalent aromatic heterocyclic groups include pyrrole diyl, franziyl, thiophene diyl, pyridine diyl, pyridazine diyl, pyrimidine diyl, pyrazole diyl, isothiazole diyl, isoxazole diyl, indole diyl, anthraquinone diyl, carbazole diyl, fluorangeyl, quinoline diyl, isoquinoline diyl, quinazoline diyl, and phthalazine diyl.

[0167] As for the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 21 carbon atoms is preferred, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is more preferred, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is even more preferred, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is even more preferred. The number of carbon atoms in the above carbon atoms does not include the number of carbon atoms of substituents. More specifically, examples of divalent non-aromatic heterocyclic groups include pyrrolinedionediyl, pyrrolinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, tetrahydrothiophenediyl, pyrrolinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0168] When the ring-forming atom X' bonded to E1 is a nitrogen atom, ring Z is a divalent heterocyclic group in which the ring-forming atom X' bonded to E1 is a nitrogen atom, and the ring-forming atoms on either side of the nitrogen atom are carbon atoms. Such a divalent heterocyclic group is a divalent heterocyclic group that contains a nitrogen atom as a ring-forming atom. As a divalent heterocyclic group containing a nitrogen atom as a ring-forming atom, a divalent heterocyclic group having 3 to 21 carbon atoms is preferred, a divalent heterocyclic group having 3 to 15 carbon atoms is more preferred, a divalent heterocyclic group having 3 to 9 carbon atoms is even more preferred, and a divalent heterocyclic group having 3 to 6 carbon atoms is even more preferred. The divalent heterocyclic group may have, for example, 1 to 5 substituents, preferably 1 to 3, more preferably 1 or 2 substituents, or it may not have any substituents. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that it does not have such substituents. On the other hand, when a divalent heterocyclic group has substituents, examples and preferred examples of such substituents are the same as substituents that may be present on heteroarylenes, which are examples of leaving groups. The carbon atom count above does not include the carbon atoms of substituents. Examples of divalent heterocyclic groups containing a nitrogen atom as a ring constituent include divalent aromatic heterocyclic groups containing a nitrogen atom as a ring constituent and divalent non-aromatic heterocyclic groups containing a nitrogen atom as a ring constituent. Examples of divalent aromatic heterocyclic groups containing a nitrogen atom as a ring constituent include pyrrole diyl, imidazole diyl, indole diyl, purine diyl, and carbazole diyl. Examples of divalent non-aromatic heterocyclic groups containing a nitrogen atom as a ring element include pyrroledionediyl, pyrrolinedionediyl, pyrrolinediyl, aziridinediyl, azetidinediyl, pyrrolidinediyl, pyrrolinediyl, imidazolidinediyl, piperidinediyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0169] Preferably, (b) the group represented by formula (i) above is (b') the following formula (i'): [ka] (Here, ring Z is a divalent ring group in which all of the ring constituent atoms bonded to E1 and the ring constituent atoms on both sides are carbon atoms. · represents a bond.) The definition, examples, and preferred examples of a divalent ring group for ring Z in the group represented by formula (i') above are the same as those for a divalent ring group for ring Z in the group represented by formula (i) above.

[0170] E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). The divalent group represented by E3 is the same as any other divalent group when the divalent group containing the electrophile represented by E contains an electrophile in addition to another divalent group.

[0171] The length of the main chain of E (a divalent group containing an electrophile) or E1 (electrophile)-E2 (above (a) or (b))-E3 (a bond or divalent group) linking L (a divalent group containing a leaving group) and B (a bioorthogonal functional group) cannot be involved in the regioselective modification of specific amino acid residues in the antibody during the reaction between the compound represented by formula (I) and the antibody. Rather, it can be involved in the distance between the antibody and the bioorthogonal functional group in the antibody produced after the reaction. The main chain of E or E1-E2-E3 refers to a chain-like structure consisting of multiple atoms linked by covalent bonds that link L and B, excluding hydrogen atoms, branched structures, and substituents. Therefore, the length of the main chain of E or E1-E2-E3 can be appropriately designed from the viewpoint of adjusting the said distance.

[0172] The length of the E or E1-E2-E3 main chain connecting L and B is not particularly limited, but may be a length consisting of three or more atoms.

[0173] If the main chain does not have a 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 structural portions, and atoms in substituents).

[0174] On the other hand, if the main chain has a structure that includes a ring structure, the number of atoms in the main chain can be conveniently counted from the viewpoint of defining the length of the main chain. Specifically, in such cases, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure that does not include a divalent ring structure (excluding hydrogen atoms, branched structural portions, and atoms in substituents), in addition to the number of atoms in the shortest path connecting two bonds in the ring structure, as described above.

[0175] Preferably, the main chain of E or E1-E2-E3 may be a chain-like structure in which the "other divalent group" in E or the "divalent group" in E3 does not contain a divalent ring structure. Therefore, the "other divalent group" in E or the "divalent group" in E3 may be a divalent linear or branched hydrocarbon group, -C(=O)-, -NR E -(R E The group may consist of a hydrogen atom (wherein represents the substituents described above), -O-, -S-, -C(=S)-, and combinations of two or more of these (e.g., 2 to 8, preferably 2 to 6, more preferably 2 to 4). The divalent linear or branched hydrocarbon group may have, for example, 1 to 5 substituents, preferably 1 to 3, more preferably 1 or 2 substituents, or it may not have any substituents. From the viewpoint of synthesizing compounds with simple chemical structures, it is preferable that such substituents are absent. On the other hand, if such a group has substituents, examples and preferred examples of such substituents are the same as the substituents that heteroarylene, which is an example of a leaving group, may have.

[0176] 1-5. Bioorthogonal functional groups (B) In equation (I), B is a bioorthothic functional group.

[0177] 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 (see, for example, 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)).

[0178] When the target of an affinity substance is an antibody (protein), the bioorthogonal functional group is a bioorthogonal functional group for the protein. A bioorthogonal functional group for a protein is a group that reacts with a specific functional group without reacting with the side chains of the 20 natural amino acid residues that make up the protein. The 20 natural amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Of these 20 naturally occurring amino acids, glycine, which lacks a side chain (i.e., a 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 the group consisting of sulfur, nitrogen, and oxygen atoms), are inactive to normal reactions. Therefore, bioorthogonal functional groups to proteins are functional groups that cannot react not only with the side chains of these amino acids, which have side chains that are inactive to normal reactions, but also with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0179] Examples of bioorthogonal functional groups that cannot react with proteins include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, any residue that has a vinylene (ethenylene) moiety, the smallest unit with an intercarbon double bond; the same applies hereafter), alkyne residues (in other words, any residue that has an ethynylene moiety, the smallest unit with an intercarbon triple bond; the same applies hereafter), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues (e.g., carbonyl residues with a fluorine, chlorine, bromine, or iodine atom at the α-position; the same applies hereafter), isonitrile residues, cydonone residues, and selenium residues. Antibodies may be proteins that cannot contain free thiols. In proteins that cannot 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 groups, alkenyl groups, alkynyl groups, cycloalkyl groups, aryl groups (e.g., phenyl group, naphthyl group), arylalkyl (aralkyl) group], acyl groups (e.g., acetyl group, propoxy group, butoxycarbonyl group such as tert-butoxycarbonyl group, benzoyl group), arylalkyloxycarbonyl group (e.g., fluorenyl methoxycarbonyl group), aryloxycarbonyl group, arylalkyl (aralkyl)oxycarbonyl group (e.g., benzyloxycarbonyl group), alkylthiol group (t-butylthio group), and arylthiol group (e.g., pyridyldithio group). Alternatively, the protected thiol residue may be a disulfide residue. In arylalkyl (aralkyl) groups and arylalkyl (aralkyl)oxycarbonyl groups, the arylalkyl group consists of one or more (e.g., two, three, four, or five) aryl atoms 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, even more preferably 1 to 10, and particularly preferably 1 to 6. In the present invention, the compound represented by formula (I) may contain one or more (e.g., two, three, or four) bioorthogonal functional groups, but preferably contains one bioorthogonal functional group.

[0180] More specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of the following: [ka] [During the ceremony, R 1f , one or more R 1g and one or more R 1h is an atom or group selected from the group consisting of (a) to (g), which is identical or different, or an electron-withdrawing group. • is a bonding hand.

[0181] The following are examples of atoms or groups selected from the group consisting of (a) to (g): (a) A hydrogen atom or a halogen atom; (b) Monovalent hydrocarbon group; (c) Aralkil; (d) Monovalent heterocyclic group; (e)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or a monovalent hydrocarbon group. ); or (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 band R c This represents a hydrogen atom or a monovalent hydrocarbon group, either identical or distinct. (g) Nitro group, sulfate group, sulfonic acid group, cyano group, or carboxyl group A group selected from the group consisting of the following. (a)~(g) contains halogen atoms, monovalent hydrocarbon groups, aralkyl groups, monovalent heterocyclic groups, and R a ~R c The definition, examples, and preferred examples of a monovalent hydrocarbon group in are the same as those in (i) to (vii) above. Particularly preferred is the atom or group selected from the group consisting of (a) to (g) being the atom or group of (a) or (b).

[0182] Preferably, the bioorthogonal functional group may be selected from the group consisting of azide residues, thiol residues, alkyne residues, maleimide residues, and disulfide residues, from the viewpoint of improving reaction efficiency, etc.

[0183] Examples of electron-withdrawing groups include those mentioned above, but halogen atoms, boronic acid residues, mesyl, tosyl, and triflates are preferred.

[0184] 1-6. Preferred structure of the compound represented by formula (I) In a preferred embodiment, the compound represented by formula (I) is the following formula (I-1): A-L1-L2-E1-E2-E3-B (I-1) [During the ceremony, A and B are the same as those in equation (I), L1 is a bond or a divalent group. L2 is a leaving group, E1 is an electrophile that (i) is linked to a leaving group and (ii) has the ability to react with a nucleophile in the antibody. E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). The leaving group has the ability to be cleaved from E1 and eliminated by a reaction between the nucleophile and the electrophile. The compound may be represented by [ ].

[0185] In formula (I-1), the leaving group represented by L2 is preferably one of (a) to (c) above. Examples and preferred examples of the leaving group represented by L2 are also the same as those described in (a) to (c) above.

[0186] In a preferred specific embodiment, the compound represented by formula (I-1) is the following: A-L1-L2-E1-XY-E3-B (I-2) [During the ceremony, A, L1, X, Y, and B are the same as those in equation (I-1), L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a divalent group. The compound may also be represented as [ ].

[0187] In another preferred specific embodiment, the compound represented by formula (I-1) is the following: [ka] [During the ceremony, A, L1, ring Z, ring constituent atom X', and B are the same as those in formula (I-1), L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group. The compound may also be represented as [E3].

[0188] Preferably, the compound represented by formula (I-3) is the following formula (I-4): [ka] [During the ceremony, A, L1, and B are the same as those in formula (I-1) above. L2 is (a) ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. Ring Z is a divalent ring group in which all of the ring constituent atoms bonded to E1 and the ring constituent atoms on both sides are carbon atoms. E3 is a bond or a divalent group. The compound may also be represented as [E3].

[0189] In the compounds represented by formulas (I-1) to (I-4) above, the definitions, examples, and preferred examples of A, L1, L2, E1, E2, E3, and B, as well as -XY-, alkyl groups with 1 to 6 carbon atoms in R1 to R5, and the group represented by formula (i) (e.g., a divalent ring group, a divalent heterocyclic group) are the same as described above. Also, the definitions, examples, and preferred examples of groups such as (a) to (c) which are L2, and ring Z (e.g., a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms), ring P, and Q (e.g., an alkyl group with 1 to 6 carbon atoms in R) are also the same as described above.

[0190] 1-7. Manufacturing method Antibody affinity substances and compounds having bioorthogonal functional groups or salts thereof can be prepared as appropriate. Antibody affinity substances and compounds having bioorthogonal functional groups or salts thereof are represented by formula (I), preferably formula (I-1), more preferably formula (I-2), (I-3), or (I-4).

[0191] As the affinity substance (A) for the antibody, any substance having any functional group can be appropriately selected. Therefore, by utilizing a reactive group that can react with the functional group, the affinity substance can be reacted with the structural unit represented by LEB to prepare the structural unit represented by ALEB. 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 even more preferably 30 minutes to 8 hours.

[0192] In the reaction system, the molar ratio (Y / X) of structural units (Y) represented by LEB to affinity substance (X) is not particularly limited, as it varies depending on the type of structural unit and affinity substance, the number of sites in the affinity substance to be modified by the structural unit, etc., but is for example 0.1 to 50, preferably 0.5 to 40, more preferably 1 to 35, even more preferably 2 to 25, and particularly preferably 3 to 15.

[0193] Confirmation of the formation of antibody affinity substances and compounds or salts thereof having bioorthogonal functional groups can be performed, depending on the specific molecular weight of the raw materials and products, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. Antibody affinity substances and compounds or salts thereof having bioorthogonal functional groups can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0194] 1-8. Others In the inventions described later (e.g., formulas (II), (III) and their sub-concept formulas, and inventions represented by substructural formulas), any symbols (e.g., A, L, E, B) and details of terms represented in relation to such symbols (e.g., definitions, examples, and preferred examples) are common to the inventions of compounds or salts represented by formula (I) or its sub-concept formulas. Furthermore, any technical elements (e.g., specific groups that can define the inventions described later (e.g., bioorthogonal functional groups, divalent groups, substituents) and specific numerical values, etc., can also be common to those described above. Therefore, these matters can be appropriately incorporated in the inventions described later without further mention. Similarly, technical elements of specific inventions described later can be appropriately incorporated as technical elements of the present invention and other inventions.

[0195] 2. Method for producing antibodies having bioorthogonal functional groups The present invention provides a method for producing an antibody having a bioorthogonal functional group or a salt thereof, comprising the following: (1) The following formula (I): ALEB (I) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to a leaving group and (ii) contains an electrophile that has the ability to react with a nucleophile in the antibody. B is a bioorthogonal functional group, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A substance that has affinity for antibodies, represented by ], and a compound having a bioorthothic functional group or a salt thereof are reacted with an antibody. Formula (II) below: Ab-EB (II) [During the ceremony, E and B are the same as those in equation (I), Ab is an antibody. The objective is to produce an antibody or a salt thereof having a bioorthogonal functional group, represented by [Ab].

[0196] The antibody used in the method for producing antibodies having bioorthogonal functional groups is the same as the antibody described above. Preferably, the antibody is a monoclonal antibody. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. The monoclonal antibody may be 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 preferred is a human antibody, a humanized antibody, or a chimeric antibody having human IgG (e.g., IgG1, IgG2, IgG3, IgG4) in its constant region.

[0197] In formula (II), Ab is the same as the antibody described above and is covalently bonded to the electrophile in E. Examples of nucleophiles in the antibody that are covalently bonded to the electrophile 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.

[0198] In formula (II), E is the same as E in formula (I) above. E can be represented as E1-E2-E3. E1, E2, and E3 are the same as those described above. As described above, E and E1-E2-E3 can be designed so as not to contain peptide moieties that have potential immunogenicity and are easily hydrolyzed in the blood. In this case, antibodies having bioorthogonal functional groups represented by formula (II) can be used to prepare antibodies with functional substances that do not have such problems.

[0199] The electrophile in E and the electrophile in E1 are covalently bonded to a nucleophile in the antibody. Examples of electrophiles covalently bonded to a nucleophile in the antibody include NH-C(=O)-, NH-SO2-, and NH-CH2- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue), OC(=O)-, O-SO2-, and O-CH2- (when the nucleophile in the antibody used for covalent bonding with E is OH in the side chain of a tyrosine, serine, or threonine residue), SC(=O)-, and S-CH2- (when the nucleophile in the antibody used for covalent bonding with E is SH in the side chain of a cysteine ​​residue). The electrophiles covalently bonded to the nucleophile in the antibody are preferably NH-C(=O)- and NH-SO2- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue), OC(=O)- and O-SO2- (when the nucleophile in the antibody used for covalent bonding with E is OH in the side chain of a tyrosine residue), more preferably NH-C(=O)- and NH-SO2- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue), and even more preferably NH-C(=O)- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue).

[0200] In equation (II), B is the same as in equation (I) above.

[0201] Preferably, the antibody having a bioorthogonal functional group produced by the production method of the present invention is an antibody having the bioorthogonal functional group regioselectively. When the affinity substance for the antibody and the compound having a bioorthogonal functional group or a salt thereof are represented by formula (I), an antibody having a bioorthogonal functional group regioselectively represented by formula (II) can be produced. The antibody having a bioorthogonal functional group regioselectively is preferably an antibody having a bioorthogonal functional group only in the constant region, and more preferably an antibody having a bioorthogonal functional group only in the Fc region.

[0202] In this specification, "regioselectivity" or "regioselectivity" means that, even though specific amino acid residues in the antibody are not concentrated in specific regions, a predetermined structural unit capable of binding to a specific amino acid residue in the antibody is concentrated in a specific region of the antibody. Therefore, expressions related to regioselectivity, such as "regioselectively present," "regioselective binding," and "regioselective binding," mean that the binding rate or retention rate of a predetermined 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 same 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 regioselective binding or retention can be achieved by the present invention, which allows for preferential reaction of a predetermined structural unit to a specific amino acid residue in the target region of the antibody by using a compound containing an affinity substance for the antibody, rather than randomly reacting a predetermined structural unit with a specific amino acid residue in the antibody.

[0203] More specifically, if an antibody (Ab) contains one or more specific amino acid residues (e.g., lysine residues, tyrosine residues, threonine residues, serine residues, cysteine ​​residues) within a target region consisting of 1 to 50 consecutive amino acid residues [e.g., (b) a region consisting of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residues at position 317 in the human IgG Fc region], and also contains five or more of the same specific amino acid residues in a non-target region other than the target region, then a substructure other than the antibody can be bound to one or more specific amino acid residues contained in the target region with regioselectivity of 30% or more. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0204] Antibodies having bioorthogonal functional groups produced by the production method of the present invention may also have bioorthogonal functional groups (i.e., structural units represented by EB) depending on the number of heavy chains. Antibodies having bioorthogonal functional groups regioselectively can be produced by first associating a compound represented by formula (I) (ALEB) with the constant region of an antibody heavy chain via an affinity substance (A) for the antibody, and then reacting the electrophile at E with a nucleophile in the side chain of a specific amino acid residue near the association site (the constant region of the same heavy chain as the antibody heavy chain). Therefore, by using an antibody having multiple antibody heavy chains (for example, 1 to 8, preferably 1 to 4, more preferably 2) in the production method of the present invention, it is possible to produce antibodies that regioselectively have multiple structural units (or multiple structural units of a sub-concept) represented by EB 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 production method of the present invention, it is possible to produce an antibody that regioselectively has two structural units represented by EB in the same target region of the two antibody heavy chains. In other words, in an antibody having bioorthogonal functional groups, the modification mode by the bioorthogonal functional groups can be made identical across multiple (e.g., two) heavy chains.

[0205] Antibodies having bioorthogonal functional groups can also have the same or different 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, the modification pattern by the bioorthogonal functional groups can be identical across multiple (e.g., 2) heavy chains in an antibody having bioorthogonal functional groups.

[0206] The production method of the present invention may also include subjecting the generated antibody to a specific treatment to produce an antibody having bioorthogonal functional groups and further modified. Such specific treatments include, for example, antibody fragmentation treatment (e.g., treatment with a specific protease such as papain or pepsin).

[0207] In the production method of the present invention, when a compound represented by formula (I) or a salt thereof is used, an antibody having a bioorthogonal functional group represented by formula (II) can be produced.

[0208] In a preferred embodiment, when the 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. Ab-E1-E2-E3-B (II-1) [During the ceremony, Ab is an antibody, E1 is an electrophile linked to a nucleophile in the antibody. E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). B is a bioorthogonal functional group.

[0209] In a preferred specific embodiment, when the compound represented by formula (I-2) is used as the compound represented by formula (I-1) in the production method of the present invention, an antibody having a bioorthogonal functional group represented by the following formula (II-2) can be produced. Ab-E1-XY-E3-B (II-2) [During the ceremony, Ab, X, Y, and B are the same as those in equation (II-1), E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a divalent group.

[0210] In another preferred specific embodiment, when the compound represented by formula (I-3) is used as the compound represented by formula (I-1) in the production method of the present invention, an antibody having a bioorthogonal functional group represented by the following formula (II-3) can be produced. [ka] [During the ceremony, Ab, ring constituent atom X', ring Z, and B are the same as those in formula (II-1), E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group.

[0211] Preferably, when the 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. [ka] [During the ceremony, Ab, ring Z, and B are the same as those in equation (II-1), E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group.

[0212] Details (e.g., definitions, examples, and preferred examples) of any symbols (e.g., E, E1, E2, E3, B) in formulas (II), (II-1), (II-2), (II-3), or (II-4), and terms represented in relation to such symbols (e.g., antibody, electrophile, bioorthogonal functional group) are the same as those of formula (I) or its sub-concepts.

[0213] Affinity compounds for antibodies, and compounds or salts thereof having bioorthogonal functional groups, can react with antibodies because they have an electrophile at E or an electrophile at E1. Such reactions can be carried out as appropriate under mild conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds). For example, such reactions can be carried out at room temperature (e.g., about 15-30°C) in a suitable reaction system, such as a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, and more preferably 6.0-8.0. 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 on such reactions, see, for example, GJLBernardes et al., Chem. Rev., 115, 2174 (2015); GJLBernardes et al., Chem. Asian. J., 4, 630 (2009); BGDavies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0214] In the reaction system, the molar ratio (Y / X) of the antibody affinity substance and the compound having a bioorthogonal functional group or its salt (Y) to the antibody (X) is not particularly limited, as it varies depending on the type of antibody affinity substance, the compound having a bioorthogonal functional group or its salt and the antibody, the number of sites in the antibody to be modified by the antibody affinity substance and the compound having a bioorthogonal functional group or its salt (e.g., DAR), etc., but is for example 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

[0215] Confirmation of the generation of antibodies having bioorthogonal functional groups can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw materials and molecular weight of the product. Confirmation of regioselectivity can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Confirmation of the number of bioorthogonal functional groups possessed by antibodies having bioorthogonal functional groups can be performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Antibodies having bioorthogonal functional groups can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0216] 3. A method for producing an antibody containing a functional substance, using an antibody affinity substance and a compound having a bioorthogonal functional group or a salt thereof. The present invention provides a method for producing an antibody having a functional substance or a salt thereof, comprising the following: (1) The following formula (I): ALEB (I) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to a leaving group and (ii) contains an electrophile that has the ability to react with a nucleophile in the antibody. B is a bioorthogonal functional group, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A substance that has affinity for antibodies, represented by ], and a compound having a bioorthothic functional group or a salt thereof are reacted with an antibody. Formula (II) below: Ab-EB (II) [During the ceremony, E and B are the same as those in formula (I) above, Ab is an antibody. To produce an antibody or salt thereof having a bioorthogonal functional group represented by ]; and (2) An antibody or salt thereof having a bioorthogonal functional group represented by formula (II) above is reacted with a functional substance via the bioorthogonal functional group, Formula (III) below: Ab-E-B'-F (III) [During the ceremony, Ab is the same as that in equation (II), E is the same as that in equation (I), B' is a divalent group that includes a moiety generated by a reaction between a functional substance and a bioorthogonal functional group. F is a functional substance. The process involves producing an antibody or a salt thereof containing a functional substance represented by [ ].

[0217] Step (1) can be carried out in the same manner as for the production of antibodies having bioorthothic functional groups.

[0218] The functional substance (F) used in step (2) is not particularly limited as long as it is a substance that confers any function to the antibody, and examples include drugs, labeling substances and stabilizers, but is preferably a drug or labeling substance. The functional substance may also be a single functional substance or a substance in which two or more functional substances are linked together.

[0219] The drug may be any drug used for any disease. Examples of such diseases include cancer (e.g., lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumors, melanoma), autoimmune and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), neurological diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood disorders (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, digestive system diseases). The drug may be a drug that treats or prevents a specific disease, or a drug that alleviates side effects associated with the use of antibodies against target proteins of a specific disease.

[0220] More specifically, the drugs are anticancer agents. Examples of anticancer agents include chemotherapeutic agents, toxins, radioisotopes, or substances containing them. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., lysine). Examples of radioisotopes include radioisotopes of the hydrogen atom (e.g., 3 H), radioactive isotopes of carbon atoms (e.g., 14 C) Radioactive isotopes of the phosphorus atom (e.g., 32P), radioactive isotopes of the sulfur atom (e.g., 35 S ), radioactive isotopes of yttrium (e.g., 90 Y), radioactive isotopes of technetium (e.g., 99m Tc), radioactive isotopes of indium (e.g., 111 In), radioactive isotopes of the iodine atom (e.g., 123 I, 125 I, 129 I, 131 I) Radioactive isotopes of samarium (e.g., 153 Sm), radioactive isotopes of rhenium (e.g., 186 Re), radioactive isotopes of astatine (e.g., 211 At), radioactive isotopes of bismuth (e.g., 212 Bi) is one example.

[0221] Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioactive isotopes (e.g., those mentioned above), or substances containing them.

[0222] Functional substances are also high molecular weight compounds, medium molecular weight compounds, or low molecular weight compounds, preferably low molecular weight compounds. Low molecular weight compounds are compounds with a molecular weight of 1500 or less. Low molecular weight compounds are natural compounds or synthetic compounds. The molecular weight of low molecular weight compounds may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of low molecular weight compounds may also be 30 or more, 40 or more, or 50 or more. Low molecular weight compounds may be drugs or labeling substances 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.

[0223] Functional substances have various functional groups depending on their structure. If a functional substance has a functional group that readily reacts with a bioorthogonal functional group, the functional group of the functional substance and the bioorthogonal functional group can be reacted as appropriate. The functional group that readily reacts with a bioorthogonal functional group may also vary depending on the specific type of bioorthogonal functional group. Those skilled in the art can appropriately select a functional group that readily reacts with a bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Examples of functional groups that readily react with a bioorthogonal functional group include, but are not limited to, azide residues when the bioorthogonal functional group is an alkyne residue, hydrazine residues when the bioorthogonal functional group is an aldehyde or ketone residue, and maleimide and disulfide residues when the bioorthogonal functional group is a thiol residue. For example, if the bioorthogonal functional group is an alkyne residue and the functional group readily reacting with the bioorthogonal functional group is an azide residue (or vice versa), the divalent group containing the moiety produced by the reaction between the functional substance and the bioorthogonal functional group may be a divalent group containing a triazole residue (which may or may not be fused with another ring); if the bioorthogonal functional group is an aldehyde residue or a ketone residue and the functional group readily reacting with the bioorthogonal functional group is a hydrazine residue (or vice versa), the function A divalent group containing a moiety formed by a reaction between a functional substance and a bioorthogonal functional group may be a divalent group containing a hydrazone residue; if the bioorthogonal functional group is a thiol residue and the functional group readily reacting with the bioorthogonal functional group is a maleimide residue or a disulfide residue (or vice versa), a divalent group containing a moiety formed by a reaction between a functional substance and a bioorthogonal functional group may be a divalent group containing a thiosuccinimide residue or a divalent group containing a disulfide residue (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195).Divalent groups containing a triazole residue (which may or may not be fused with another ring), a hydrazone residue, a thiosuccinimide residue, or a disulfide residue are preferred examples of divalent groups that include a moiety generated by a reaction between a functional substance and a bioorthothic functional group.

[0224] On the other hand, if a functional substance does not have a functional group that readily reacts with a bioorthogonal functional group, a derivatized version of the functional substance that has the desired functional group can be used. For example, if the functional substance is a soluble protein, a derivatized version of the soluble protein that has a functional group not naturally present can be used.

[0225] Derivatization is common technical knowledge in the art (e.g., International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be carried out using a crosslinking agent as described above. Alternatively, derivatization may be carried out using a specific linker having the desired functional group. For example, such a linker may be capable of separating the functional substance and the antibody by cleavage in a suitable 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)), and linkers that can be cleaved at locally acidic sites present in the body (e.g., U.S. Patent Nos. 5,622,929, 5,122,368; 5,824,805). Linkers may also be self-immolative (e.g., International Publication No. 02 / 083180, International Publication No. 04 / 043493, International Publication No. 05 / 112919). In this invention, derivatized functional substances can also be simply referred to as "functional substances."

[0226] In formula (III), Ab is the same as the antibody described above and is covalently bonded to the electrophile in E. Examples of nucleophiles in the antibody that are covalently bonded to the electrophile 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.

[0227] In formula (III), E is the same as E in formula (I) above. E can be represented as E1-E2-E3. E1, E2, and E3 are the same as those described above. As described above, E and E1-E2-E3 can be designed so as not to contain peptide portions that have potential immunogenicity and are easily hydrolyzed in the blood. In this case, antibodies having the functional substance represented by formula (III) can be suitably used as pharmaceuticals.

[0228] The electrophile in E and the electrophile in E1 are covalently bonded to a nucleophile in the antibody. Examples of electrophiles covalently bonded to a nucleophile in the antibody include NH-C(=O)-, NH-SO2-, and NH-CH2- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue), OC(=O)-, O-SO2-, and O-CH2- (when the nucleophile in the antibody used for covalent bonding with E is OH in the side chain of a tyrosine, serine, or threonine residue), SC(=O)-, and S-CH2- (when the nucleophile in the antibody used for covalent bonding with E is SH in the side chain of a cysteine ​​residue). The electrophiles covalently bonded to the nucleophile in the antibody are preferably NH-C(=O)- and NH-SO2- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue), OC(=O)- and O-SO2- (when the nucleophile in the antibody used for covalent bonding with E is OH in the side chain of a tyrosine residue), more preferably NH-C(=O)- and NH-SO2- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue), and even more preferably NH-C(=O)- (when the nucleophile in the antibody used for covalent bonding with E is NH2 in the side chain of a lysine residue).

[0229] In certain embodiments, if the functional substance has a functional group that readily reacts with a bioorthogonal functional group, or if it has been derivatized to have a functional group that readily reacts with a bioorthogonal functional group, the functional group that readily reacts with a bioorthogonal functional group may be a group selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene residues, halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isonitrile residues, cydonone residues, and selenium residues.

[0230] Furthermore, in certain embodiments, if the functional substance has a functional group that readily reacts with a bioorthogonal functional group, or if it has been 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 may be a group selected from the group consisting of the groups represented below. [ka] [During the ceremony, R 1f , one or more R 1g and one or more R 1h (i) is an atom or group selected from the group consisting of (i) to (vii) above, either identical or different, or an electron-withdrawing group, and (ii) is a bonding site for the functional material.

[0231] The divalent group represented by B' in formula (III), which includes a portion produced by a reaction between a functional substance and a bioorthogonal functional group, may be (1) a divalent group containing a triazole residue, a hydrazone residue, or a thiosuccinimide residue, which are the residues mentioned in the preferred example above, or (2) a divalent group containing a residue selected from the group consisting of, for example, a disulfide residue (which is the residue mentioned in the preferred example above), an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoamidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinaldiol residue, a selenium residue, an aromatic ring-containing residue with an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, an unsaturated bond-containing chain residue, or a glycosyl residue.

[0232] Divalent groups, which include a moiety formed by a reaction between a functional substance and a bioorthogonal functional group, are also, but are not particularly limited, examples below: [ka] [Here, the wavy lines perpendicular to the bonds indicate the bonds formed by the reaction.] Multiple R 2a , multiple R 2b , and multiple R 2c These are, either identical or different, hydrogen atoms or the aforementioned substituents. J is -CH2-, -O-, or -S-, r is any integer between 1 and 4. ○ (white circle) indicates a connection to the F side, and ● (black circle) indicates a connection to the B' side. If the chemical structure is asymmetric with respect to the cleavage region, ● may indicate binding to the B' side portion and ○ may indicate binding to the F side portion. It may also be a divalent group containing a residue corresponding to any one chemical structure selected from the group consisting of ].

[0233] Preferably, the antibody having a functional substance produced in step (2) of the production method of the present invention is an antibody that regioselectively possesses the functional substance. If the antibody having a regioselectively orthogonal functional group is represented by formula (II), then an antibody having a regioselectively possessing a functional substance represented by formula (III) can be produced. The antibody having a regioselectively possessing a functional substance is preferably an antibody having the functional substance only in the constant region, and more preferably an antibody having the functional substance only in the Fc region.

[0234] If an antibody (Ab) contains one or more specific amino acid residues (e.g., lysine residues, tyrosine residues, threonine residues, serine residues, cysteine ​​residues) within a target region consisting of 1 to 50 consecutive amino acid residues [e.g., (b) a region consisting of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residues at position 317 in the human IgG Fc region], and also contains five or more of the same specific amino acid residues in a non-target region other than the target region, then a substructure other than the antibody can be bound to one or more specific amino acid residues contained in the target region with regioselectivity of 30% or more. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0235] Antibodies having functional substances produced by the production method of the present invention may also have functional substances (i.e., structural units represented as E-B'-F) depending on the number of heavy chains. This is because antibodies having functional substances can be produced from antibodies having bioorthogonal functional groups that can have structural units represented as EB depending on the number of heavy chains. Therefore, by using antibodies having multiple antibody heavy chains (e.g., 1 to 8, preferably 1 to 4, more preferably 2) in the production method of the present invention, it is possible to produce antibodies that regioselectively have multiple structural units (or multiple structural units of a sub-concept) represented as E-B'-F in the same target region of the multiple antibody heavy chains. For example, by using antibodies having two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab')2 antibody, Fc region protein, Fc fusion protein) in the production method of the present invention, it is possible to produce antibodies that regioselectively have two structural units represented as E-B'-F in the same target region of the two antibody heavy chains. In other words, in antibodies containing functional substances, the modification mode by the functional substance can be made identical across multiple (e.g., two) heavy chains.

[0236] Antibodies containing functional substances may also have the same or different functional substances (e.g., structural units represented as 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, the modification pattern by the functional substance can be made identical across multiple (e.g., 2) heavy chains in the antibody containing the functional substance.

[0237] The production method of the present invention may also include subjecting the generated antibody to a specific treatment to produce an antibody that has a functional substance and is further modified. Such specific treatments include, for example, antibody fragmentation (e.g., treatment with a specific protease such as papain or pepsin).

[0238] In the manufacturing method of the present invention, when a compound represented by formula (I) or a salt thereof is used, an antibody having a bioorthogonal functional group represented by formula (II) can be produced in step (1), and then an antibody having a functional material group represented by formula (III) can be produced in step (2).

[0239] In a preferred embodiment, when the 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 bioorthothic functional group represented by formula (II-1) can be produced in step (1), and then an antibody having a functional material group represented by the following formula (III-1) can be produced in step (2). Ab-E1-E2-E3-B'-F (III-1) [During the ceremony, Ab is an antibody, E1 is an electrophile linked to a nucleophile in the antibody. E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). B' is a divalent group that includes a moiety generated by a reaction between a functional substance and a bioorthogonal functional group. F is a functional substance.

[0240] In a preferred particular embodiment, when the 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 formula (II-2) can be produced in step (1), and then an antibody having a functional material group represented by the following formula (III-2) can be produced in step (2). Ab-E1-XY-E3-B'-F (III-2) [During the ceremony, Ab, X, Y, B', and F are the same as those in formula (III-1) above. E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a divalent group.

[0241] In another preferred specific embodiment, when the compound represented by formula (I-3) is used as the compound represented by formula (I-1) in the production method of the present invention, an antibody having the bioorthogonal functional group represented by formula (II-3) can be produced in step (1), and then an antibody having the functional material group represented by the following formula (III-3) can be produced in step (2). [ka] [During the ceremony, Ab, ring constituent atom X', ring Z, B', and F are the same as those in formula (III-1), E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group.

[0242] Preferably, in the manufacturing method of the present invention, when the compound represented by formula (I-4) is used as the compound represented by formula (I-3), an antibody having a bioorthogonal functional group represented by the following formula (II-4) can be produced in step (1), and then an antibody having a functional substance group represented by the following formula (III-4) can be produced in step (2). [ka] [During the ceremony, The rings Ab, Z, B', and F are the same as those in equation (III-1), E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group.

[0243] Details (e.g., definitions, examples, and preferred examples) of any symbols (e.g., E, E1, E2, E3, B) in formulas (III), (III-1), (III-2), (III-3), or (III-4), and terms represented in relation to such symbols (e.g., antibody, electrophile, bioorthogonal functional group) are the same as those of formula (I) or its sub-concepts.

[0244] Antibodies having bioorthogonal functional groups can react with functional substances via these bioorthogonal functional groups. Such reactions can be carried out under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) as described above.

[0245] In the reaction system, the molar ratio (Z / Y) of the functional substance (Z) to the antibody (Y) having a bioorthotropic functional group is not particularly limited, as it varies depending on the type of bioorthotropic functional group, the functional substance, and the antibody, as well as the number of sites in the antibody to be modified (e.g., DAR). However, it is, for example, 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

[0246] Confirmation of the generation of antibodies containing functional substances can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw materials and molecular weight of the product. Confirmation of regioselectivity can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Confirmation of the number of functional substances contained in antibodies containing functional substances can be performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Antibodies containing functional substances can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0247] 4. Antibodies or salts thereof that regioselectively possess bioorthogonal functional groups or functional substances The present invention provides antibodies or salts thereof that regioselectively have bioorthogonal functional groups or functional substances.

[0248] An antibody or salt thereof having a bioorthogonal functional group in a regioselective manner is an antibody having a bioorthogonal functional group represented by formula (II-1) in a regioselective manner. Preferably, as an antibody having a bioorthogonal functional group represented by formula (II-1), an antibody having a bioorthogonal functional group represented by formula (II-2) or (II-3) in a regioselective manner is provided. More preferably, as an antibody having a bioorthogonal functional group represented by formula (II-3), an antibody having a bioorthogonal functional group represented by formula (II-4) in a regioselective manner is provided. Details (e.g., definitions, examples, and preferred examples) of any symbols in formula (II), (II-1), (II-2), (II-3), or (II-4) (e.g., E, E1, E2, E3, B), and terms expressed in relation to such symbols (e.g., antibody, electrophile, bioorthogonal functional group) are the same as those of formula (I) or its sub-concepts.

[0249] An antibody or salt thereof having a functional substance regioselectively is an antibody having a functional substance represented by formula (III-1) regioselectively. Preferably, as an antibody having a functional substance represented by formula (III-1) regioselectively, an antibody having a functional substance represented by formula (III-2) or (III-3) regioselectively is provided. More preferably, as an antibody having a functional substance represented by formula (III-3) regioselectively, an antibody having a functional substance represented by formula (III-4) regioselectively is provided. Details (e.g., definitions, examples, and preferred examples) of any symbols in formulas (III), (III-1), (III-2), (III-3), or (III-4) (e.g., E, E1, E2, E3, B) and terms expressed in relation to such symbols (e.g., antibody, electrophile, bioorthogonal functional group) are the same as those of formula (I) or its sub-concepts.

[0250] Antibodies having bioorthogonal functional groups or functional substances regioselectively are the same as the antibodies 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 preferred are human antibodies, humanized antibodies, or chimeric antibodies having human IgG (e.g., IgG1, IgG2, IgG3, IgG4) in the constant region.

[0251] Antibodies having bioorthogonal functional groups or functional substances in a regioselective manner are preferably antibodies having bioorthogonal functional groups or functional substances only in the constant region of the antibody, and more preferably antibodies having bioorthogonal functional groups or functional substances only in the Fc region of the antibody.

[0252] An antibody having a bioorthogonal functional group or functional substance regioselectively can have the bioorthogonal functional group or functional substance with regioselectivity of 30% or more at one or more specific amino acid residues in the target region, if it contains one or more specific amino acid residues (e.g., lysine residues, tyrosine residues, threonine residues, serine residues, cysteine ​​residues) in a target region consisting of 1 to 50 consecutive amino acid residues [e.g., (a) a region consisting of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residues at position 317 in the human IgG Fc region] and also contains five or more of the specific amino acid residues in a non-target region other than the target region. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0253] Antibodies having regioselectively bioorthogonal functional groups or functional substances can also have, depending on the number of heavy chains, bioorthogonal functional groups (i.e., structural units represented by EB) or functional substances (i.e., structural units represented by E-B'-F). When an antibody having regioselectively bioorthogonal functional groups or functional substances has multiple antibody heavy chains (e.g., 1 to 8, preferably 1 to 4, more preferably 2), the bioorthogonal functional groups or functional substances can be regioselectively present in the same target region of the multiple antibody heavy chains. In other words, in an antibody having regioselectively bioorthogonal functional groups or functional substances, the modification pattern by the bioorthogonal functional groups or functional substances can be made identical across multiple (e.g., 2) heavy chains.

[0254] Antibodies having regioselectively bioorthogonal functional groups or functional substances may also have homogeneous or heterogeneous bioorthogonal functional groups (e.g., structural units represented by EB) or homogeneous or heterogeneous 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, the modification pattern by the bioorthogonal functional groups or functional substances can be made identical across multiple (e.g., 2) heavy chains in antibodies having regioselectively bioorthogonal functional groups or functional substances.

[0255] 5. Compounds or salts thereof having affinity substances for antibodies and functional substances. The present invention provides compounds having an affinity substance for antibodies and a functional substance represented by formula (IV), or salts thereof. ALEF (IV) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, F is a functional substance, The leaving group has the ability to be cleaved from E and eliminated by a reaction between the nucleophile and the electrophile.

[0256] The definitions, examples, and preferred examples of the antibody-affinity substance (A), the divalent group containing a leaving group (L), the divalent group containing an electrophile (E), and the functional substance (F) in compounds or salts thereof that have an antibody-affinity substance and a functional substance are the same as those described above. Therefore, in compounds or salts thereof that have an antibody-affinity substance and a functional substance, A, L, and E can be specified in the same way as A, L, and E in compounds or salts thereof that have an antibody-affinity substance and a bioorthothic functional group (see, for example, formula (I)). Also, in compounds or salts thereof that have an antibody-affinity substance and a functional substance, F can be specified in the same way as F in antibodies or salts thereof that have a functional substance (see, for example, formula (III)).

[0257] In a preferred embodiment, the compound represented by formula (IV) is the following formula (IV-1): A-L1-L2-E1-E2-E3-F (IV-1) [During the ceremony, A and F are the same as those in equation (IV), L1 is a bond or a divalent group. L2 is a leaving group, E1 is an electrophile that (i) is linked to a leaving group and (ii) has the ability to react with a nucleophile in the antibody. E2 is (a)-XY-[where X bonded to E1 is C(R1)(R2) (where R1 and R2 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), N(R3) (where R3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E3 is C(R4)(R5) (where R4 and R5 are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)], or (b) the following formula (i): [ka] (Here, ring Z is a group represented as follows: (where ring Z is a divalent ring group in which all of the ring constituent atoms X' bonded to E1 and the ring constituent atoms on both sides of it are carbon atoms, or a divalent heterocyclic ring group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on both sides of the nitrogen atom are carbon atoms. · represents a bond.) E3 is a divalent group when E2 is -XY-, and a bond or a divalent group when E2 is the group represented by formula (i). The leaving group has the ability to be cleaved and eliminated from E1 by a reaction between the nucleophile and the electrophile. The compound may also be represented by [ ].

[0258] In formula (IV-1), the definition, examples, and preferred examples of the leaving group represented by L2 are the same as those described in (a) to (c) above in "1-3. Divalent groups containing a leaving group (L)".

[0259] In a preferred specific embodiment, the compound represented by formula (IV-1) is the following: A-L1-L2-E1-XY-E3-F (IV-2) [During the ceremony, A, L1, X, Y, and F are the same as those in equation (IV-1), L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a divalent group. The compound may also be represented as [ ].

[0260] In another preferred specific embodiment, the compound represented by formula (IV-1) is the following: [ka] [During the ceremony, A, L1, ring Z, ring constituent atoms X' and F are the same as those in formula (IV-1) above. L2 is (a) Ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. E3 is a bond or a divalent group. The compound may also be represented as [E3].

[0261] Preferably, the compound represented by formula (IV-3) is the following formula (IV-4): [ka] [During the ceremony, A, L1, and F are the same as those in formula (IV-1) above. L2 is (a) ring PQ- [wherein ring P is a group selected from the group consisting of arylene which may be substituted with an electron-withdrawing group, heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, 2,6-diketopyrrolidine which may be fused, 2-ketopyrrolidine which may be fused, 2-ketopyrperidin which may be fused, and 2-pyridone, and Q is a group selected from the group consisting of -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).] (b) heteroarrenes, or (c)-Q- [where Q is a group selected from the group consisting of -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)], E1 is a group selected from the group consisting of -C(=O)-, -SO2-, and -CH2-. Ring Z is a divalent ring group in which all of the ring constituent atoms bonded to E1 and the ring constituent atoms on both sides are carbon atoms. E3 is a bond or a divalent group. The compound may also be represented as [E3].

[0262] In the compounds represented by formulas (IV-1) to (IV-4) above, the definitions, examples, and preferred examples of A, L1, L2, E1, E2, E3, and F, as well as -XY-, alkyl groups with 1 to 6 carbon atoms in R1 to R5, and the group represented by formula (i) (e.g., a divalent ring group, a divalent heterocyclic group) are the same as described above. Also, the definitions, examples, and preferred examples of groups such as (a) to (c) which are L2, and ring Z (e.g., a divalent ring group in which the ring constituent atom X' bonded to E1 and all of the ring constituent atoms on either side of it are carbon atoms, or a divalent heterocyclic group in which the ring constituent atom X' bonded to E1 is a nitrogen atom and the ring constituent atoms on either side of the nitrogen atom are carbon atoms), ring P, and Q (e.g., an alkyl group with 1 to 6 carbon atoms in R) are also the same as described above.

[0263] Compounds or salts thereof having an affinity substance for antibodies and functional substances can be appropriately prepared by reacting a compound or salt thereof having an affinity substance for antibodies and a bioorthogonal functional group with the functional substance as described above via the bioorthogonal functional group. Such reactions 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 even more preferably 30 minutes to 8 hours.

[0264] In the reaction system, the molar ratio (Y / X) of the functional substance (X), the affinity substance for the antibody, and the compound having a bioorthothic functional group or its salt is not particularly limited, as it varies depending on the type of structural unit and affinity substance, the number of sites in the affinity substance to be modified by the structural unit, etc., but is for example 0.01 to 100, preferably 0.05 to 20, and more preferably 0.1 to 10.

[0265] Confirmation of the formation of compounds or salts containing affinity substances for antibodies and functional substances depends on the specific molecular weight of the raw materials and products, but can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. Compounds or salts containing affinity substances for antibodies and functional substances can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0266] 6. A method for producing an antibody containing a functional substance, using a compound or salt thereof that has affinity for the antibody and a functional substance. The present invention provides a method for producing an antibody having a functional substance or a salt thereof, comprising the following: Formula (IV): ALEF (IV) [During the ceremony, A is an affinity substance for antibodies, L is a divalent group containing a leaving group, E is a divalent group that (i) is linked to the leaving group and (ii) contains an electrophile capable of reacting with the nucleophile in the antibody, F is a functional substance, The leaving group has the ability to be cleaved and removed from E by a reaction between the nucleophile and the electrophile. A compound or salt thereof having an affinity substance for an antibody and a functional substance, represented by ], is reacted with an antibody. The following equation (V): Ab-EF (V) [During the ceremony, Ab is an antibody, E and F are the same as those in formula (IV) above. To produce an antibody or a salt thereof having a functional substance represented by ].

[0267] Preferably, the antibody having a functional substance produced by the production method of the present invention is an antibody that regioselectively contains the functional substance. In this case, an antibody having a functional substance regioselectively represented by formula (V) can be produced. The antibody having a functional substance regioselectively is preferably an antibody that has the functional substance only in the constant region, and more preferably an antibody that has the functional substance only in the Fc region.

[0268] If an antibody (Ab) contains one or more specific amino acid residues (e.g., lysine residues, tyrosine residues, threonine residues, serine residues, cysteine ​​residues) within a target region consisting of 1 to 50 consecutive amino acid residues [e.g., (b) a region consisting of amino acid residues at positions 246 to 248 in the human IgG Fc region, (b) a region consisting of amino acid residues at positions 288 to 290 in the human IgG Fc region, or (c) a region consisting of amino acid residues at position 317 in the human IgG Fc region], and also contains five or more of the same specific amino acid residues in a non-target region other than the target region, then a substructure other than the antibody can be bound to one or more specific amino acid residues contained in the target region with regioselectivity of 30% or more. The positional selectivity may be preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0269] Antibodies having functional substances produced by the production method of the present invention may also have functional substances (i.e., structural units represented by EF) depending on the number of heavy chains. Therefore, by using antibodies having multiple antibody heavy chains (e.g., 1 to 8, preferably 1 to 4, more preferably 2) in the production method of the present invention, it is possible to produce antibodies that regioselectively have multiple structural units (or multiple structural units of a sub-concept) represented by EF in the same target region of multiple antibody heavy chains. For example, by using antibodies having two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab')2 antibody, Fc region protein, Fc fusion protein) in the production method of the present invention, it is possible to produce antibodies that regioselectively have two structural units represented by EF in the same target region of the two antibody heavy chains. In other words, in antibodies having functional substances, the modification mode by the functional substance can be made identical across multiple (e.g., 2) heavy chains.

[0270] Antibodies containing functional substances may also have the same or different functional substances (e.g., structural units represented by EF) 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 functional substances may have the same modification pattern by the functional substance across multiple (e.g., 2) heavy chains.

[0271] The production method of the present invention may also include subjecting the generated antibody to a specific treatment to produce an antibody that has a functional substance and is further modified. Such specific treatments include, for example, antibody fragmentation (e.g., treatment with a specific protease such as papain or pepsin).

[0272] In the manufacturing method of the present invention, when a compound represented by formula (IV) or a salt thereof is used, an antibody having the functional substance represented by formula (V) can be produced.

[0273] In a preferred embodiment, when the 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. Ab-E1-E2-E3-F (V-1) [During the ceremony, Ab is an antibody, E1, E2, E3, and F are the same as those in formula (IV-1) above.

[0274] In a preferred specific embodiment, when the 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. Ab-E1-XY-E3-F (V-2) [During the ceremony, Ab is an antibody, E1, X, Y, E3, and F are the same as those in equation (IV-2) above.

[0275] In another preferred specific embodiment, when the 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. [ka] [During the ceremony, Ab is an antibody, E1, ring Z, ring constituent atom X', E3, and F are the same as those in formula (IV-3) above.

[0276] Preferably, when the 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. [ka] [During the ceremony, Ab is an antibody, E1, ring Z, E3, and F are the same as those in equation (IV-4) above.

[0277] Compounds or salts thereof that have affinity substances for antibodies and functional substances can react with antibodies because they have an electrophile at E or an electrophile at E1. Such reactions can be carried out as appropriate under conditions (mild conditions) that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds). For example, such reactions can be carried out at room temperature (e.g., about 15-30°C) in a suitable reaction system, such as a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, and more preferably 6.0-8.0. 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 on such reactions, see, for example, GJLBernardes et al., Chem. Rev., 115, 2174 (2015); GJLBernardes et al., Chem. Asian. J., 4, 630 (2009); BGDavies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0278] In the reaction system, the molar ratio (Y / X) of the antibody (X) to the compound or salt (Y) having an affinity substance for the antibody and a functional substance is not particularly limited, as it varies depending on the type of antibody, the affinity substance for the antibody and the compound or salt having a functional group, the number of sites in the antibody to be modified by the antibody (e.g., DAR) and the compound or salt having a bioorthogonal functional group. However, it is for example 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

[0279] Confirmation of the generation of antibodies containing functional substances can be performed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw materials and molecular weight of the product. Confirmation of regioselectivity can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. Confirmation of the number of functional groups possessed by antibodies containing functional groups can be performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Antibodies containing functional groups can be purified as appropriate by any method such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0280] 7. Salt 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, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and salts with acidic amino acids (e.g., aspartic acid, glutamic acid). Preferably, the salt is a salt with an inorganic acid (e.g., hydrogen chloride) or an organic acid (e.g., trifluoroacetic acid).

[0281] 8.Applications The compounds of the present invention having an affinity for antibodies and a bioorthogonal functional group, or a salt thereof, are useful, for example, for the regioselective modification of antibodies by a bioorthogonal functional group. Accordingly, the present invention provides a regioselective modification reagent for antibodies by a bioorthogonal functional group, comprising an affinity for antibodies and a compound having a bioorthogonal functional group, or a salt thereof. In the regioselective modification reagent for antibodies by a bioorthogonal functional group, the details of the affinity for antibodies and the compound having a bioorthogonal functional group, or a salt thereof (e.g., definitions, examples, and preferred examples) are the same as described above.

[0282] The compounds or salts thereof of the present invention, which have an affinity substance for antibodies and a functional substance, are useful, for example, for the regioselective modification of antibodies by functional substances. Accordingly, the present invention provides a regioselective modification reagent for antibodies by functional substances, comprising a compound or salt thereof having an affinity substance for antibodies and a functional substance. In the regioselective modification reagent for antibodies by functional substances, the details (e.g., definitions, examples, and preferred examples) of the compounds or salts thereof having an affinity substance for antibodies and a functional substance are the same as described above.

[0283] Details of the regioselective modification of the antibody modified in the regioselective modification reagent of the present invention (e.g., definition, examples, and preferred examples) are the same as those described above.

[0284] The regioselective modification reagent of the present invention may be provided in the form of a composition further comprising other components. Such other components include, for example, solutions and stabilizers (e.g., antioxidants, preservatives). As the solution, an aqueous solution is preferred. Examples of aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline) and buffer solutions (e.g., aqueous phosphoric acid solution, Tris-hydrochloride buffer, carbonic acid-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), but buffer solutions are preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The regioselective modification reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[0285] Antibodies or salts thereof that possess regioselectively orthogonal functional groups are useful, for example, as intermediates in the preparation of antibodies or salts thereof that possess regioselectively functional substances.

[0286] Antibodies or salts thereof that regioselectively contain functional substances are useful, for example, as pharmaceuticals or reagents (e.g., diagnostic agents, research reagents), particularly as pharmaceuticals. It has been reported that changing the number and binding site of drugs in an antibody-drug conjugate (ADC) alters pharmacokinetics, drug release rate, and efficacy. For these reasons, it is necessary to control the number and position of conjugated drugs in next-generation ADCs. It is believed that if the number and position are constant, the expected efficacy, variations in conjugated drugs, and lot-to-lot differences, or regulation issues, will be resolved. The antibody or salt thereof of the present invention that regioselectively contains functional substances can solve these regulation problems. Therefore, the antibody or salt thereof of the present invention that regioselectively contains functional substances may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the antibody or salt thereof that regioselectively contains functional substances. Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizine ammonium salt, glycine, and 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; and base waxes such as cocoa butter, polyethylene glycol, and kerosene.The antibody or salt thereof of the present invention, which regioselectively contains a functional substance, may also have any modifications (e.g., PEGylation) to achieve stability.

[0287] Suitable formulations for oral administration include liquid preparations in which an effective amount of ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of ligand as a solid or granule; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution of an effective amount of the active ingredient is dispersed in a suitable dispersion medium and emulsified.

[0288] Pharmaceutical compositions are suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Suitable pharmaceutical compositions for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Also, aqueous and non-aqueous sterile suspensions are examples, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0289] The dosage of a pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the target animal species, the target animal's drug tolerance, body weight, age, etc., but can be set as appropriate. [Examples]

[0290] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0291] [Example 1: Synthesis of IgG1 Fc affinity substance] (1-1) Synthesis of affinity peptides for antibodies The peptides listed below, which are affinity substances for antibodies, were all prepared by the same method. Solid-phase peptide synthesis using Rink amide resin via the Fmoc method was performed to prepare compounds with the N-terminus capped with an acetyl group (compounds 1, 2, 32-53) and compounds with the N-terminus capped with 3-(triphenylmethylthio)propionic acid (compounds 3, 31, 54). These compounds were then cleaved from the resin and deprotected by stirring in a solution of trifluoroacetic acid:water:triisopropylsilane:ethanedithiol = 94:2.5:1.0:2.5 for 3 hours. The resin was removed by filtration, diethyl ether was added for precipitation, and the diethyl ether was removed by decantation to obtain the peptides as crude crystals. These were purified by preparative HPLC to obtain the affinity peptides.

[0292] Compounds 35-53, which are peptides containing disulfide bonds within the molecule (excluding those containing methionine), were synthesized by first obtaining linear precursor peptides using the method described above, and then synthesizing them using the method described below. Several tens of milligrams of the obtained precursor were dissolved in 1 mL of DMSO, 100 μL of NH3 / MeOH and 100 μL of H2O2 were added, and the mixture was stirred overnight. After confirming that the reaction was complete by LC-MS, the mixture was purified by preparative HPLC to obtain the target affinity peptides.

[0293] Compounds 33 and 34, which are peptides containing disulfide bonds (methionine), were synthesized by first obtaining linear precursor peptides using the method described above, and then synthesizing them using the method described below. Several tens of milligrams of the obtained precursor 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. After confirming that the reaction was complete by LC-MS, the mixture was purified by preparative HPLC to obtain the target affinity peptides.

[0294] [ka]

[0295] MS (ESI) m / z: z = 3 1392 [M + 3H] 3+ , z = 4 1044 [M + 4H] 4+

[0296] [Chemical formula]

[0297] MS (ESI) m / z: z = 3 1392 [M + 3H] 3+ , z = 4 1044 [M + 4H] 4+

[0298] [Chemical formula]

[0299] MS (ESI) m / z: z = 3 1478 [M + 3H] 3+ , z = 4 1108 [M + 4H] 4+

[0300] [Chemical formula]

[0301] MS (ESI) m / z: z = 2 1892 [M + 2H] 2+ , Z = 3 1262 [M + 3H] 3+ , Z = 4 946 [M + 4H] 4+ , z = 5 757 [M + 5H] 5+

[0302] [Chemical formula]

[0303] MS (ESI) m / z: z = 3 1401 [M + 3H] 3+ , Z = 4 1051 [M + 4H] 4+ , z = 5 841 [M + 5H] 5+

[0304] [Chemical formula]

[0305] MS (ESI) m / z: z = 3 1426 [M + 3H] 3+ , Z = 4 1070 [M + 4H] 4+ , z = 5 859 [M + 5H] 5+

[0306] [Chemical formula]

[0307] MS (ESI) m / z: z = 3 1417 [M + 3H] 3+ , Z = 4 1063 [M + 4H] 4+ , z = 5 851 [M + 5H] 5+

[0308] [Chemical formula]

[0309] MS (ESI) m / z: z = 3 1425 [M + 3H] 3+ , Z = 4 1069 [M + 4H] 4+ , z = 5 855 [M + 5H] 5+

[0310] [Chemical formula]

[0311] MS (ESI) m / z: z = 1 2090 [M + 1H] + , Z = 2 1045 [M + 2H] 2+ , Z = 3 697 [M + 3H] 3+

[0312] [[ID=6​​​​​​

[0313] MS (ESI) m / z: z = 1 2074 [M + 1H] + , Z = 2 1037 [M + 2H] 2+ , Z = 3 692 [M + 3H] 3+

[0314]

Chem.

[0315] MS (ESI) m / z: z = 1 2061 [M + 1H] + , Z = 2 1031 [M + 2H] 2+ , Z = 3 687 [M + 3H] 3+

[0316]

Chem.

[0317] MS (ESI) m / z: z = 1 2032 [M + 1H] + , Z = 2 1016 [M + 2H] 2+ , Z = 3 678 [M + 3H] 3+

[0318]

Chem.

[0319] MS (ESI) m / z: z = 1 2089 [M + 1H] + , Z = 2 1044 [M + 2H] 2+ , Z = 3 696 [M + 3H] 3+

[0320]

Chem.

[0321] MS (ESI) m / z: z = 1 2088 [M + 1H] +, Z = 2 1044 [M + 2H] 2+ , Z = 3 696 [M + 3H] 3+

[0322] [Chemical formula]

[0323] MS (ESI) m / z: z = 1 1561 [M + H] + , Z = 2 781 [M + 2H] 2+

[0324] [Chemical formula]

[0325] MS (ESI) m / z: z = 1 1548 [M + 1H] + , Z = 2 774 [M + 2H] 2+

[0326] [Chemical formula]

[0327] MS (ESI) m / z: z = 2 1059 [M + 2H] 2+ , Z = 3 706 [M + 3H] 3+

[0328] [Chemical formula]

[0329] MS (ESI) m / z: z = 2 1074 [M + 2H] 2+ , Z = 3 716 [M + 3H] 3+

[0330] <000...It seems the text is incomplete at the end. If you can provide the full text, I can give a more complete translation.​​​​​​MS (ESI) m / z: z = 2 1081 [M + 2H] 2+ , z = 3 721 [M + 3H] 3+ , z = 4 541 [M + 4H] 4+

[0332]

Chem.

[0333] MS (ESI) m / z: z = 2 1085 [M + 2H] 2+ , z = 3 723 [M + 3H] 3+ , z = 4 543 [M + 4H] 4+

[0334]

Chem.

[0335] MS (ESI) m / z: z = 2 1045 [M + 2H] 2+ , z = 3 697 [M + 3H] 3+

[0336]

Chem.

[0337] MS (ESI) m / z: z = 1 1345 [M + 1H] + , z = 2 673 [M + 2H] 2+

[0338]

Chem.

[0339] MS (ESI) m / z: z = 2 1052 [M + 2H] 2+ , z = 3 702 [M + 3H] 3+

[0340]

Chem.

化

Chem.

[0341] MS (ESI)m / z:z=2 1073 [M+2H] 2+ Z=3715 [M+3H] 3+

[0342]

change

[0343] MS (ESI)m / z:z=2 1073 [M+2H] 2+ Z=3716 [M+3H] 3+

[0344]

change

[0345] MS (ESI)m / z:z=2 966 [M+2H] 2+ Z=3644 [M+3H] 3+

[0346]

change

[0347] MS (ESI)m / z:z=3 1381 [M+3H] 3+ Z=4 1036 [M+4H] 4+ z=5 829 [M+5H] 5+

[0348] (1-2) Synthesis of Antibody Antibody Affinity Biotechnology The peptide-azide adducts (compounds 4 and 5) shown below were synthesized as follows. Using amino acids in which only the Lys residue, which will later be functionalized, was protected with an mtt group, a peptide capped with an acetyl group at the N-terminus was prepared by solid-phase peptide synthesis using a Rink amide resin by the Fmoc method. The mixture was stirred for 1 hour in a solution of dichloromethane:trifluoroacetic acid:triisopropylsilane = 90:5:5 to deprotect only the mtt group. After washing the resin with DMF, it was added in a solution of triethylamine (50 molar equivalents), azidoacetyl acid NHS ester (10 molar equivalents), and 4 mL of DMF, and stirred for 16 hours. After removing the solution, the peptide was cleaved from the resin and deprotected by stirring for 1 hour in a solution of trifluoroacetic acid:water:triisopropylsilane = 95:2.5:2.5. The resin was removed by filtration, precipitation was performed by adding diethyl ether, and 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, which is the product.

[0349] [ka]

[0350] MS(ESI)m / z:z=3 1401[M+3H] 3+ ,z=4 1051[M+4H] 4+

[0351] [ka]

[0352] MS(ESI)m / z:z=3 1420[M+3H] 3+ ,z=4 1065[M+4H] 4+

[0353] [Example 2: Synthesis of antibody-modified linker and linkage with IgG1 Fc affinity peptide azide adduct] (2-1) Synthesis of imidazolylcarbonyl compounds (2-1-1) Synthesis of imidazolyl carbonyl compounds (compound 6) [ka]

[0354] 3-Butyn-1-ol (0.100 g, 1.32 mmol) and carbonyldiimidazole (263 mg, 1.62 mmol) were dissolved in THF solvent and stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate, washed with water and saline solution, and then sodium sulfate was added and allowed to stand for 5 minutes. Sodium sulfate was removed by filtration, and the crude product was obtained by concentration under reduced pressure, after which it was purified by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain 1H-imidazole-1-carboxylic acid-3-butynyl ester (0.180 g, 1.10 mmol), corresponding to compound 6.

[0355] 1 H NMR(400MHz,Chloroform-d) δ 2.08(t,J=2.7Hz,1H),2.73(td,J=6.6,2.7Hz,2H),4.54(t,J=6.6Hz,2H),7.11(s,1H),7.43(s,1H),8.18(s,1H).

[0356] MS(ESI) m / z:165[M+Na] +

[0357] (2-1-2) Synthesis of imidazolyl carbonyl compounds (compound 7) [ka]

[0358] 9-decine-1-ol (0.100 g, 0.745 mmol) and carbonyldiimidazole (148 mg, 0.916 mmol) were dissolved in THF solvent and stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate, washed with water and saline solution, and then sodium sulfate was added and allowed to stand for 5 minutes. Sodium sulfate was removed by filtration, and the crude product was obtained by concentration under reduced pressure, after which it was purified by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain 1H-imidazole-1-carboxylic acid-9-butynyl ester (0.152 g, 0.612 mmol), corresponding to compound 7.

[0359] 1 H NMR(400MHz,Chloroform-d) δ 1.33-1.50(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).

[0360] MS(ESI) m / z:271[M+Na] +

[0361] (2-1-3) Synthesis of imidazolyl carbonyl compounds (compound 9) [ka]

[0362] N,N'-dicyclohexylcarbodiimide (113 mg, 0.546 mmol), 1-hydroxybenzotriazole monohydrate (78.1 mg, 0.546 mmol), and 4-pentic acid (53.6 mg, 0.546 mmol) were dissolved in dichloromethane solvent and stirred at room temperature for 1 hour. Then, 12-amino-dodecanol (0.100 g, 0.497 mmol) was added and the mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate, washed with water and saline solution, and then sodium sulfate was added and allowed to stand for 5 minutes. Sodium sulfate was removed by filtration, and the mixture was concentrated under reduced pressure to obtain 12-(penti-4-in-1-oxo)aminododecane-1-ol (0.102 g, 0.363 mmol), which corresponds to compound 8.

[0363] MS(ESI) m / z:281[M+H] +

[0364] [ka]

[0365] 12-(penti-4-inamide)dodecane-1-ol (25.0 mg, 0.089 mmol) and carbonyldiimidazole (29.1 mg, 0.178 mmol) were dissolved in THF solvent and stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate, washed with water and saline solution, and then sodium sulfate was added and allowed to stand for 5 minutes. Sodium sulfate was removed by filtration, and the crude product was obtained by concentration under reduced pressure, after which it was purified by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain 1H-imidazole-1-carboxylic acid-12-(penti-4-in-1-oxo)aminododecanyl ester (12.0 mg, 0.032 mmol), which corresponds to compound 9.

[0366] 1H NMR(400MHz,Chloroform-d) δ 1.23-1.50(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).

[0367] MS(ESI) m / z:398[M+Na] +

[0368] (2-2) Synthesis of imidazolylcarbonyl compounds and linkage with affinity peptide azide derivatives 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 all linked in the same manner. The peptide azide was dissolved in 100 mM phosphate buffer (pH 7.0), and aminoguanidine hydrochloride (20 molar equivalents), sodium ascorbate (20 molar equivalents), and a 100 mg / mL solution of imidazolyl carbonyl compound (3 molar equivalents) in dimethyl sulfoxide were added. To this reaction mixture, aqueous solutions of copper sulfate monohydrate (4 molar equivalents) and tris(3-hydroxypropyltriazolylmethyl)amine (20 molar equivalents), which were prepared separately, were added and the mixture was stirred. After monitoring the reaction using LC-MS and confirming the disappearance of the starting material, the mixture was concentrated and diluted with water four times using 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.

[0369] [ka]

[0370] MS (ESI)m / z:z=3 1455[M+3H] 3+ ,z=4 1092[M+4H] 4+

[0371] [ka]

[0372] MS (ESI)m / z:z=3 1483[M+3H] 3+ ,z=4 1113[M+4H] 4+

[0373] [ka]

[0374] MS (ESI)m / z:z=3 1525[M+3H] 3+ ,z=4 1144[M+4H] 4+

[0375] [Reference Example 1: Verification of differences in reactivity due to differences in the number of atoms from affinity peptides to antibody modification groups (electrophiles) using model experiments] (1-1) Synthesis of IgG antibody trastuzumab-peptide complex 20 μg of the 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 Example 2 (2-2) were added relative to the antibody, and the mixture was stirred at 37°C for 4 hours. The reaction was stopped by removing the peptide reagent by ultrafiltration (Amicon Ultra, 3K MWCO) to obtain the IgG antibody trastuzumab-peptide complex.

[0376] (1-2) Analysis of IgG antibody trastuzumab-peptide complex by SDS-PAGE Figure 2 shows the results of analyzing three types of IgG antibody trastuzumab-peptide conjugates obtained in (1-1) using three peptide-imidazolyl carbonyl compounds (compounds 10, 11, and 12) by SDS-PAGE (Mini-PROTEAN TGX gel, 4-20%, Bio-RAD; under reducing conditions; stained with Coomasie Brilliant Blue G-250 Stain). From these results, it was found that the antibody modification reaction proceeded when compounds 10 and 11 were used, but did not proceed when compound 12 was used.

[0377] [Example 3: Synthesis of an antibody affinity reagent containing maleimide] (3-1) Synthesis of Protected Thiol Compounds (3-1-1) Synthesis of Protected Thiol Compound (Compound 14) [ka]

[0378] 3-(triphenylmethylthio)propionic acid (100 mg, 0.287 mmol) was dissolved in THF, and isobutyl chlorocarbonate (42.7 μL, 0.316 mmol) and N-methylmorpholine (69.4 μL, 0.631 mmol) were added at 0°C and the mixture was stirred for 30 minutes to prepare the corresponding mixed acid anhydride. 5-aminovaleric acid (33.6 mg, 0.287 mmol) was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, and the THF solution of the aforementioned mixed acid 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 collected. 6 M aqueous hydrochloric acid solution was added to the aqueous phase to adjust the pH of the system to 3.0, followed by liquid-liquid extraction with ethyl acetate. The organic phase was washed with saline solution, and anhydrous magnesium sulfate was added and allowed to stand for 5 minutes. Magnesium sulfate was removed by filtration, and the crude product was obtained by concentration under reduced pressure, after which it was purified by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain 5-[3-(triphenylmethyl)sulfanyl-propyl-1-oxo]aminohexanoic acid (110 mg, 0.246 mmol), which corresponds to compound 13.

[0379] 1 H NMR(400MHz,Chloroform-d) δ 1.66(m,3H),2.03(t,J=7.3Hz,2H),2.39(t,J=7.3Hz,2H),2.52(t,J=7.3Hz,2 H),3.22(q,J=6.6Hz,2H),5.37(s,1H),7.19-7.36(m,9H),7.40-7.49(m,6H).

[0380] MS(ESI) m / z:470[M+Na] +

[0381] [ka]

[0382] 5-(3-triphenylmethylpropyl-1-oxo)aminohexanoic acid (20.2 mg, 0.045 mmol) was dissolved in THF, and isobutyl chlorocarbonate (6.70 μL, 0.050 mmol) and N-methylmorpholine (10.9 μL, 0.0991 mmol) were added at 0°C and the mixture was stirred for 30 minutes to prepare the corresponding mixed acid anhydride. Propargylamine (16.6 μL, 0.226 mmol) was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, and the THF solution of the aforementioned mixed acid 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 collected. 6 M aqueous hydrochloric acid solution was added to the aqueous phase to adjust the pH of the system to 3.0, and then liquid-liquid extraction with ethyl acetate was performed. After washing the organic phase with saline solution, anhydrous magnesium sulfate was added and the mixture was allowed to stand for 5 minutes. Magnesium sulfate was removed by filtration, and the crude product was obtained by concentration under reduced pressure, followed by purification by silica gel column chromatography. The fraction containing the product was recovered and concentrated under reduced pressure to obtain N-propynyl-5-(3-triphenylmethylsulfanyl-propyl-1-oxo)amino-hexaneamide (20.0 mg, 0.041 mmol), corresponding to compound 14.

[0383] 1 H NMR(400MHz,Chloroform-d) δ 1.52(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).

[0384] MS(ESI) m / z:507[M+H] +

[0385] (3-1-2) Synthesis of Protected Thiol Compounds (Compound 15) [ka]

[0386] 3-(triphenylmethylthio)propionic acid (100 mg, 0.287 mmol) was dissolved in THF, and isobutyl chlorocarbonate (42.7 μL, 0.316 mmol) and N-methylmorpholine (69.4 μL, 0.631 mmol) were added at 0°C and the mixture was stirred for 30 minutes to prepare the corresponding mixed acid anhydride. Propargylamine (105 μL, 1.43 mmol) was dissolved in 1 M aqueous sodium hydroxide solution at room temperature, and the THF solution of the aforementioned mixed acid 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 collected. 6 M aqueous hydrochloric acid solution was added to the aqueous phase to adjust the pH of the system to 3.0, and then liquid-liquid extraction was performed with ethyl acetate. After washing the organic phase with saline solution, anhydrous magnesium sulfate was added and the mixture was allowed to stand for 5 minutes. By removing magnesium sulfate by filtration and concentrating under reduced pressure, N-propynyl-3-triphenylmethylsulfanil-propanamide (110 mg, 0.286 mmol), corresponding to compound 15, was obtained.

[0387] 1H NMR(400MHz,Chloroform-d) δ 2.01(t,J=7.1Hz,2H),2.24(t,J=2.8Hz,1H),2.53(t,J=7.6Hz,2H),3.99(m,2H),5.40(brs,1H),7.19-7.35(m,9H),7.40-7.49(m,6H).

[0388] MS(ESI) m / z:408[M+Na] +

[0389] (3-2) Linking affinity peptides and protected thiol compounds (3-2-1) Peptide-protected thiol conjugate (compound 16) synthesis [ka]

[0390] Compound 5 synthesized in Example 1 was dissolved in N,N'-dimethylformamide, and 3-(triphenylmethylthio)propionic acid (5 molar equivalents), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (15 molar equivalents), and 1-hydroxybenzotriazole (15 molar equivalents) 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. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the peptide-protected thiol conjugate (compound 16).

[0391] MS (ESI)m / z:z=3 1502[M+3H] 3+ ,z=4 1125[M+4H] 4+

[0392] (3-2-2) Synthesis of peptide-protected thiol conjugates (compounds 17, 18) The affinity peptide azide (compound 5) synthesized in Example 1 and the protected thiol compounds (compounds 17, 18) synthesized in (4-1-1) and (4-1-2) were linked in the same manner. The peptide azide was dissolved in 100 mM phosphate buffer (pH 7.0), and aminoguanidine hydrochloride (20 molar equivalents), sodium ascorbate (20 molar equivalents), and a 100 mg / mL solution of the protected thiol compound (3 molar equivalents) in dimethyl sulfoxide were added. To this reaction mixture, a separately prepared aqueous solution of copper sulfate monohydrate (4 molar equivalents) and tris(3-hydroxypropyltriazolylmethyl)amine (20 molar equivalents) was added and the mixture was stirred. After monitoring the reaction using LC-MS and confirming the disappearance of the starting material, the mixture was concentrated and diluted with water four times using ultrafiltration (Amicon Ultra-4, 3K MWCO). The resulting aqueous solution was freeze-dried to obtain peptide-protected thiol conjugates (compounds 17, 18).

[0393] [ka]

[0394] MS(ESI) m / z:z=3 1548[M+3H] 3+ ,z=4 1161[M+4H] 4+

[0395] [ka]

[0396] MS(ESI) m / z:z=3 1581[M+3H] 3+ ,z=4 1186[M+4H] 4+

[0397] (3-3) Synthesis of peptide-protected thiol conjugates (compounds 19, 20, 21) The affinity peptide-protected thiol conjugates (compounds 16, 17, 18) synthesized in (3-2) were stirred in a trifluoroacetic acid:dichloromethane = 1:1 solution for 1 hour to remove the triphenylmethyl group. After confirming the end of the reaction by LC-MS, the product was purified by preparative HPLC. The fraction containing the target product was identified by ESI-MS, and then freeze-dried to obtain the thiol-peptide conjugates (compounds 19, 20, 21).

[0398] [ka]

[0399] MS(ESI) m / z: z=3 1454[M+3H] 3+ ,z=4 1091[M+4H] 4+

[0400] [ka]

[0401] MS(ESI) m / z:z=3 1468[M+3H] 3+ ,z=4 1111[M+4H] 4+

[0402] [ka]

[0403] m / z: z=3 1501[M+3H] 3+ ,z=4 1126[M+4H] 4+

[0404] (3-4) Synthesis of antibody affinity peptide reagents containing maleimide (1) (Compounds 22, 23, 24) The thiol-peptide conjugates (compounds 19, 20, 21) synthesized in (3-3) were dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of N-hydroxymaleimide (20 molar equivalents) was added, and the mixture was stirred for 1 hour. After confirming the disappearance of the starting material by LC-MS, trans-4-[(2,5-dihydro-1H-pyrrolic-1-yl)methyl)cyclohexanecarboxylic acid (40 molar equivalents), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (30 molar equivalents), and 1-hydroxybenzotriazole (30 molar equivalents) were added, and the mixture was stirred for 4 hours. After confirming the termination of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the maleimide-affinity peptide reagents (compounds 22, 23, 24).

[0405] [ka]

[0406] MS(ESI) m / z: z=3 1532[M+3H] 3+ ,z=4 1150[M+4H] 4+

[0407] [ka]

[0408] MS(ESI) m / z: z=3 1578[M+3H] 3+ ,z=4 1184[M+4H] 4+

[0409] [ka]

[0410] MS(ESI) m / z:z=3 1611[M+3H] 3+ ,z=4 1208[M+4H] 4+

[0411] (3-5) Synthesis of antibody affinity peptide reagents containing maleimide (2) (Compounds 56, 58) (3-5-1) Synthesis of N-hydroxysuccinimide-peptide linkage (compound 55) Compound 3 synthesized in (1-1) was dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of triethylamine (1 equivalent) and N-hydroxymaleimide (1.2 equivalents) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the completion of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the product, an N-hydroxysuccinimide-peptide conjugate (compound 55).

[0412] [ka]

[0413] MS (ESI)m / z:z=3 1429 [M+3H] 3+ , Z=4 1072 [M+4H] 4+ , z=5 857 [M+5H] 5+ , z=6 715 [M+6H] 6+

[0414] (3-5-2) Synthesis of antibody affinity peptide reagent containing maleimide (Compound 56) The N-hydroxysuccinimide-peptide conjugate obtained in (3-5-1), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-succinimidyl (20 equivalents), was dissolved in dimethyl sulfoxide, and triethylamine (8 equivalents) was added. After stirring at room temperature for 2 hours, the reaction was confirmed by LC-MS. The mixture was purified by preparative HPLC, and the fraction containing the target product was confirmed by ESI-MS. The mixture was then freeze-dried to obtain an antibody affinity peptide reagent (compound 56) containing the target maleimide.

[0415] [ka]

[0416] MS (ESI)m / z:z=3 1502 [M+3H] 3+ , Z=4 1126 [M+4H] 4+ , z=5 901 [M+5H] 5+ , z=6 751 [M+6H] 6+

[0417] (3-5-3) Synthesis of N-hydroxysuccinimide-peptide linkage (compound 57) The antibody affinity peptide (compound 36) synthesized in Example 1 was dissolved in dimethyl sulfoxide, and 2-iminothiolane hydrochloride (10 molar equivalents) and diisopropylethylamine (15 molar equivalents) were added, and the mixture was stirred for 1 hour. After confirming the disappearance of the starting material by LC-MS, a dimethyl sulfoxide solution of N-hydroxymaleimide (20 molar equivalents) was added, and the mixture was stirred for 1 hour. After confirming the progress of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the product, a hydroxymaleimide-affinity peptide (compound 57).

[0418] [ka]

[0419] MS (ESI)m / z:z=2 1152 [M+2H] 2+ , Z=3 769 [M+3H] 3+

[0420] (3-5-4) Synthesis of antibody affinity peptide reagents containing maleimide (Compound 58) The N-hydroxysuccinimide-peptide conjugate (H1) obtained in (3-5-2), 4-[(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)methyl]bezoic acid (20 equivalents) was dissolved in dimethyl sulfoxide, and triethylamine (8 equivalents) was added. After stirring at room temperature for 2 hours, the reaction was confirmed by LC-MS. The mixture was purified by preparative HPLC, and the fraction containing the target product was confirmed by ESI-MS. The mixture was then freeze-dried to obtain an antibody affinity peptide reagent (compound 58) containing the target maleimide.

[0421] [ka]

[0422] MS (ESI)m / z:z=2 1259 [M+2H] 2+ , Z=3 840 [M+3H] 3+

[0423] [Example 4: Regioselective maleimide introduction and analysis of the anti-HER2 IgG antibody trastuzumab] (4-1) Synthesis of thiol-containing peptide reagents Compound 25, described below, was synthesized by solid-phase peptide synthesis using 2-chlorotrityllase resin via the Fmoc method. After solid-phase synthesis, the peptide was cleaved from the resin and deprotected by stirring for 1 hour in a solution of trifluoroacetic acid:water:triisopropylsilane = 95:2.5:2.5. The resin was removed by filtration, precipitation was performed by adding diethyl ether, and the diethyl ether was removed by decantation to obtain the peptide as crude crystals. This was purified by preparative HPLC to obtain the product, compound 25, described below. Compound 25 is not an affinity peptide, but a model peptide compound that should be introduced via the maleimide group to antibodies that regioselectively possess the bioorthogonal functional group maleimide, as an alternative to a drug.

[0424] [ka]

[0425] MS (ESI) m / z: z=2 1482[M+2H] 2+ ,z=3 988[M+3H] 3+

[0426] (4-2) Site-specific modification of the IgG antibody trastuzumab using maleimide-affinity peptide reagents The maleimide-affinity peptide reagents (compounds 22, 23, 24) synthesized in (4-4) of Example 4 were dissolved in N,N'-dimethylformamide to a concentration of 2.18 mM. 20.0 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 14.8 μL of 50 mM HEPES buffer (pH 7.2), and the peptide reagents (10-40 molar equivalents) were added to the antibody. The mixture was stirred at 37°C for 4 hours. The reaction mixture was replaced with 20 mM acetate buffer (pH 4.5) by ultrafiltration (Amicon Ultra, 10K MWCO). The affinity peptide reagents were removed by repeating this procedure three times to obtain the IgG antibody trastuzumab-maleimide modified product.

[0427] (4-3) Conjugation of IgG antibody trastuzumab-maleimide modified compound with thiol-containing peptide reagent The IgG antibody trastuzumab-maleimide modified compound synthesized in Example 5 (5-2) was replaced with 50 mM HEPES buffer (pH 7.2) by ultrafiltration (Amicon Ultra, 10K MWCO) to neutralize the pH of the system. A 10 mg / mL aqueous solution of compound 25 (80 molar equivalents relative to the antibody) was added, and the mixture was stirred for 16 hours. The reaction was stopped by removing the peptide reagent by ultrafiltration (Amicon Ultra, 10K MWCO) to obtain the IgG antibody trastuzumab-peptide complex.

[0428] (4-4) Analysis of IgG antibody trastuzumab-peptide complex by SDS-PAGE Figure 3 shows the results of analyzing the IgG antibody trastuzumab-peptide complex obtained in (4-3) by SDS-PAGE (Mini-PROTEAN TGX gel, 4-20%, Bio-RAD; under reducing conditions; stained with Coomasie Brilliant Blue G-250 Stain). From these results, it was found that in the regioselective introduction reaction of maleimide (5-2), the antibody modification reaction proceeded when compounds 22 and 23 were used, but did not proceed when compound 24 was used.

[0429] (4-5) Confirmation of heavy chain selectivity of IgG antibody trastuzumab-maleimide modified by ESI-TOFMS analysis under reducing conditions. (4-5-1) Synthesis of IgG antibody trastuzumab-maleimide modified compound (1) The affinity peptide reagent (compound 56) containing maleimide synthesized in Example 3 (3-5-2) was dissolved in N,N'-dimethylformamide to a concentration of 2.0 mM. 250 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 230 μL of 50 mM MHE PES buffer (pH 7.2), and 20 molar equivalents of the 2.0 mM peptide reagent were added relative to the antibody. The mixture was stirred at 37°C for 16 hours. The IgG antibody trastuzumab-maleimide modified product was obtained by concentration by ultrafiltration (Amicon Ultra, 10k MWCO).

[0430] (4-5-2) Confirmation of heavy chain selectivity of trastuzumab-maleimide modified compound by ESI-TOFMS analysis under reducing conditions (1) To the trastuzumab-maleimide modified product synthesized in (4-5-1), 2.0 μL of 7 mM tris(2-carboxyethyl)phosphine hydrochloride solution (100 equivalents relative to the antibody) was added and left at room temperature for 20 minutes. Mass measurement by ESI-TOFMS revealed heavy chain peaks at 50596 and 50757, and a light chain peak at 23439 for the trastuzumab used as a control. For the reaction product, maleimide reacted with tris(2-carboxyethyl)phosphine was introduced into the heavy chain at 51062 and 51224, and a light chain peak was observed at 23439, the same as the starting material. The ESI-TOFMS analysis results are shown in Figure 4.

[0431] (4-5-3) Synthesis of IgG antibody trastuzumab-maleimide modified compounds (2) Similar to (4-5-1), an affinity peptide reagent (compound 58) containing the maleimide synthesized in (3-5-4) of Example 3 was used to obtain the IgG antibody trastuzumab-azide modified product.

[0432] (4-5-4) Confirmation of heavy chain selectivity of trastuzumab-maleimide modified compound by ESI-TOFMS analysis under reducing conditions (2) The same procedure as in (4-5-2) was followed, and the mass was measured by ESI-TOFMS. Trastuzumab, the starting material used as a control, showed heavy chain peaks at 50602 and 50764, and a light chain peak at 23443. The reaction products showed a light chain peak at 50818 and 50979, in which maleimide reacted with N-acetylcysteine ​​was introduced into the heavy chain, and at 23443, the same as the starting material.

[0433] [Example 5: Synthesis of antibody modification reagent containing azide] (5-1) Synthesis of antibody modification reagents (comparative examples) having a heteroatom at the β-position of the antibody modification group (electrophile) [ka]

[0434] The antibody affinity peptide (compound 2) synthesized in Example 1 was dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of N-hydroxymaleimide (20 molar equivalents) was added, and the mixture was stirred for 1 hour. After confirming the disappearance of the starting material by LC-MS, 11-azido-3,6,9-trioxaundecanoic acid (40 molar equivalents), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (32 molar equivalents), and 1-hydroxybenzotriazole (32 molar equivalents) were added, and the mixture was stirred for 2 hours. After confirming the completion of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the azido-PEG-affinity peptide reagent (compound 26).

[0435] MS (ESI)m / z:z=3 1501[M+3H] 3+ ,z=4 1125[M+4H] 4+

[0436] (5-2) Synthesis of antibody modification reagents having a carbon atom or heteroatom at the β-position of the antibody modification group (electrophile) (5-2-1) Synthesis of antibody-modified reagents (compounds 27, 59, 60) by linking azide reagents with affinity peptides. [ka]

[0437] The antibody affinity peptide (compound 3) synthesized in Example 1 was dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of N-hydroxymaleimide (20 molar equivalents) was added, and the mixture was stirred for 1 hour. After confirming the disappearance of the starting material by LC-MS, 6-azidohexanoic acid (40 molar equivalents), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (32 molar equivalents), and 1-hydroxybenzotriazole (32 molar equivalents) were added, and the mixture was stirred for 2 hours. After confirming the end of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the azido-alkyl-affinity peptide reagent (compound 27).

[0438] MS (ESI)m / z:z=3 1475[M+3H] 3+ ,z=4 1107[M+4H] 4+

[0439] Using a similar method, the N-hydroxysuccinimide-peptide conjugate (compound 57) synthesized in Example 3 was used to obtain an azido-alkyl-affinity peptide reagent (compound 59).

[0440] [ka]

[0441] MS (ESI)m / z:z=2 1222 [M+2H] 2+ , Z=3 815 [M+3H] 3+

[0442] The synthesis was carried out similarly by replacing 6-azidohexanoic acid with azidoacetic acid to obtain an azido-alkyl-affinity peptide reagent (compound 60). Compound 60 is a comparative example compound having a heteroatom at the β-position of the antibody-modifying group (electrophile). [ka]

[0443] MS (ESI)m / z:z=2 1193 [M+2H] 2+ , Z=3 796 [M+3H] 3+

[0444] (5-2-2) Synthesis of antibody-modified reagents (compounds 28, 61) by linking azide reagents with affinity peptides [ka]

[0445] The antibody affinity peptide (compound 3) synthesized in Example 1 was dissolved in dimethyl sulfoxide, and a dimethyl sulfoxide solution of N-hydroxymaleimide (20 molar equivalents) was added, and the mixture was stirred for 1 hour. After confirming the disappearance of the starting material by LC-MS, 4-azidobenzoic acid (40 molar equivalents), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (32 molar equivalents), and 1-hydroxybenzotriazole (32 molar equivalents) were added, and the mixture was stirred for 2 hours. After confirming the end of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the azidophenyl affinity peptide reagent (compound 28).

[0446] MS (ESI)m / z:z=3 1478[M+3H] 3+ ,z=4 1108[M+4H] 4+

[0447] Using a similar method, an azidophenyl-affinity peptide reagent (compound 61) was obtained using the antibody affinity peptide (compound 31) synthesized in Example 1.

[0448] [ka]

[0449] MS (ESI)m / z:z=3 1348 [M+3H] 3+ , Z=4 1011 [M+4H] 4+

[0450] (5-2-3) Synthesis of antibody-modified reagents (compounds 29, 62-83) by linking azide reagents with affinity peptides [ka]

[0451] The antibody affinity peptide (compound 3) synthesized in Example 1 was dissolved in dimethyl sulfoxide, and 2-iminothiolane hydrochloride (10 molar equivalents) and diisopropylethylamine (15 molar equivalents) were added, and the mixture was stirred for 1 hour. After confirming the disappearance of the starting material by LC-MS, a dimethyl sulfoxide solution of N-hydroxymaleimide (20 molar equivalents) was added, and the mixture was stirred for 1 hour. After confirming the progress of the reaction by LC-MS, 4-azidobenzoic acid (40 molar equivalents), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (32 molar equivalents), and 1-hydroxybenzotriazole (32 molar equivalents) were added, and the mixture was stirred for 2 hours. After confirming the termination of the reaction by LC-MS, the mixture was purified by preparative HPLC. After confirming the fraction containing the target product by ESI-MS, it was freeze-dried to obtain the azidophenyl affinity peptide reagent (compound 29).

[0452] MS (ESI)m / z:z=3 1512[M+3H] 3+ ,z=4 1134[M+4H] 4+

[0453] Using a similar method, antibody-modifying reagents were synthesized by linking the antibody-affinity peptides (compounds 32-53) synthesized in Example 1 with the azide reagents shown below (compounds 62-83).

[0454] The antibody modification reagent synthesized using the antibody affinity peptide (compound 32) synthesized in Example 1 is as follows: [ka]

[0455] MS (ESI)m / z:z=3 1521[M+3H] 3+ ,z=4 1141[M+4H] 4+

[0456] The antibody modification reagent synthesized using the antibody affinity peptide (compound 33) synthesized in Example 1 is as follows: [ka]

[0457] MS (ESI)m / z:z=3 1546 [M+3H] 3+ , Z=4 1159 [M+4H] 4+

[0458] The antibody modification reagent synthesized using the antibody affinity peptide (compound 34) synthesized in Example 1 is as follows: [ka]

[0459] MS (ESI)m / z:z=3 1537 [M+3H] 3+ , Z=4 1153 [M+4H] 4+

[0460] The antibody modification reagent synthesized using the antibody affinity peptide (compound 35) synthesized in Example 1 is as follows: [ka]

[0461] MS (ESI)m / z:z=3 1545 [M+3H] 3+ , Z=4 1159 [M+4H] 4+

[0462] The antibody modification reagent synthesized using the antibody affinity peptide (compound 36) synthesized in Example 1 is as follows: [ka]

[0463] MS (ESI)m / z: z=2 1225[M+H] + ,z=3 817[M+H] +

[0464] The antibody modification reagent synthesized using the antibody affinity peptide (compound 37) synthesized in Example 1 is as follows: [ka]

[0465] MS (ESI)m / z:z=2 1217 [M+2H] 2+ ,z=3 812 [M+3H] 3+

[0466] The antibody modification reagent synthesized using the antibody affinity peptide (compound 38) synthesized in Example 1 is as follows: [ka]

[0467] MS (ESI)m / z:z=2 1211 [M+2H] 2+ ,z=3 807 [M+3H] 3+

[0468] The antibody modification reagent synthesized using the antibody affinity peptide (compound 39) synthesized in Example 1 is as follows: [ka]

[0469] MS (ESI)m / z:z=2 1196 [M+2H] 2+ ,z=3 798 [M+3H] 3+

[0470] The antibody modification reagent synthesized using the antibody affinity peptide (compound 40) synthesized in Example 1 is as follows: [ka]

[0471] MS (ESI)m / z:z=2 1224 [M+2H] 2+ ,z=3 817 [M+3H] 3+

[0472] The antibody modification reagent synthesized using the antibody affinity peptide (compound 41) synthesized in Example 1 is as follows: [ka]

[0473] MS (ESI)m / z:z=2 1224 [M+2H] 2+ ,z=3 817 [M+3H] 3+

[0474] The antibody modification reagent synthesized using the antibody affinity peptide (compound 42) synthesized in Example 1 is as follows: [ka]

[0475] MS (ESI)m / z:z=2 961 [M+2H] 2+

[0476] The antibody modification reagent synthesized using the antibody affinity peptide (compound 43) synthesized in Example 1 is as follows: [ka]

[0477] MS (ESI)m / z:z=1 1909[M+H] + ,z=3 955[M+H] +

[0478] The antibody modification reagent synthesized using the antibody affinity peptide (compound 44) ​​synthesized in Example 1 is as follows: [ka]

[0479] MS (ESI)m / z:z=2 1239 [M+2H] 2+ ,z=3 827 [M+3H] 3+

[0480] The antibody modification reagent synthesized using the antibody affinity peptide (compound 45) synthesized in Example 1 is as follows: [ka]

[0481] MS (ESI)m / z:z=2 1253 [M+2H] 2+ ,z=3 836 [M+3H] 3+

[0482] The antibody modification reagent synthesized using the antibody affinity peptide (compound 46) synthesized in Example 1 is as follows: [ka]

[0483] MS (ESI)m / z:z=2 1260 [M+2H] 2+,z=3 841 [M+3H] 3+

[0484] The antibody modification reagent synthesized using the antibody affinity peptide (compound 47) synthesized in Example 1 is as follows: [ka]

[0485] MS (ESI)m / z:z=2 1265[M+H] + ,z=3 844[M+H] +

[0486] The antibody modification reagent synthesized using the antibody affinity peptide (compound 48) synthesized in Example 1 is as follows: [ka]

[0487] MS (ESI)m / z:z=2 1225 [M+2H] 2+ ,z=3 817 [M+3H] 3+

[0488] The antibody modification reagent synthesized using the antibody affinity peptide (compound 49) synthesized in Example 1 is as follows: [ka]

[0489] MS (ESI)m / z:z=2 853 [M+2H] 2+

[0490] The antibody modification reagent synthesized using the antibody affinity peptide (compound 50) synthesized in Example 1 is as follows: [ka]

[0491] MS (ESI)m / z:z=2 1232 [M+2H] 2+ ,z=3 822 [M+3H] 3+

[0492] The antibody modification reagent synthesized using the antibody affinity peptide (compound 51) synthesized in Example 1 is as follows: [ka]

[0493] MS (ESI)m / z:z=2 1253 [M+2H] 2+ ,z=3 836 [M+3H] 3+

[0494] The antibody modification reagent synthesized using the antibody affinity peptide (compound 52) synthesized in Example 1 is as follows: [ka]

[0495] MS (ESI)m / z:z=2 1254 [M+2H] 2+ ,z=3 836 [M+3H] 3+

[0496] The antibody modification reagent synthesized using the antibody affinity peptide (compound 53) synthesized in Example 1 is as follows: [ka]

[0497] MS (ESI)m / z:z=2 1147 [M+2H] 2+ ,z=3 764 [M+3H] 3+

[0498] [Example 6: Site-specific modification of IgG1 Fc using IgG1 Fc affinity peptide reagent and analysis by ESI-TOFMS] (6-1) Antibody modification reaction with antibody modification reagents having a carbon atom at the β position of the antibody modification group (electrophile) (6-1-1) Antibody modification reaction using affinity peptide reagents (compound 27, compound 59) (azide-modified antibodies 1, 2) The affinity peptide reagent (compound 27) containing the azide synthesized in Example 5 was dissolved in N,N'-dimethylformamide to a concentration of 0.22 mM. 200 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 1200 μL of 50 mM MEPES buffer (pH 7.2), and 20 molar equivalents of the 0.22 mM peptide reagent were added relative to the antibody. The mixture was stirred at 37°C for 16 hours. 50 μL of 10 mg / mL Lys aqueous solution was added to the reaction mixture, and the reaction was stopped by stirring for 30 minutes. The reaction mixture was purified using Protein A (Aspire Protein A Tips, Thermo), and the eluate was replaced with 9.57 mM PBS buffer (pH 7.0) and concentrated by ultrafiltration (Amicon Ultra, 10K MWCO) to obtain the IgG antibody trastuzumab-azide modified compound (azide-modified antibody 1).

[0499] Using a similar method, an antibody modification reaction was performed using an affinity peptide reagent (compound 59) containing azide to obtain an IgG antibody trastuzumab-azide modified product (azide-modified antibody 2).

[0500] (6-1-2) Antibody modification reaction using affinity peptide reagents (compound 28, compound 61) (azide-modified antibodies 3, 4) The affinity peptide reagent (compound 28) containing the azide synthesized in Example 5 was dissolved in N,N'-dimethylformamide to a concentration of 0.22 mM. 200 μg of the anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was dissolved in 1200 μL of 50 mM MHE PES buffer (pH 7.2), and 20 molar equivalents of the 0.22 mM peptide reagent were added relative to the antibody. The mixture was stirred at 37°C for 16 hours. 50 μL of 10 mg / mL Lys aqueous solution was added to the reaction mixture, and the reaction was stopped by stirring for 30 minutes. The reaction mixture was purified using Protein A (Aspire Protein A Tips, Thermo), and the eluate was replaced with 9.57 mM PBS buffer (pH 7.0) and concentrated by ultrafiltration (Amicon Ultra, 10K MWCO) to obtain the IgG antibody trastuzumab-azide modified c...

Claims

1. Formula (II) below: Ab-E-B (II) [During the ceremony, Ab is an antibody, E is a divalent group consisting of -C(=O)- linked to an amino group in the side chain of a lysine residue in the constant region of the antibody, and B is a bioorthogonal functional group, wherein the bioorthogonal functional group is an alkene residue, or E comprises (a) -C(=O)- linked to an amino group in the side chain of a lysine residue in the constant region of the antibody, (b) -CH2- linked to the -C(=O)-, and (c) a linear divalent group linked to the -CH2-, wherein the linear divalent group of (c) is a divalent group that does not contain a peptide moiety, and is a combination of (i) a linear divalent hydrocarbon group and (ii) one heteroatom selected from the group consisting of -O- and -S-, and B is a bioorthogonal functional group, wherein the bioorthogonal functional group is an azide residue or an alkene residue. The antibody or a salt thereof having a structural unit represented by E-B at any of the following positions (1) to (4): (1) Lysine residues at position 246 or 248 in human IgG Fc according to Eu numbering; (2) Lysine residues at position 288 or 290 in human IgG Fc according to Eu numbering; (3) The lysine residue at position 317 in human IgG Fc according to Eu numbering; or (4) (i) a lysine residue at position 246 or 248 in human IgG Fc according to Eu numbering, and (ii) a lysine residue at position 288 or 290 in human IgG Fc according to Eu numbering.

2. The antibody or a salt thereof according to claim 1, wherein the linear divalent hydrocarbon group is a linear alkylene.

3. The antibody or salt thereof according to claim 2, wherein the linear alkylene is an alkylene having 1 to 12 carbon atoms.

4. The antibody or salt thereof according to claim 2, wherein the linear alkylene is an alkylene having 1 to 6 carbon atoms.

5. The antibody or a salt thereof according to claim 2, wherein the linear alkylene is an alkylene having 1 to 4 carbon atoms.

6. The bioorthogonal functional group is as follows: 【Chemistry 1】 (Here, R １ｆ and R １ｇ A is a hydrogen atom, and a dot is a bonding hand. The antibody or a salt thereof according to any one of claims 1 to 5, corresponding to any one chemical structure selected from ).

7. The antibody or a salt thereof according to any one of claims 1 to 6, wherein the antibody is an IgG antibody.

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

9. (a) The antibody or salt thereof according to any one of claims 1 to 8, wherein the -C(=O)- linked to the amino group in the side chain of a lysine residue in the constant region of the antibody is the -C(=O)- linked to the amino group in the side chain of a lysine residue in the CH2 region of the antibody.

10. (a) The antibody or salt thereof according to any one of claims 1 to 9, wherein the -C(=O)- linked to the amino group in the side chain of a lysine residue in the constant region of the antibody is the -C(=O)- linked to the amino group in the side chain of a lysine residue at position 246 or 248, a lysine residue at position 288 or 290, or a lysine residue at position 317, according to Eu numbering in human IgG Fc.

11. The antibody or a salt thereof according to any one of claims 1 to 10, wherein the antibody has a plurality of structural units represented by E-B.

12. Formula (III): Ab-E-B'-F (III) [During the ceremony, Ab is an antibody, E is a divalent group consisting of -C(=O)- linked to an amino group in the side chain of a lysine residue in the constant region of the antibody, and B' is a divalent group including a portion produced by a reaction between a functional substance and a bioorthogonal functional group, wherein the bioorthogonal functional group is an alkene residue. ,or E consists of (a) -C(=O)- linked to an amino group in the side chain of a lysine residue in the constant region of the antibody, (b) -CH2- linked to the -C(=O)-, and (c) a linear divalent group linked to the -CH2-, wherein the linear divalent group of (c) is a divalent group that does not contain a peptide moiety, and is a combination of (i) a linear divalent hydrocarbon group and (ii) one heteroatom selected from the group consisting of -O- and -S-, and B' is a divalent group that includes a moiety produced by a reaction between a functional substance and a bioorthogonal functional group, wherein the bioorthogonal functional group is an azide residue or an alkene residue. F includes a drug, labeling agent, or stabilizer as a functional substance. An antibody or salt thereof having a functional substance regioselectively represented by ] The antibody or a salt thereof having a structural unit represented by E-B at any of the following positions (1) to (4): (1) Lysine residues at position 246 or 248 in human IgG Fc according to Eu numbering; (2) Lysine residues at position 288 or 290 in human IgG Fc according to Eu numbering; (3) The lysine residue at position 317 in human IgG Fc according to Eu numbering; or (4) (i) a lysine residue at position 246 or 248 in human IgG Fc according to Eu numbering, and (ii) a lysine residue at position 288 or 290 in human IgG Fc according to Eu numbering.

13. The antibody according to claim 12, wherein the antibody has a plurality of structural units represented by E-B'-F, or a salt thereof.

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