Affinity substance for soluble protein, compound having cleavable moiety and reactive group or salt thereof

Abstract

To provide a technique enabling modification of a soluble protein and in particular regioselective modification of a soluble protein.SOLUTION: The present invention provides a compound having an affinity substance to a soluble protein, a cleavable portion, and a reactive group, represented by the following formula (I): A-L-B-R (I) [where, A is an affinity substance to a soluble protein, L is a cleavable linker which is a divalent group comprising a cleavable portion, B is (a) a divalent group comprising a bioorthogonal functional group or (b) a divalent group comprising no bioorthogonal functional group, and R is a reactive group to the soluble protein] or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an affinity substance for a soluble protein, a compound having a cleavable moiety and a reactive group, or a salt thereof, and the like. [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 a drug (e.g., an anticancer drug) is conjugated to an antibody, and have direct cytocidal activity against cancer cells and the like. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)), jointly developed by Immunogene and Roche (Non-Patent Documents 1 to 3).

[0003] Since the beginning of their development, ADCs, including T-DM1, have faced the problem of heterogeneity. Specifically, because small molecule drugs are randomly conjugated to the approximately 70–80 Lys residues in an antibody, the drug-antibody ratio (DAR) and conjugation position are not consistent. This random conjugation method typically results in a DAR ranging from 0 to 8, resulting in the production of multiple drugs with different numbers of drugs attached. In recent years, it has been reported that varying the number and position of drugs attached to an ADC can affect pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs require control of the number and position of conjugated drugs. It is believed that a consistent number and position would achieve the expected efficacy and resolve issues such as variation in conjugated drugs and lot-to-lot differences, or so-called regulatory issues (Non-Patent Document 4).

[0004] Methods for site-selective modification of antibodies have been studied worldwide, but most of these involve genetic engineering or enzyme-based modification. While genetic engineering modification methods can control site and number selectivity, problems have been noted, such as a decrease in the expression efficiency of the antibody itself (reducing the overall yield when preparing ADCs). Another problem is that it takes a long time to establish an antibody expression system (Non-Patent Documents 5-7).

[0005] Recently, a method for chemically modifying proteins in a noisy environment such as inside a cell using small molecule probes has been reported. This method is used for identifying receptors in imaging and repositioning of small molecule drugs. Furthermore, in the field of chemical biology, organic chemical protein modification methods using synthetic small molecule probes have attracted attention (Non-Patent Documents 8-10).

[0006] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed. Regioselective modification of antibodies has been successfully achieved by reacting a peptide reagent in which an NHS-activated ester and a drug are linked to a peptide (i.e., a method for producing an ADC via a linker containing a peptide moiety). This method is the world's first to successfully modify the Fc region of an antibody with a drug using chemical synthesis, and has proven to be practically satisfactory (reaction time: 30 minutes, yield: 70% (for a DAR of 1), and regioselectivity: 100%). It has been demonstrated that the DAR can be controlled at 2 by adding approximately 5 equivalents of the peptide reagent, making it a groundbreaking technology in that it also allows control of the modification site (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] STLaughlin et al.,Science 2008;320,664 [Non-Patent Document 9] AESpeers et al.,ChemBioChem 2004;5,41 [Non-Patent Document 10] Y.Takaoka et al.,Angew.Chem.Int.Ed. 2013;52,4088 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to develop a technique that enables the modification of soluble proteins, in particular the site-selective modification of soluble proteins. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have discovered that compounds developed based on a novel and original design concept, which have the following structural features: (1) an affinity substance for a soluble protein; (2) a reactive group for an amino acid residue constituting the soluble protein; and (3) a cleavable moiety between the affinity substance and the reactive group; and (4) a structural unit bearing a bioorthogonal functional group on the reactive group side (i.e., a structural unit containing a bioorthogonal functional group and a reactive group) can be generated by cleavage at the cleavable moiety. These compounds are useful for site-specific modification of soluble proteins (e.g., Figures 1-1, 1-2, 1-3, and 2). The present inventors have also discovered that such compounds can be used to prepare soluble proteins (e.g., antibody-drug conjugates (ADCs)) that regioselectively carry functional substances (e.g., drugs) without using a peptide moiety as a linker. Avoiding the use of linkers containing peptide moieties, which are potentially immunogenic and susceptible to hydrolysis in blood, is desirable for the clinical application of ADCs. In other words, the method developed by the present inventors is the first in the world to successfully modify the antibody Fc region with a drug regioselectively by chemical synthesis without using a linker containing a peptide moiety. The present inventors have also succeeded in developing various compounds possessing the structural features (1) to (4) above (e.g., Figures 1-1, 1-2, 1-3, and 2), thereby completing the present invention.

[0011] That is, the present invention is as follows.

[0012] In a first embodiment, the present invention provides a reagent for regioselectively modifying a soluble protein, comprising a substance with affinity for the soluble protein, a compound or a salt thereof having a cleavable moiety and a reactive group, and the compound or a salt thereof. [1] The following formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; and R is a group reactive to the soluble protein.] A compound or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, and a reactive group, [2] The compound of [1] or a salt thereof, wherein L is (i) a cleavable linker that is a divalent group containing a cleavable moiety that has the ability to generate a bioorthogonal functional group on the reactive group side upon cleavage, or (ii) a cleavable linker that is a divalent group containing a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side upon cleavage. [3] The compound or salt thereof according to [2], wherein L is the cleavable linker (i). [4] The compound or salt thereof according to [2] or [3], wherein L is the cleavable linker of (i) above, and B is the divalent group of (b) above. [5] The compound or salt thereof according to [2], wherein L is the cleavable linker (ii) above, and B is the divalent group (a) above. [6] The compound or salt thereof according to any one of [1] to [5], wherein the substance having affinity for a soluble protein is a peptide. [7] The compound or salt thereof according to [6], wherein the peptide is a peptide capable of binding to the Fc region of a monoclonal antibody. [8] The compound or salt thereof according to [7], wherein the binding peptide is a binding peptide for the Fc region of IgG. [9] The compound according to any one of [1] to [8] or a salt thereof, wherein the affinity substance comprises any one Fc region protein selected from the group consisting of the following (A) to (C), and is an affinity substance for an antibody having antigen-binding ability: (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1; (B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues have been inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) An Fc region protein comprising an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 1.

[10] The binding peptide is represented by the following formula (i): (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 94) (i) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, and W is a tryptophan residue. The compound or salt thereof according to any one of [7] to [9], which is a peptide or a salt thereof, comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by the following formula:

[11] The binding peptide is represented by the following formula (i-1): (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 95) (i-1) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a glutamic acid residue or an asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue. The compound or salt thereof according to any one of [7] to [9], which is a peptide or a salt thereof, comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by the following formula:

[12] The binding peptide is represented by the following formula (i-2): (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 96) (i-2) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is an arginine residue or a leucine residue, G is a glycine residue, Xaa2 is a lysine, glutamine, or aspartic acid residue; L is a leucine residue, V is a valine residue, and and W is a tryptophan residue.], and is a peptide or a salt thereof capable of binding to human IgG and / or rabbit IgG.

[13] The binding peptide is represented by the following formula (v): (X 1-3 )-C-(Xaa3)-(xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 102) (v) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 can be a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or or an aspartic acid residue, L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa5 is a threonine or lysine residue, Xaa6 is a tyrosine residue, a lysine residue, or none; and and Xaa7 is a histidine residue, a lysine residue, or none.], and is a peptide or a salt thereof capable of binding to human IgG and / or rabbit IgG.

[14] The binding peptide is represented by the following formula (vi): DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 103) (vi) D is an aspartic acid residue, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa5 is a threonine or lysine residue, Xaa6 is a tyrosine residue, a lysine residue, or none; Xaa7 is a histidine residue, a lysine residue, or is absent.], and is a peptide or a salt thereof characterized in that it contains an amino acid sequence consisting of 13 to 15 amino acid residues represented by the formula (I) and is capable of binding to human IgG and / or rabbit IgG.

[15] The binding peptide is represented by the following formula (vii): DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 104) (vii) [During the ceremony, D is an aspartic acid residue, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, and and T is a threonine residue], and is capable of binding to human IgG and / or rabbit IgG, or a salt thereof.

[16] The binding peptide is represented by the following formula (viii): RGNC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 105) (viii) [During the ceremony, R is an arginine residue, G is a glycine residue, N is an asparagine residue, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa5 is a threonine or lysine residue, Xaa6 is a tyrosine residue, a lysine residue, or none; and Xaa7 is a histidine residue, a lysine residue, or is absent.], and is a peptide or a salt thereof characterized in that it contains an amino acid sequence consisting of 13 to 15 amino acid residues represented by the formula (I) and is capable of binding to human IgG and / or rabbit IgG.

[17] The compound or salt thereof according to any one of [7] to

[16] , wherein the binding peptide is capable of binding to human IgG.

[18] The binding peptide comprises: (a) any amino acid residue in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 92) substituted with one amino acid residue selected from the group consisting of lysine residues, aspartic acid residues, glutamic acid residues, 2-aminosuberic acid residues, and diaminopropionic acid residues; and (b) A compound or a salt thereof according to any one of [7] to [9], which is an affinity peptide or a salt thereof, comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 92.

[19] The compound according to any one of [1] to

[18] or a salt thereof, wherein the cleavable moiety is a moiety that can be cleaved by any of (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) leaving the compound when a cleavable linker containing a self-degrading cleavable moiety is used.

[20] A compound according to any one of [1] to

[19] , wherein the cleavable moiety is selected from the group consisting of disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues. Or its salt.

[21] The compound according to any one of [2] to

[20] , or a salt thereof, wherein the cleavable moiety (i) is selected from the group consisting of a disulfide residue, an ester residue, an acetal residue, a ketal residue, an imine residue, and a vicinal diol residue.

[22] The compound according to any one of [2] to

[20] , or a salt thereof, wherein the cleavable moiety (ii) is selected from the group consisting of an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a phosphoramidate residue, an aconityl residue, a trityl residue, an azo residue, a vicinal diol residue, a selenium residue, an aromatic ring-containing residue having an electron-withdrawing group, a coumarin-containing residue, a sulfone-containing residue, an unsaturated bond-containing chain residue, and a glycosyl residue.

[23] The cleavable moiety is: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Multiple R 2a , multiple R 2b , and multiple R 2c are the same or different, (i) a hydrogen atom or a halogen atom; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v)R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or a monovalent hydrocarbon group; (vi)NR d R e -, NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R eare the same or different hydrogen atoms. represents a monovalent hydrocarbon group; or (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group selected from the group consisting of J is -CH2-, -O-, or -S-; r is any integer from 1 to 4, ○ (open circle) indicates a bond to A, and ● (filled circle) indicates a bond to B. When the chemical structure is asymmetric with respect to the cleavage site, ● may represent a bond to A and ○ may represent a bond to B.].

[24] The cleavable moiety of (i) is: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; R 2a is the same as

[23] , ○ (open circle) indicates a bond to A, and ● (filled circle) indicates a bond to B. When the chemical structure is asymmetric with respect to the cleavage site, ● may represent a bond to A and ○ may represent a bond to B.], a compound or a salt thereof according to any one of [2] to

[19] ,

[21] , and

[23] , which corresponds to any one chemical structure selected from the group consisting of:

[25] The cleavable moiety of (ii) is: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; R 2b , R 2c ,J,r are the same as in

[23] , ○ (open circle) indicates a bond to A, and ● (filled circle) indicates a bond to B. When the chemical structure is asymmetric with respect to the cleavage site, ● may represent a bond to A and ○ may represent a bond to B.], a compound or a salt thereof according to any one of [2] to

[19] ,

[22] , and

[23] , which corresponds to any one chemical structure selected from the group consisting of:

[26] L is the following formula (L1) to (L3): La-C-Lb (L1) La-C (L2) C-Lb (L3) [During the ceremony, La and Lb are each a divalent group; C is a cleavable moiety.] The compound or salt thereof according to any one of [1] to

[25] ,

[27] The La and Lb are the following (La') and (Lb'), respectively: [ka] [During the ceremony, p and p' are the same or different and are any integers from 0 to 10, q and q' are the same or different and each represents an integer of 0 to 10; X and X' may be the same or different and represent a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R 1b is absent, and X' is a nitrogen atom, R 1b’ does not exist. If X is a single bond, R 1a and R 1b is absent and X' is a single bond, R 1a’ and R 1b’ does not exist), R 1a , R 1b , R 1a’ and R 1b’ are the same or different and are an atom or group selected from the group consisting of (i) to (vii).] The compound of

[26] , or a salt thereof.

[28] A compound according to any one of [1] to

[27] , or a salt thereof, wherein the divalent group containing a bioorthogonal functional group is selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene 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 groups, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[29] A compound according to any one of [1] to

[27] , or a salt thereof, wherein the divalent group containing a bioorthogonal functional group is 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, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[30] The bioorthogonal functional group is one of the following: [ka] [During the ceremony, R 1f , single or multiple R 1g and one or more R 1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups, A compound or a salt thereof according to any one of [1] to

[29] , wherein the compound is any one of the following:

[31] The divalent group of (b) is an optionally substituted alkylene, an optionally substituted cycloalkylene, an optionally substituted aryl, an optionally substituted divalent heterocyclic group, -NR a -(R aA compound or a salt thereof according to any one of [1] to

[30] , wherein - is selected from the group consisting of -O-, or a combination of two or more thereof.

[32] B is represented by the following formula (B-1): [ka] [During the ceremony, Y is -NH-, -O-, -CH2-, or the following formula (B-2): [ka] (In the formula, V and V' are the same or different and each represent -NH-, -O-, -CH2-, or a single bond; V1 is a divalent group containing a bioorthogonal functional group; s is an integer between 0 and 10, The circle and the circle in formula (B-2) are in the same orientation as the circle and the circle in formula (B-1), respectively. Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH-), In formula (B-1), ○ (white circle) indicates a bond to the L-side moiety, and ● (black circle) indicates a bond to the R-side moiety.], a compound or a salt thereof according to any one of [1] to

[31] .

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

[32] , wherein the reactive group is a group reactive specifically with the side chain of any one of a lysine residue, a tyrosine residue, and a tryptophan residue.

[34] The compound or salt thereof according to

[33] , wherein the reactive group is a group reactive specifically with the side chain of a lysine residue.

[35] The reactive group is one of the following: [ka] [Here, R 5a and R 5cis an atom or group selected from the group consisting of (i) to (vii) above. and R 5b is an electron-withdrawing group, j is an integer from 1 to 5, and k is an integer of 1 to 4.]. The compound or salt thereof according to any one of [1] to

[34] , which corresponds to any one chemical structure selected from the group consisting of:

[36] A compound according to any one of [1] to

[35] , or a salt thereof, wherein the main chain connecting A and R has 4 to 20 atoms.

[37] A compound or a salt thereof according to any one of [1] to

[36] , wherein the main chain connecting A and R does not contain a ring structure.

[38] A compound or a salt thereof according to any one of [1] to

[37] , wherein the partial structure represented by LB does not contain a peptide moiety.

[39] The compound represented by formula (I) is the following (I'): A-B2-L'-B1-R (I') [During the ceremony, A and R are the same as those in formula (I). L' is a cleavable linker, which is a divalent group that includes a cleavable moiety; B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; B1 and B2 may have a symmetric structure with L' at the center.] The compound or a salt thereof according to any one of [1] to

[38] , which is a compound represented by the formula:

[40] The compound represented by the formula (I') is the following (I''): [ka] [During the ceremony, A and R are the same as those in formula (I) described in [1], C is a cleavable moiety, p, p', q, q', X, X', R 1a , R 1a’ , R 1b , and R 1b’are the same as those of formulas (La') and (Lb') described in

[27] , Y and Y' are the same or different and are the same as Y in formula (B-1) described in

[32] , Z and Z' may be the same or different and are the same as Z in the formula (B-1).

[41] The following formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; and R is a group reactive to the soluble protein.]. A reagent for site-selective modification of a soluble protein, comprising a substance having affinity for the soluble protein, a compound having a cleavable moiety and a reactive group, or a salt thereof,

[42] The following formula (I): ALBR (I) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; and R is a reactive group specific to the side chain of a lysine residue.] A compound or a salt thereof having an affinity substance for an antibody, a cleavable moiety and a reactive group,

[43] The following formula (I): The following formula (I): ALBR (I) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; and R is a reactive group specific to the side chain of a lysine residue.] A reagent for site-selective modification of an antibody, comprising a substance with affinity for an antibody, a compound having a cleavable moiety and a reactive group, or a salt thereof,

[0013] Second, the present invention provides an affinity substance for a soluble protein, a soluble protein having a cleavable portion, or a salt thereof, and a method for producing the same. (Affinity substances for soluble proteins, and soluble proteins or salts thereof having cleavable moieties)

[44] The following formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein.] and a soluble protein or a salt thereof having an affinity substance for a soluble protein and a cleavable portion.

[45] The soluble protein or a salt thereof according to

[44] , wherein the soluble protein is a monoclonal antibody.

[46] The soluble protein or salt thereof according to

[44] or

[45] , wherein the soluble protein is an IgG antibody.

[47] The soluble protein or salt thereof according to any one of

[44] to

[46] , wherein the soluble protein is derived from a human.

[48] ​​The soluble protein or salt thereof according to any one of

[44] to

[47] , wherein the soluble protein comprises any one Fc domain protein selected from the group consisting of the following (A) to (C), and is an antibody having antigen-binding ability: (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1; (B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues have been inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) An Fc region protein comprising an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 1.

[49] A soluble protein comprising one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and five or more of the specific amino acid residues in a non-target region other than the target region; A soluble protein or a salt thereof according to any one of

[44] to

[48] , wherein a structural unit represented by ALB-R' is bound to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more.

[50] The soluble protein or salt thereof according to

[49] , wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues.

[51] The soluble protein or a salt thereof according to

[50] , wherein the target region is a region consisting of 1 to 3 consecutive amino acid residues.

[52] The soluble protein or salt thereof according to

[51] , wherein the target region is (a) a region consisting of amino acid residues at positions 246 to 248 in the Fc region of human IgG, (b) a region consisting of amino acid residues at positions 288 to 290 in the Fc region of human IgG, or (c) a region consisting of amino acid residue at position 317 in the Fc region of human IgG.

[53] The soluble protein or salt thereof according to any one of

[49] to

[52] , wherein the position selectivity is 50% or more.

[54] The soluble protein or salt thereof according to

[53] , wherein the position selectivity is 70% or more.

[55] The soluble protein or salt thereof according to

[54] , wherein the position selectivity is 90% or more.

[56] The soluble protein or salt thereof according to any one of

[49] to

[55] , wherein the specific amino acid residue does not contain any amino acid residue of the same type as the specific amino acid residue other than the specific amino acid residue located at the specific position in a region extending from the specific amino acid located at the specific position to a number a (where a is any integer from 1 to 10) of amino acid residues away from the specific amino acid located at the specific position on the N-terminal and C-terminal sides, respectively.

[57] The soluble protein is a multimeric protein containing multiple monomeric proteins; The soluble protein or salt thereof according to any one of

[44] to

[56] , wherein T has a structural unit represented by ALB-R' in a plurality of target regions corresponding to a plurality of monomeric proteins, so that the multimeric protein has a plurality of structural units represented by ALB-R'.

[58] The soluble protein is an antibody comprising multiple heavy chains; T is represented by ALB-R' in a corresponding plurality of target regions in a plurality of heavy chains. The soluble protein or salt thereof according to any one of

[44] to

[57] , wherein the antibody has a plurality of structural units represented by ALB-R' as a result of the structural unit being represented by ALB-R'.

[59] The soluble protein or salt thereof according to

[58] , wherein the number of heavy chains is two.

[60] The soluble protein or salt thereof according to any one of

[44] to

[59] , wherein the moiety generated by the reaction between the soluble protein and the reactive group is a moiety generated by the reaction of a reactive group specific to the side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue with a lysine residue, a tyrosine residue, or a tryptophan residue.

[61] The soluble protein or salt thereof according to any one of

[44] to

[60] , wherein the moiety generated by the reaction between the soluble protein and the reactive group is a moiety generated by the reaction between a lysine residue and a reactive group specific to the side chain of the lysine residue.

[62] The moiety produced by the reaction is: [ka] [wherein a black circle (●) indicates a bond to the T-side moiety, and a white circle (○) indicates a bond to the B-side moiety. A straight line perpendicular to the bond indicates a bond formed by the reaction.] A soluble protein or a salt thereof according to any one of

[44] to

[61] , which corresponds to any one chemical structure selected from the group consisting of:

[63] A soluble protein or a salt thereof according to any one of

[44] to

[62] , wherein the main chain connecting A and R' has 4 to 20 atoms.

[64] A soluble protein or a salt thereof according to any one of

[44] to

[63] , wherein the main chain connecting A and R does not contain a ring structure.

[65] A soluble protein or a salt thereof according to any one of

[44] to

[64] , wherein the partial structure represented by LB does not contain a peptide moiety.

[66] The compound represented by formula (II) is the following (II'): A-B2-L'-B1-R'-T (II') [During the ceremony, A, R' and T are the same as those in formula (II). L' is a cleavable linker, which is a divalent group that includes a cleavable moiety; B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; B1 and B2 may have a symmetric structure with L' at the center. The soluble protein or salt thereof according to any one of

[44] to

[65] , wherein B1 and B2 are compounds represented by the formula:

[67] The compound represented by the formula (II') is the following (II''): [ka] [During the ceremony, A, R′, and T are the same as those in formula (II) described in

[44] , C is a cleavable moiety, p and p' are the same or different and are any integers from 0 to 10, q and q' are the same or different and each represents an integer of 0 to 10; X and X' may be the same or different and represent a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R 1b is absent, and X' is a nitrogen atom, R 1b’ does not exist. If X is a single bond, R 1a and R 1b is absent and X' is a single bond, R 1a’ and R 1b’ does not exist), R 1a , R 1b , R 1a’ and R 1b’ are the same or different, (i) a hydrogen atom or a halogen atom; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v)R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or a monovalent hydrocarbon group; (vi)NR d R e -, NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; or (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group selected from the group consisting of Y and Y' are the same or different and are the same as Y in formula (B-1) described in

[32] , Z and Z' may be the same or different and are the same as Z in the formula (B-1).

[68] The following formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety resulting from the reaction between an antibody and a reactive group specific for the side chain of a lysine residue; T is an antibody.] and an antibody or a salt thereof having an affinity substance for the antibody and a cleavable moiety, as represented by the formula:

[0014] (Method for producing a substance with affinity for a soluble protein and a soluble protein or a salt thereof having a cleavable moiety)

[69] A method for producing a soluble protein or a salt thereof having an affinity substance for a soluble protein and a cleavable portion, comprising: The following formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; and R is a reactive group to the soluble protein.], by reacting a compound or a salt thereof having an affinity substance for the soluble protein, a cleavable moiety and a reactive group, with the soluble protein, The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I) above; R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and producing a soluble protein or a salt thereof having an affinity substance for the soluble protein represented by the formula:

[70] The method of

[69] , wherein the soluble protein is an antibody and the reactive group is a reactive group specific for the side chain of a lysine residue.

[0015] Third, the present invention provides a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, and a method for producing the same. (A complex having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, or a salt thereof)

[71] The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; F is a functional substance, R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] A complex or a salt thereof, comprising a substance having affinity for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, the complex being represented by the formula (I) above.

[72] The conjugate or salt thereof according to

[71] , wherein the soluble protein is a monoclonal antibody.

[73] The conjugate or salt thereof of

[71] or

[72] , wherein the soluble protein is an IgG antibody.

[74] A conjugate or a salt thereof according to any one of

[71] to

[73] , wherein the soluble protein is derived from a human.

[75] The conjugate or salt thereof according to any one of

[71] to

[74] , wherein the soluble protein comprises any one Fc domain protein selected from the group consisting of the following (A) to (C), and is an antibody having antigen-binding ability: (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1; (B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues have been inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) An Fc region protein comprising an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 1.

[76] A soluble protein comprising one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and five or more of the specific amino acid residues in a non-target region other than the target region; A complex or a salt thereof according to any one of

[71] to

[75] , wherein a structural unit represented by AL-B'(-F)-R' is bound to one or more specific amino acid residues contained in the target region with a position selectivity of 30% or more.

[77] The method according to

[76] , wherein the specific amino acid residue does not contain any amino acid residue identical to the specific amino acid residue other than the specific amino acid residue at the specific position in a region extending from the specific amino acid at the specific position to a number a (where a is any integer from 1 to 10) of amino acid residues away from the N-terminal and C-terminal ends of the specific amino acid residue at the specific position. A complex or a salt thereof.

[78] The soluble protein is a multimeric protein containing multiple monomeric proteins; The complex or salt thereof according to any one of

[71] to

[77] , wherein T has a structural unit represented by AL-B'(-F)-R' in a plurality of corresponding target regions in a plurality of monomeric proteins, so that the multimeric protein has a plurality of structural units represented by AL-B'(-F)-R'.

[79] The soluble protein is an antibody comprising multiple heavy chains; The conjugate or salt thereof according to any one of

[71] to

[78] , wherein T has a structural unit represented by AL-B'(-F)-R' in a plurality of corresponding target regions in a plurality of heavy chains, and as a result, the antibody has a plurality of structural units represented by AL-B'(-F)-R'.

[80] A complex or salt thereof according to any one of

[71] to

[79] , wherein the divalent group containing a moiety generated by a reaction between the functional substance and the bioorthogonal functional group is a divalent group containing a reactive moiety selected from the group consisting of disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[81] The moiety produced by the reaction is: [ka] [wherein the wavy lines perpendicular to the bonds represent bonds formed by the reaction, Multiple R 2a , multiple R 2b , and multiple R 2c are the same or different, (i) a hydrogen atom or a halogen atom; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v)R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or a monovalent hydrocarbon group; (vi)NR d R e -, NR d R e -C(=O)-, NR dR e -C(=O)-O- or R d -C(=O)-NR e -(R d and R e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; or (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group selected from the group consisting of J is -CH2-, -O-, or -S-; r is any integer from 1 to 4, ○ (open circle) indicates a bond to A, and ● (filled circle) indicates a bond to B. When the chemical structure is asymmetric with respect to the cleavage site, ● may represent a bond to A and ○ may represent a bond to B.].

[82] A conjugate or salt thereof according to any one of

[71] to

[81] , wherein the moiety generated by the reaction between a soluble protein and a reactive group is a moiety generated by the reaction between a lysine residue, a tyrosine residue, or a tryptophan residue and a reactive group specific to the side chain of any one of the lysine residue, tyrosine residue, or tryptophan residue.

[83] A conjugate or a salt thereof according to any one of

[71] to

[82] , wherein the moiety generated by the reaction between the soluble protein and the reactive group is a moiety generated by the reaction between a lysine residue and a reactive group specific to the side chain of the lysine residue.

[84] The moiety produced by the reaction is: [ka] [wherein ● (black circle) indicates a bond to the T-side moiety, and ○ (white circle) indicates a bond to the B-side moiety]. A complex or salt thereof according to any one of

[71] to

[83] , which corresponds to any one chemical structure selected from the group consisting of:

[85] A complex or a salt thereof according to any one of

[71] to

[84] , wherein the functional substance is a drug or a labeling substance.

[86] A complex or a salt thereof according to any one of

[71] to

[85] , wherein the functional substance is a low molecular weight compound.

[87] The conjugate of

[85] or

[86] or a salt thereof, wherein the drug is an anticancer drug.

[88] A complex or a salt thereof according to any one of

[71] to

[87] , wherein the main chain connecting A and R' has 4 to 20 atoms.

[89] A complex or a salt thereof according to any one of

[71] to

[88] , wherein the main chain connecting A and R does not contain a ring structure.

[90] The partial structure represented by LB does not contain a peptide moiety, and any of

[71] to

[89] A complex of either of these or a salt thereof.

[91] The compound represented by formula (III) is represented by the following formula (III'): A-B2'(-F2)-L'-B1'(-F1)-R'-T (III') [During the ceremony, A, R′, and T are the same as those in formula (II), L' is a cleavable linker, which is a divalent group that includes a cleavable moiety; B1' and B2' are the same or different and are divalent groups containing a moiety generated by a reaction between a functional substance and a bioorthogonal functional group; F1 and F2 are the same or different functional substances; B1' (-F1) and B2' (-F2) may have a symmetric structure with L' at the center.] A complex or a salt thereof according to any one of

[71] to

[90] ,

[92] The compound represented by formula (III') is the following (III''): [ka] [During the ceremony, A, R′, and T are the same as those in formula (III) described in

[71] , C is a cleavable moiety, p and p' are the same or different and are any integers from 0 to 10, q and q' are the same or different and each represents an integer of 0 to 10; X and X' may be the same or different and represent a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R 1b is absent, and X' is a nitrogen atom, R 1b’ does not exist. If X is a single bond, R 1a and R 1b is absent and X' is a single bond, R 1a’ and R 1b’ does not exist), R 1a , R 1b , R 1a’ and R 1b’ are the same or different and are selected from the group consisting of (i) to (vii), Y and Y' are the same or different and represent a residue in which one hydrogen atom has been removed from Y of formula (B-1) described in

[32] , Z and Z′ are the same or different and are the same as Z in formula (B-1), F and F' may be the same or different and each represent a functional substance.

[93] The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; F is a functional substance, R' is a moiety resulting from the reaction between an antibody and a reactive group specific for the side chain of a lysine residue; and T is an antibody.] A complex or a salt thereof comprising an affinity substance, a functional substance, and an antibody, the complex being represented by the formula:

[0016] (Affinity substances for soluble proteins, cleavable moieties, functional substances and soluble proteins (Method for producing a complex having the above properties or a salt thereof)

[94] A method for producing a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, comprising: The following formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and a soluble protein or a salt thereof having a cleavable portion are reacted with a functional substance to form a soluble protein. The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A, L, R′, and T are the same as those in formula (II), B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; and F is a functional substance.].

[95] The method of

[94] , wherein the soluble protein is an antibody and the reactive group is a reactive group specific for the side chain of a lysine residue.

[96] A method for producing a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; and R is a group reactive to the soluble protein, or a salt thereof, is reacted with the soluble protein to form a compound represented by the formula (I): The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and producing a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable portion, (B) reacting the substance having affinity for the soluble protein and the soluble protein or its salt having a cleavable portion with a functional substance, The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A and L are the same as those in formula (I), R′ and T are the same as those in formula (II), B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; and F is a functional substance.].

[0017] Fourth, the present invention provides a method for producing soluble proteins with bioorthogonal functional groups.

[97] A method for producing a soluble protein or a salt thereof having a bioorthogonal functional group, comprising: The following formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. ] and a soluble protein or a salt thereof having a cleavable portion, The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), and L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group.

[98] The method of

[97] , wherein L is (i) a cleavable linker that is a divalent group containing a cleavable moiety that has the ability to generate a bioorthogonal functional group on the reactive group side upon cleavage, or (ii) a cleavable linker that is a divalent group containing a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on the reactive group side upon cleavage.

[99] L is the cleavable linker (i), L1 is (i') a monovalent group containing a bioorthogonal functional group; The method according to

[98] , wherein B is the divalent group (a) or (b).

[100] L is the cleavable linker (i), L1 is (i') a monovalent group containing a bioorthogonal functional group, The method according to

[98] or

[99] , wherein B is the divalent group of (b).

[101] L is the cleavable linker (ii) above, L1 is (i') a monovalent group that does not contain a bioorthogonal functional group, The method according to

[98] , wherein B is the divalent group (a).

[102] The method according to any one of

[97] to

[101] , wherein the soluble protein is an antibody and the reactive group is a reactive group specific to the side chain of a lysine residue.

[103] A method for producing a soluble protein or a salt thereof having a bioorthogonal functional group, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; and R is a group reactive to the soluble protein, or a salt thereof, is reacted with the soluble protein to form a compound represented by the formula (I): The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and producing a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable portion, (B) cleaving the cleavable portion of the soluble protein or its salt, which has an affinity substance for the soluble protein and a cleavable portion; The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), and L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group.

[0018] Fifth, the present invention provides a method for producing a soluble protein or a salt thereof having a functional substance.

[104] A method for producing a soluble protein or a salt thereof having a functional substance, comprising: The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; F is a functional substance, R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. ], or by cleaving the cleavable portion of a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable portion, a functional substance, and a soluble protein, The following formula (IV): L1-B-R'-T (IV) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. A soluble protein or a salt thereof having a bioorthogonal functional group, represented by the formula (I), is reacted with a functional substance to form a soluble protein. The following formula (V): F-(L1-B)'-R'-T (V) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; The structural unit represented by (L1-B)' is a functional substance and the structural unit represented by (i') and (a) a divalent structural unit comprising a moiety formed by reaction with one or both of the bioorthogonal functional groups, F is a functional substance, R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.].

[105] Cleavage of the cleavable portion of the complex or its salt, which comprises an affinity substance for the soluble protein, a cleavable portion, a functional substance, and a soluble protein, The following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; wherein B', F, R' and T are the same as those in formula (III).

[106] The soluble protein or its salt having a bioorthogonal functional group is reacted with one or two functional substances, The following formula (V2): F-L1'-B-R'-T (V2) [During the ceremony, B, R′, and T are the same as those in formula (IV), L1' is a divalent group containing a moiety generated by a reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; F is a functional substance; or The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (IV), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; wherein Fa and Fb are the same or different functional substances.]. The method of

[104] , which is a method comprising producing a soluble protein or a salt thereof having a functional substance represented by the formula:

[107] The method according to any one of

[104] to

[106] , wherein the soluble protein is an antibody and the reactive group is a reactive group specific to the side chain of a lysine residue.

[108] A method for producing a soluble protein or a salt thereof having a functional substance, comprising: (A) Formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and a soluble protein or a salt thereof having a cleavable portion are reacted with a functional substance to form a soluble protein. The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A, L, R′, and T are the same as those in formula (II), B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; F is a functional substance; and forming a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, the complex being represented by the formula: (B) cleaving the cleavable portion of the complex or its salt, which comprises an affinity substance for the soluble protein, a cleavable portion, a functional substance, and a soluble protein; The following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; wherein B', F, R' and T are the same as those in formula (III).

[109] A method for producing a soluble protein or a salt thereof having a functional substance, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; and R is a reactive group to the soluble protein.], by reacting a compound or a salt thereof having an affinity substance for the soluble protein, a cleavable moiety and a reactive group, with the soluble protein, The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable portion; (B) reacting the substance having affinity for the soluble protein and the soluble protein or its salt having a cleavable portion with a functional substance, The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A and L are the same as those in formula (I), R′ and T are the same as those in formula (II), B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; F is a functional substance; and forming a complex or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein, the complex being represented by the formula: (C) cleaving the cleavable portion of the complex or its salt, which comprises the affinity substance for the soluble protein, the cleavable portion, the functional substance, and the soluble protein; The following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; wherein B', F, R' and T are the same as those in formula (III).

[110] A method for producing a soluble protein or a salt thereof having a functional substance, comprising: (A) Formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. ] and a soluble protein or a salt thereof having a cleavable portion, The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. (B) reacting the soluble protein or its salt having the bioorthogonal functional group with one or more functional substances, The following formula (V2) F-L1'-B-R'-T (V2) [During the ceremony, B, R′, and T are the same as those in formula (IV), L1' is a divalent group containing a moiety generated by a reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; F is a functional substance; or The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (IV), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; and Fa and Fb are the same or different functional substances.

[111] A method for producing a soluble protein or a salt thereof having a functional substance, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; and R is a group reactive to the soluble protein, or a salt thereof, is reacted with the soluble protein to form a compound represented by the formula (I): The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; and T is a soluble protein.] and a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable portion; (B) cleaving the cleavable portion of the soluble protein or its salt, which has an affinity substance for the soluble protein and a cleavable portion; The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. (C) reacting the soluble protein or its salt having the bioorthogonal functional group with one or more functional substances, The following formula (V2) F-L1'-B-R'-T (V2) [During the ceremony, B, R′, and T are the same as those in formula (IV), L1' is a divalent group containing a moiety generated by a reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; F is a functional substance; or The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (IV), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; and Fa and Fb are the same or different functional substances.

[0019] Sixth, the present invention provides a soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, and a method for producing the same. (Soluble protein or salt thereof having a bioorthogonal functional group regioselectively) [1] A soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region; and A soluble protein or a salt thereof having a bioorthogonal functional group regioselectively bound to one or more specific amino acid residues contained in the target region via a linker that does not contain a peptide moiety with a regioselectivity of 30% or more. [2] The following formula (IV): L1-B-R'-T (IV) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, wherein a structural unit represented by L1-B-R' is bound to one or more specific amino acid residues contained in the target region of the soluble protein with a regioselectivity of 30% or more. [3] The soluble protein or salt thereof according to [1] or [2], wherein the soluble protein is a monoclonal antibody. [4] The soluble protein or salt thereof according to any one of [1] to [3], wherein the soluble protein is an IgG antibody. [5] The soluble protein or salt thereof according to any one of [1] to [4], wherein the soluble protein is derived from a human. [6] The soluble protein or salt thereof according to any one of [1] to [5], wherein the soluble protein comprises any one Fc domain protein selected from the group consisting of the following (A) to (C) and is an antibody having antigen-binding ability: (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1; (B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues have been inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) An Fc region protein comprising an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 1. [7] The soluble protein or salt thereof according to any one of [1] to [6], wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues. [8] The soluble protein or salt thereof according to any one of [1] to [7], wherein the target region is a region consisting of 1 to 3 consecutive amino acid residues. [9] The soluble protein or salt thereof according to [8], wherein the target region is (a) a region consisting of amino acid residues at positions 246 to 248 in the Fc region of human IgG, (b) a region consisting of amino acid residues at positions 288 to 290 in the Fc region of human IgG, or (c) a region consisting of amino acid residue at position 317 in the Fc region of human IgG.

[10] The soluble protein or salt thereof according to any one of [1] to [9], wherein the regioselectivity is 50% or more.

[11] The soluble protein or salt thereof according to

[10] , wherein the regioselectivity is 70% or more.

[12] The soluble protein or salt thereof according to

[11] , wherein the regioselectivity is 90% or more.

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

[12] , wherein the specific amino acid residue does not contain any amino acid residue of the same type as the specific amino acid residue other than the specific amino acid residue located at the specific position, in regions extending from the specific amino acid located at the specific position to a number a (where a is any integer from 1 to 10) of amino acid residues away from the specific amino acid located at the specific position on the N-terminal and C-terminal sides, respectively.

[14] The soluble protein is a multimeric protein containing multiple monomeric proteins; The soluble protein or salt thereof according to any one of [1], [3] to

[13] , wherein the multimeric protein has bioorthogonal functional groups at specific amino acid residues present at the positions in a plurality of monomeric proteins contained in the multimeric protein, resulting in the multimeric protein having multiple bioorthogonal functional groups.

[15] The soluble protein is an antibody comprising multiple heavy chains; The soluble protein or salt thereof according to any one of [1], [3] to

[14] , wherein the antibody has multiple bioorthogonal functional groups at multiple specific amino acid residues present at the aforementioned positions in multiple heavy chains contained in the antibody.

[16] The soluble protein is a multimeric protein containing multiple monomeric proteins; The soluble protein or salt thereof according to any one of [2] to

[13] , wherein T has a structural unit represented by ALB-R' in a plurality of target regions corresponding to a plurality of monomeric proteins, resulting in a multimeric protein having a plurality of structural units represented by ALB-R'.

[17] The soluble protein is an antibody comprising multiple heavy chains; The soluble protein or salt thereof according to any one of [2] to

[13] and

[16] , wherein T has a structural unit represented by ALB-R' in a plurality of corresponding target regions in a plurality of heavy chains, resulting in an antibody having a plurality of structural units represented by ALB-R'.

[18] The soluble protein or salt thereof according to

[15] or

[17] , wherein the number of heavy chains is two.

[19] L1 is represented by the following formulas (L1-1) to (L1-2): C1-Lb (L1-1) C1 (L1-2) [During the ceremony, Lb is a divalent group, C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group.] A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[18] , wherein the soluble protein is represented by any one of the following:

[20] The Lb is represented by the following formula (Lb'): [ka] [During the ceremony, p is an integer between 0 and 10; q is an integer from 0 to 10, X is a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R 1b does not exist. If X is a single bond, R 1a and R 1b does not exist), R 1a , and R 1b are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents. A soluble protein or a salt thereof according to

[19] , wherein ○ (open circle) indicates binding to C1, and ● (filled circle) indicates binding to B.

[21] A soluble protein or a salt thereof according to any one of [1] to

[20] , wherein the divalent group containing a bioorthogonal functional group is a divalent group containing in its main chain a bioorthogonal functional group selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene 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 groups, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[22] The divalent group containing a bioorthogonal functional group is an azide residue, an aldehyde residue, a thiol residue, an alkyne residue, an alkene residue, a halogen residue, a tetrazine residue, a nitrone residue, a hydroxylamine residue, a nitrile residue, a hydrazine residue, a ketone residue, a boronic acid residue, a cyanobenzothiazole residue, an allyl residue, a phosphine residue, a maleimide residue, a disulfide residue, an α-halocarbonyl residue, an isonitrile residue, a sydnone residue, or a selenium residue.

[20] A soluble protein or a salt thereof according to any one of [1] to

[20] , wherein the soluble protein is a divalent group having a bioorthogonal functional group selected from the group consisting of:

[23] The bioorthogonal functional group is one of the following: [ka] [During the ceremony, R 1f , single or multiple R 1g and one or more R 1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups, 22. A soluble protein or a salt thereof according to any one of [1] to

[22] , wherein the soluble protein or salt thereof is one of the following:

[24] The divalent group of (b) is an optionally substituted alkylene, an optionally substituted cycloalkylene, an optionally substituted aryl, an optionally substituted divalent heterocyclic group, -NR a -(R a A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[23] , wherein - is selected from the group consisting of -O-, -O-, or a combination of two or more thereof.

[25] B is represented by the following formula (B-1): [ka] [During the ceremony, Y is -NH-, -O-, -CH2-, or the following formula (B-2): [ka] (In the formula, V and V' may be the same or different and each represent -NH-, -O-, -CH2-, or a single bond. and V1 is a divalent group containing a bioorthogonal functional group; s is an integer between 0 and 10, The circle and the circle in formula (B-2) are in the same orientation as the circle and the circle in formula (B-1), respectively. Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH-), In formula (B-1), ○ (open circle) indicates a bond to the L-side moiety, and ● (filled circle) indicates a bond to the R-side moiety. A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[24] , represented by the formula (B-1).

[26] A soluble protein or a salt thereof according to any one of [1] to

[25] , wherein the bioorthogonal functional group is bound to the soluble protein via the side chain of one of a lysine residue, a tyrosine residue, or a tryptophan residue.

[27] The soluble protein or salt thereof according to

[26] , wherein the bioorthogonal functional group is bound to the soluble protein via the side chain of a lysine residue.

[28] The soluble protein or salt thereof according to any one of [2] to

[13] and

[16] to

[26] , wherein the reactive group is a group specifically reactive with the side chain of any one of a lysine residue, a tyrosine residue, and a tryptophan residue.

[29] The soluble protein or salt thereof according to any one of [2] to

[13] and

[16] to

[28] , wherein the reactive group is a group that is reactive specifically with the side chain of a lysine residue.

[30] The reactive group is one of the following: [ka] [Here, R 5a and R 5c is an atom or group selected from the group consisting of (i) to (vii) above. and R 5b is an electron-withdrawing group, j is an integer from 1 to 5, and k is an integer of 1 to 4.]. A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[29] , which corresponds to any one chemical structure selected from the group consisting of:

[31] A soluble protein or a salt thereof according to any one of [1] to

[30] , wherein the bioorthogonal functional group is bound to the soluble protein via a linker having 2 to 10 atoms in the main chain connecting the bioorthogonal functional group and the side chain of a specific amino acid residue.

[32] The main chain connecting the bioorthogonal functional group and the side chain of a specific amino acid residue contains a ring structure. The soluble protein or a salt thereof according to any one of [1] to

[31] , wherein a bioorthogonal functional group is bound to the soluble protein via a linker that does not bind to the soluble protein.

[33] A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[32] , wherein the number of atoms in the main chain connecting the L1 terminal portion and R' is 2 to 10.

[34] A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[33] , wherein the main chain connecting A and R does not contain a ring structure.

[35] A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[34] , wherein the partial structure represented by L1-B does not contain a peptide moiety.

[36] The soluble protein represented by the formula (IV) is the following (IV'): [ka] [During the ceremony, C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group. p, q, X, R 1a and R 1b is the same as that of the formula (Lb'), Y and Z are the same as above, wherein R' and T are the same as those in the formula (IV). ] A soluble protein or a salt thereof according to any one of [2] to

[13] and

[16] to

[35] ,

[37] A soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region; and The bioorthogonal functional group is bound to one or more specific amino acid residues contained in the target region with a site selectivity of 30% or more via a linker that does not contain a peptide moiety; The bioorthogonal functional group is attached to a soluble protein via the side chain of a lysine residue, the soluble protein is an antibody comprising multiple heavy chains; A soluble protein or a salt thereof having bioorthogonal functional groups regioselectively, wherein the antibody has bioorthogonal functional groups at multiple specific amino acid residues present at the aforementioned positions in multiple heavy chains contained in the antibody, resulting in the antibody having multiple bioorthogonal functional groups.

[38] The following formula (IV): L1-B-R'-T (IV) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety resulting from the reaction between an antibody and a reactive group specific for the side chain of a lysine residue; T is a soluble protein, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, wherein a structural unit represented by L1-B-R' is bound to one or more specific amino acid residues contained in the target region of the soluble protein with a regioselectivity of 30% or more.

[0020] (Method for producing a soluble protein or its salt having a bioorthogonal functional group regioselectively) The present invention also provides a method for producing a soluble protein or a salt thereof that has a bioorthogonal functional group regioselectively and that is identified by formula (IV) or a subformula thereof, among the soluble proteins [1] to

[38] above.

[39] A method for producing a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, comprising: The following formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein, the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable moiety in a site-selective manner, wherein a structural unit represented by L1-B-R' binds to one or more specific amino acid residues contained in the target region of the soluble protein with a site-selectivity of 30% or more, The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. A method comprising producing a soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, wherein a structural unit represented by L1-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more.

[0021] Preferably, the method of

[39] above may be as follows.

[40] The method of

[39] , wherein the soluble protein is an antibody and the reactive group is a reactive group specific for the side chain of a lysine residue.

[41] A method for producing a soluble protein or a salt thereof regioselectively having a bioorthogonal functional group, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; and R is a group reactive to the soluble protein.] or a salt thereof is reacted with a soluble protein (wherein the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region), to form a soluble protein. , The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. a structural unit represented by L1-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, and a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable moiety site-selectively; (B) cleaving the cleavable portion of the soluble protein or its salt, which has an affinity substance for the soluble protein and a cleavable portion; The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. A method comprising producing a soluble protein or a salt thereof, wherein a structural unit represented by L1-B-R' regioselectively has a bioorthogonal functional group that is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more.

[0022] Seventh, the present invention provides a soluble protein or a salt thereof having a functional substance regioselectively thereon, and a method for producing the same. (Soluble protein or salt thereof having a functional substance site-selectively) [1] A soluble protein or a salt thereof having a functional substance regioselectively, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region; and A soluble protein or a salt thereof having a functional substance regioselectively bound to one or more specific amino acid residues contained in the target region via a linker that does not contain a peptide moiety with a regioselectivity of 30% or more. [2] The following formula (V): F-(L1-B)'-R'-T (V) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; The structural unit represented by (L1-B)' is a divalent structural unit containing a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a), F is a functional substance, R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A soluble protein or a salt thereof having a functional substance regioselectively, in which a structural unit represented by F-(L1-B)'-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more. [3] The soluble protein or a salt thereof is represented by the following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1, F, R' and T are the same as those in formula (V), and B' is a divalent group containing a moiety generated by a reaction between the functional substance and the bioorthogonal functional group.], and the structural unit represented by L1-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more, or a salt thereof. [4] The soluble protein or a salt thereof is represented by the following formula (V2): F-L1'-B-R'-T (V2) [During the ceremony, F, B, R′, and T are the same as those in formula (V), and L1' is a divalent group containing a moiety generated by a reaction between the functional substance and (i') a monovalent group containing a bioorthogonal functional group. A soluble protein or a salt thereof having a functional substance regioselectively, wherein the structural unit represented by F-L1'-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more. The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (V), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; wherein Fa and Fb are the same or different functional substances.], and the structural unit represented by Fa-L1'-B'(-Fb)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, or a salt thereof. [5] The soluble protein or salt thereof according to any one of [1] to [4], wherein the soluble protein is a monoclonal antibody. [6] The soluble protein or salt thereof according to any one of [1] to [5], wherein the soluble protein is an IgG antibody. [7] The soluble protein or salt thereof according to any one of [1] to [6], wherein the soluble protein is derived from a human. [8] The soluble protein or salt thereof according to any one of [1] to [7], wherein the soluble protein comprises any one Fc domain protein selected from the group consisting of the following (A) to (C), and is an antibody having antigen-binding ability: (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1; (B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues have been inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) An Fc region protein comprising an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 1. [9] The soluble protein or salt thereof according to any one of [1] to [8], wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues.

[10] The target region is a region consisting of 1 to 3 consecutive amino acid residues. [9] A soluble protein or a salt thereof.

[11] The soluble protein or salt thereof according to

[10] , wherein the target region is (a) a region consisting of amino acid residues at positions 246 to 248 in the Fc region of human IgG, (b) a region consisting of amino acid residues at positions 288 to 290 in the Fc region of human IgG, or (c) a region consisting of amino acid residue at position 317 in the Fc region of human IgG.

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

[11] , wherein the regioselectivity is 50% or more.

[13] The soluble protein or salt thereof according to

[12] , wherein the position selectivity is 70% or more.

[14] The soluble protein or salt thereof according to

[13] , wherein the regioselectivity is 90% or more.

[15] The soluble protein or salt thereof according to any one of [1] to

[14] , wherein the specific amino acid residue does not contain any amino acid residue of the same type as the specific amino acid residue other than the specific amino acid residue located at the specific position, in regions extending from the specific amino acid located at the specific position to a number a (where a is any integer from 1 to 10) of amino acid residues away from the specific amino acid located at the specific position on the N-terminal and C-terminal sides, respectively.

[16] The soluble protein is a multimeric protein containing multiple monomeric proteins; The soluble protein or salt thereof according to any one of [1], [5] to

[15] , wherein the multimeric protein has functional substances at specific amino acid residues present at the positions in the monomeric proteins contained in the multimeric protein, resulting in the multimeric protein having multiple functional substances.

[17] The soluble protein is an antibody comprising multiple heavy chains; The soluble protein or salt thereof according to any one of [1], [5] to

[16] , wherein the antibody has functional substances at multiple specific amino acid residues present at the aforementioned positions in multiple heavy chains contained in the antibody, resulting in the antibody having multiple functional substances.

[18] The soluble protein is a multimeric protein containing multiple monomeric proteins; The soluble protein or salt thereof according to any one of [2] to

[15] , wherein T has a structural unit represented by ALB-R' in a plurality of target regions corresponding to a plurality of monomeric proteins, so that the multimeric protein has a plurality of structural units represented by ALB-R'.

[19] The soluble protein is an antibody comprising multiple heavy chains; The soluble protein or salt thereof according to any one of [2] to

[15] and

[18] , wherein T has a structural unit represented by ALB-R' in a plurality of corresponding target regions in a plurality of heavy chains, so that the antibody has a plurality of structural units represented by ALB-R'.

[20] The soluble protein or salt thereof according to

[17] or

[19] , wherein the number of heavy chains is two.

[21] L1 is represented by the following formulas (L1-1) to (L1-2): C1-Lb (L1-1) C1 (L1-2) [During the ceremony, Lb is a divalent group, C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group.] A soluble protein or a salt thereof according to any one of [2] to

[15] ,

[18] to

[20] , wherein the soluble protein is represented by any one of the following:

[22] The Lb is represented by the following formula (Lb'): [ka] [During the ceremony, p is an integer between 0 and 10; q is an integer from 0 to 10, X is a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R 1b does not exist. If X is a single bond, R 1a and R 1b does not exist), R 1a , and R 1b are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents. A soluble protein or a salt thereof according to

[21] , wherein ○ (open circle) indicates binding to C1, and ● (filled circle) indicates binding to B.

[23] A soluble protein or a salt thereof according to any one of [1] to

[22] , wherein the divalent group containing a bioorthogonal functional group is a divalent group containing in its main chain a bioorthogonal functional group selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene 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 groups, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[24] A soluble protein or a salt thereof according to any one of [1] to

[22] , wherein the divalent group containing a bioorthogonal functional group is a divalent group containing a bioorthogonal functional group in a side chain 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, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[25] The bioorthogonal functional group is one of the following: [ka] [During the ceremony, R 1f , single or multiple R 1g and one or more R 1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups, A soluble protein or a salt thereof according to any one of [1] to

[24] , wherein the soluble protein or salt thereof is one of the following:

[26] The divalent group of (b) is an optionally substituted alkylene, an optionally substituted cycloalkylene, an optionally substituted aryl, an optionally substituted divalent heterocyclic group, -NR a -(R aA soluble protein or a salt thereof according to any one of [1] to

[25] , wherein - is selected from the group consisting of -O-, -O-, or a combination of two or more thereof.

[27] B is represented by the following formula (B-1): [ka] [During the ceremony, Y is -NH-, -O-, -CH2-, or the following formula (B-2): [ka] (In the formula, V and V' are the same or different and each represent -NH-, -O-, -CH2-, or a single bond; V1 is a divalent group containing a bioorthogonal functional group; s is an integer between 0 and 10, The circle and the circle in formula (B-2) are in the same orientation as the circle and the circle in formula (B-1), respectively. Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH-), In formula (B-1), ○ (open circle) indicates a bond to the L-side moiety, and ● (filled circle) indicates a bond to the R-side moiety. A soluble protein or a salt thereof according to any one of [2] to

[15] and

[18] to

[26] , represented by the formula (B-1).

[28] The soluble protein or salt thereof according to any one of [2] to

[15] and

[18] to

[27] , wherein the reactive group is a group specifically reactive with the side chain of any one of a lysine residue, a tyrosine residue, or a tryptophan residue.

[29] The soluble protein or salt thereof according to any one of [2] to

[15] and

[18] to

[28] , wherein the reactive group is a group reactive specifically with the side chain of a lysine residue.

[30] The reactive group is one of the following: [ka] [Here, R 5a and R 5c is an atom or group selected from the group consisting of (i) to (vii) above. and R 5b is an electron-withdrawing group, j is an integer from 1 to 5, and k is an integer of 1 to 4.]. A soluble protein or a salt thereof according to any one of [2] to

[15] and

[18] to

[29] , which corresponds to any one chemical structure selected from the group consisting of:

[31] A soluble protein or a salt thereof according to any one of [1] to

[30] , wherein a bioorthogonal functional group is bound to the soluble protein via a linker having 2 to 10 atoms in the main chain connecting the functional substance and the side chain of a specific amino acid residue.

[32] A soluble protein or a salt thereof according to any one of [1] to

[31] , wherein a bioorthogonal functional group is bound to the soluble protein via a linker whose main chain connecting the functional substance and the side chain of a specific amino acid residue does not contain a ring structure.

[33] A soluble protein or a salt thereof according to any one of [2] to

[15] and

[18] to

[32] , wherein the number of atoms in the main chain connecting the L1 terminal portion and R' is 2 to 10.

[34] A soluble protein or a salt thereof according to any one of [2] to

[15] and

[18] to

[33] , wherein the main chain connecting A and R does not contain a ring structure.

[35] A soluble protein or a salt thereof according to any one of [2] to

[15] and

[18] to

[34] , wherein the partial structure represented by L1-B does not contain a peptide moiety.

[36] The soluble protein or salt thereof having a functional substance regioselectively, represented by the formulas (V1), (V2), and (V3), is preferably the following (V1′), (V2′), and (V3′), respectively: [ka] [During the ceremony, C1 is a bioorthogonal functional group or a group other than a bioorthogonal functional group. p, q, X, R 1aand R 1b is the same as that of the formula (Lb'), Y' is a residue obtained by removing one hydrogen atom from Y in formula (B-1), Z is the same as that in formula (B-1), F, R′, and T are the same as those in formula (V). [ka] [During the ceremony, C1' is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. p, q, X, R 1a and R 1b is the same as that of the formula (Lb'), Y and Z are the same as those in formula (B-1), F, R' and T are the same as those in formula (V) above; or [ka] [During the ceremony, C1' is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. p, q, X, R 1a and R 1b is the same as that of the formula (Lb'), Y' is a residue obtained by removing one hydrogen atom from Y in formula (B-1), Z is the same as that in formula (B-1), Fa and Fb are the same or different functional substances, wherein R' and T are the same as those in the formula (V). A soluble protein or a salt thereof according to any one of [2] to

[15] and

[18] to

[35] , which is a soluble protein or a salt thereof having a functional substance regioselectively, represented by the formula (V).

[37] A soluble protein or a salt thereof having a functional substance regioselectively, The soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region; and a functional substance is bound to one or more specific amino acid residues contained in the target region with a site selectivity of 30% or more via a linker that does not contain a peptide moiety; The functional substance is bound to the soluble protein via the side chain of the lysine residue, the soluble protein is an antibody comprising multiple heavy chains; A soluble protein or a salt thereof having functional substances site-selectively, wherein the antibody has functional substances at multiple specific amino acid residues present at the aforementioned positions in multiple heavy chains contained in the antibody, resulting in the antibody having multiple functional substances.

[38] The following formula (V): F-(L1-B)'-R'-T (V) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; The structural unit represented by (L1-B)' is a divalent structural unit containing a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a), F is a functional substance, R' is a moiety formed by the reaction between a lysine residue of an antibody and a reactive group specific for the side chain of a lysine residue; T is an antibody, The antibody contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A soluble protein or a salt thereof having a functional substance regioselectively, wherein a structural unit represented by F-(L1-B)'-R' is bound to one or more specific amino acid residues contained in the target region of the soluble protein with a regioselectivity of 30% or more.

[39] The soluble protein or a salt thereof is represented by the following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1, F, R' and T are the same as those in formula (V), and B' is a divalent group containing a moiety generated by a reaction between the functional substance and the bioorthogonal functional group.], and the structural unit represented by L1-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more, or a salt thereof.

[40] The soluble protein or a salt thereof is represented by the following formula (V2): F-L1'-B-R'-T (V2) [During the ceremony, F, B, R′, and T are the same as those in formula (V), and L1' is a divalent group containing a moiety generated by a reaction between the functional substance and (i') a monovalent group containing a bioorthogonal functional group. A soluble protein or a salt thereof having a functional substance regioselectively, wherein the structural unit represented by F-L1'-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more. The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (V), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; wherein Fa and Fb are the same or different functional substances.], and the structural unit represented by Fa-L1'-B'(-Fb)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, or a salt thereof.

[0023] (Method for producing a soluble protein or its salt having a functional substance site-selectively) The present invention also relates to a soluble protein represented by formula (V) or The present invention provides a method for producing a soluble protein or a salt thereof that has a functional substance regioselectively and that needs to be specified by a sub-formula.

[41] A method for producing a soluble protein or a salt thereof having a functional substance regioselectively, comprising: The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; F is a functional substance, R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein, the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region; and a structural unit represented by AL-B'(-F)-R' binds with a site selectivity of 30% or more to one or more specific amino acid residues contained in the target region of the soluble protein, by cleaving the cleavable portion of a complex or a salt thereof having an affinity substance for the soluble protein, a cleavable portion, a functional substance, and a soluble protein in a site-selective manner, or The following formula (IV): L1-B-R'-T (IV) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein that contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and that contains five or more of the specific amino acid residues in a non-target region other than the target region. A soluble protein or a salt thereof that has a bioorthogonal functional group regioselectively, wherein the structural unit represented by AL-B'(-F)-R' is bound to one or more specific amino acid residues contained in the target region of the soluble protein with a regioselectivity of 30% or more, is reacted with a functional substance, The following formula (V): F-(L1-B)'-R'-T (V) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; The structural unit represented by (L1-B)' is a divalent structural unit containing a moiety generated by a reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a), F is a functional substance, wherein R' and T are the same as those in formula (III) or (IV), and the structural unit represented by F-(L1-B)'-R' is bonded to one or more specific amino acid residues contained in the target region of the soluble protein with a site selectivity of 30% or more, thereby producing a soluble protein or a salt thereof having a functional substance site-selectively. Including, a method.

[0024] Preferably, the method of

[41] above may be as follows.

[42] A method for producing a soluble protein comprising regioselectively cleaving the cleavable portion of a complex or a salt thereof, the complex comprising an affinity substance for the soluble protein, a cleavable portion, a functional substance, and a soluble protein, The following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; wherein B', F, R', and T are the same as those in formula (III). ], and a structural unit represented by L1-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more, or a salt thereof.

[43] A method for producing a soluble protein or a salt thereof having a bioorthogonal functional group regioselectively, by reacting the soluble protein or a salt thereof with one or two functional substances, The following formula (V2): F-L1'-B-R'-T (V2) [During the ceremony, B, R′, and T are the same as those in formula (IV), L1' is a divalent group containing a moiety generated by a reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; and F is a functional substance. ], and a structural unit represented by F-L1'-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more; or The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (IV), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; wherein Fa and Fb are the same or different functional substances.], and a structural unit represented by Fa-L1'-B'(-Fb)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, or a salt thereof.

[44] The method according to any one of

[41] to

[43] , wherein the soluble protein is an antibody and the reactive group is a reactive group specific to the side chain of a lysine residue.

[45] A method for producing a soluble protein or a salt thereof having a functional substance regioselectively, comprising: (A) Formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein, the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A structural unit represented by ALB-R' binds to one or more specific amino acid residues contained in the target region of the soluble protein with a site selectivity of 30% or more, and the soluble protein or its salt having a site-selective cleavable moiety is reacted with a functional substance to produce a soluble protein. The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A, L, R′, and T are the same as those in formula (II), B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; and F is a functional substance. ], and a structural unit represented by AL-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, thereby producing a complex or a salt thereof having an affinity substance for the soluble protein, a cleavable moiety, a functional substance, and a soluble protein in a site-selective manner; (B) cleaving the cleavable portion of the complex or its salt, which site-selectively comprises a substance with affinity for the soluble protein, a cleavable portion, a functional substance, and a soluble protein; The following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; wherein B', F, R', and T are the same as those in formula (III). ], and a structural unit represented by L1-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more, or a salt thereof.

[46] A method for producing a soluble protein or a salt thereof having a functional substance regioselectively, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; and R is a reactive group for the soluble protein.], a compound or a salt thereof having a substance with affinity for a soluble protein, a cleavable moiety, and a reactive group regioselectively, with a soluble protein (wherein the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region), The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. ], and a structural unit represented by ALB-R' binds to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, thereby producing a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable moiety site-selectively; (B) reacting the soluble protein or its salt having an affinity substance for the soluble protein and a site-selectively cleavable portion with a functional substance, The following formula (III): AL-B'(-F)-R'-T (III) [During the ceremony, A and L are the same as those in formula (I), R′ and T are the same as those in formula (II), B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; and F is a functional substance. ], and a structural unit represented by AL-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, thereby producing a complex or a salt thereof having an affinity substance for the soluble protein, a cleavable moiety, a functional substance, and a soluble protein in a site-selective manner; (C) cleaving the cleavable portion of the complex or its salt, which site-selectively comprises a substance with affinity for the soluble protein, a cleavable portion, a functional substance, and a soluble protein; The following formula (V1): L1-B'(-F)-R'-T (V1) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; wherein B', F, R', and T are the same as those in formula (III). ], and a structural unit represented by L1-B'(-F)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a regioselectivity of 30% or more, or a salt thereof.

[47] A method for producing a soluble protein or a salt thereof having a functional substance regioselectively, comprising: (A) Formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein, the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region. A structural unit represented by ALB-R' binds to one or more specific amino acid residues contained in the target region of the soluble protein with a site selectivity of 30% or more, and the cleavable portion of a soluble protein or a salt thereof having an affinity substance for the soluble protein and a site selectivity for the cleavable portion, The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. ], and the structural unit represented by L1-B-R' is bound to one or more specific amino acid residues contained in the target region of the soluble protein with a regioselectivity of 30% or more, to produce a soluble protein or a salt thereof having a bioorthogonal functional group regioselectively; (B) reacting the soluble protein or its salt regioselectively having the bioorthogonal functional group with one or more functional substances, The following formula (V2) F-L1'-B-R'-T (V2) [During the ceremony, B, R′, and T are the same as those in formula (IV), L1' is a divalent group containing a moiety generated by a reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; and F is a functional substance.], and a structural unit represented by F-L1'-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, or a salt thereof; The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (IV), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; wherein Fa and Fb are the same or different functional substances, and a structural unit represented by Fa-L1'-B'(-Fb)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more,

[48] ​​A method for producing a soluble protein or a salt thereof having a functional substance regioselectively, comprising: (A) Formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (a) a divalent group not containing a bioorthogonal functional group; and R is a group reactive to the soluble protein.] or a salt thereof is reacted with a soluble protein (wherein the soluble protein contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and contains five or more of the specific amino acid residues in a non-target region other than the target region), to thereby produce a soluble protein. The following formula (II): ALB-R'-T (II) [During the ceremony, A, L, and B are the same as those in formula (I). R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein. ], and a structural unit represented by ALB-R' binds to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, thereby producing a soluble protein or a salt thereof having an affinity substance for the soluble protein and a cleavable moiety site-selectively; (B) cleaving the cleavable portion of the soluble protein or its salt, which has an affinity substance for the soluble protein and a cleavable portion site-selectively; The following formula (IV): L1-B-R'-T (IV) [During the ceremony, B, R′, and T are the same as those in formula (II), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. ], and the structural unit represented by L1-B-R' is bound to one or more specific amino acid residues contained in the target region of the soluble protein with a regioselectivity of 30% or more, to produce a soluble protein or a salt thereof having a bioorthogonal functional group regioselectively; (C) reacting the soluble protein or its salt regioselectively having the bioorthogonal functional group with one or more functional substances, The following formula (V2): F-L1'-B-R'-T (V2) [During the ceremony, B, R′, and T are the same as those in formula (IV), L1' is a divalent group containing a moiety generated by a reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; and F is a functional substance.], and a structural unit represented by F-L1'-B-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, or a salt thereof; The following formula (V3): Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, R′ and T are the same as those in formula (IV), L1' is the same as that in formula (V2) above, B' is a divalent group containing a moiety formed by reaction between a functional substance and a bioorthogonal functional group; wherein Fa and Fb are the same or different functional substances, and a structural unit represented by Fa-L1'-B'(-Fb)-R' is bound to one or more specific amino acid residues contained in a target region of the soluble protein with a site selectivity of 30% or more, [Effects of the Invention]

[0025] (I) The compound of the present invention or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety and a reactive group is useful, for example, for the site-selective modification of a soluble protein. (II) The soluble protein of the present invention or a salt thereof having an affinity substance for a soluble protein and a cleavable moiety (regioselectively), (III) the complex of the present invention or a salt thereof having an affinity substance for a soluble protein, a cleavable moiety, a functional substance, and a soluble protein (regioselectively), and (IV) the soluble protein of the present invention having a bioorthogonal functional group (regioselectively) are useful, for example, as intermediates for preparing a soluble protein or a salt thereof having a functional substance (regioselectively). (V) The soluble protein of the present invention having a functional substance (regioselectively) or a salt thereof is useful, for example, as a pharmaceutical or a reagent (e.g., a diagnostic agent, a research reagent). In particular, when the soluble protein is an antibody, the antibody of the present invention having a functional substance (regioselectively) or a salt thereof is suitable for these uses. [Brief explanation of the drawings]

[0026] [Figure 1-1] Figure 1-1 is a schematic diagram (part 1) illustrating the concept of regioselective modification of a soluble protein (e.g., an antibody) with a compound of the present invention (a compound having an affinity substance for a soluble protein, a cleavable moiety, and a reactive group: ALBR(I)). First, the compound of the present invention associates with a soluble protein (T), such as an antibody, via an affinity substance for the soluble protein (A). Next, the compound of the present invention reacts with the side chain of a specific amino acid residue (the side chain of a lysine residue in the figure) in the target region near the site of association between the affinity substance and the soluble protein via the reactive group (R) (an activated ester in the figure), to generate a conjugate of the compound of the present invention and the soluble protein (a soluble protein having a structural unit containing an affinity substance for a soluble protein and a cleavable moiety in a regioselective manner: ALB-R'-T(II)). [Figure 1-2] Figure 1-2 is a schematic diagram (part 2) illustrating the concept of site-selective modification of soluble proteins (e.g., antibodies) with the compounds of the present invention. Cleavage of the cleavable moiety in the linker (L) generates a soluble protein (e.g., antibody) site-specifically modified with a bioorthogonal functional group. [Figure 1-3] Figure 1-3 is a schematic diagram (part 3) illustrating the concept of site-selective modification of soluble proteins (e.g., antibodies) with the compounds of the present invention. Reaction of a bioorthogonal functional group with a functional substance (e.g., a drug) produces a soluble protein (e.g., an antibody) site-specifically modified with the functional substance. [Figure 2] Figure 2 shows the relationship between the various inventions of the present invention (salts are omitted). In reaction (1), a compound having an affinity substance for a soluble protein, a cleavable moiety, and a reactive group is reacted with the soluble protein to produce a soluble protein having an affinity substance for the soluble protein and a cleavable moiety. In reaction (2), the affinity substance for the soluble protein and the soluble protein having a cleavable moiety are reacted with a functional substance to produce a complex having the affinity substance for the soluble protein, the cleavable moiety, the functional substance, and the soluble protein. In reaction (3), the cleavable moiety of the complex having the affinity substance for the soluble protein, the cleavable moiety, the functional substance, and the soluble protein is cleaved to produce a soluble protein having the functional substance (at this time, the affinity substance-containing moiety is produced as a by-product). In reaction (4), the affinity substance for the soluble protein and the cleavable moiety of the soluble protein having a cleavable moiety are cleaved to produce a soluble protein having a bioorthogonal functional group (at this time, the affinity substance-containing moiety is produced as a by-product). In reaction (5), a soluble protein bearing a bioorthogonal functional group is reacted with a functional substance to produce a soluble protein bearing the functional substance. Reactions (2) and (5) can be carried out in a similar manner. Reactions (3) and (5) can also be carried out in a similar manner. [Figure 3]Figure 3 shows HIC-HPLC analysis (detection: 225 nm) of the anti-HER2 IgG antibody trastuzumab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS-activated). Samples were reacted under the following conditions: a: 12 equivalents of trastuzumab + peptide reagent (solvent replaced with Amicon 10K after reaction); b: 12 equivalents of trastuzumab + peptide reagent; c: 6 equivalents of trastuzumab + peptide reagent; d: raw trastuzumab; e: peptide reagent only; f: DMF only. [Figure 4] Figure 4 shows HIC-HPLC analysis (detection: 280 nm) of the anti-HER2 IgG antibody trastuzumab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS activated). The sample was reacted under the same conditions as in Figure 1. [Figure 5] Figure 5 shows HIC-HPLC analysis (detection: 225 nm) of the anti-CD20 antibody rituximab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS-activated). Samples were reacted under the following conditions: a: 12 equivalents of rituximab + peptide reagent (solvent replaced with Amicon 10K after reaction); b: 12 equivalents of rituximab + peptide reagent; c: 6 equivalents of rituximab + peptide reagent; d: rituximab raw material; e: peptide reagent only; f: DMF only. [Figure 6] Figure 6 shows HIC-HPLC analysis (detection: 280 nm) of the anti-CD20 antibody rituximab specifically modified with a peptide reagent (peptide disulfide linker conjugate-NHS-activated). Samples were reacted under the following conditions: a: 12 equivalents of rituximab + peptide reagent (solvent replaced with Amicon 10K after reaction); b: 12 equivalents of rituximab + peptide reagent; c: 6 equivalents of rituximab + peptide reagent; d: rituximab raw material; e: peptide reagent only; f: DMF only. [Figure 7]Figure 7 shows RP-HPLC analysis (detection at 225 nm and 280 nm) of the products obtained by linker cleavage and reoxidation of trastuzumab-peptide conjugates. a: Trastuzumab; b: Trastuzumab + 6 equivalents of peptide reagent; c: Trastuzumab reduced with DTT; d: Trastuzumab reduced with D,L-dithiothreitol at a pH of 8.0 and an antibody concentration of 72 μM; e: d after 3 hours of reoxidation with dehydroascorbic acid; f: d after 24 hours of reoxidation with dehydroascorbic acid. [Figure 8] FIG. 8 shows (1) the amino acid sequence of the heavy chain of trastuzumab whose sugar chains have been cleaved with PNGase, (2) the amino acid sequence of the IgG1 Fc region whose sugar chains have been cleaved with PNGase, and (3) the amino acid sequence of the light chain of trastuzumab. [Figure 9] FIG. 9 shows the MS spectrum (m / z 998.14796, trivalent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 5) containing a modification site (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid) on a lysine residue resulting from trypsin digestion of trastuzumab. [Figure 10] FIG. 10 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 5) containing a modification site (a thiol-introduced form (+146.004 Da) that has been carboxymethylated with iodoacetic acid) at a lysine residue resulting from trypsin digestion of trastuzumab. [Figure 11] FIG. 11 shows the MS spectrum (m / z 929.49651, divalent) of a peptide fragment of LLGGPSVFLFPPKPKD (SEQ ID NO: 6) containing a modification site on a lysine residue by Glu-C digestion of trastuzumab (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid). [Figure 12]FIG. 12 shows the CID spectrum of a peptide fragment of LLGGPSVFLFPPKPKD (SEQ ID NO: 6) containing a modification site (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid) at a lysine residue resulting from Glu-C digestion of trastuzumab. [Figure 13] FIG. 13 shows the MS spectrum (m / z 994.12546, trivalent) of a peptide fragment of THTCPPCPAPEAEGAPSVFLFPPKPK (SEQ ID NO: 7) containing a modification site (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid) on a lysine residue resulting from trypsin digestion of IgG1 Fc. [Figure 14] FIG. 14 shows the CID spectrum of a peptide fragment of THTCPPCPAPEAEGAPSVFLFPPKPK (SEQ ID NO: 7) containing a modification site (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid) at a lysine residue obtained by trypsin digestion of IgG1 Fc. [Figure 15] FIG. 15 shows the MS spectrum (m / z 892.12624, trivalent) of a peptide fragment of GAPSVFLFPPKPKKDTLMISRTPE (SEQ ID NO: 8) containing a modification site (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid) on a lysine residue resulting from Glu-C digestion of IgG1 Fc. [Figure 16] FIG. 16 shows the CID spectrum of a peptide fragment of GAPSVFLFPPKPKKDTLMISRTPE (SEQ ID NO: 8) containing a modification site (a thiol-introduced product (+146.004 Da) that has been carboxymethylated with iodoacetic acid) at a lysine residue upon Glu-C digestion of IgG1 Fc. [Figure 17] FIG. 17 shows the consensus amino acid sequence (SEQ ID NO: 1) of the Fc region in the heavy chain of trastuzumab and the IgG1 Fc region. [Figure 18]Figure 18 shows the amino acid sequence and modifications identified for modified trastuzumab (IgG1). The gray portion represents the identified amino acid sequence. The identified modifications are noted above the amino acid sequence. Site-selective azide-introduced modifications were confirmed only at the two boxed lysine residues (positions 246 and 248 according to EU numbering). [Figure 19] Figure 19 shows the modification sites of azide-introduced trastuzumab. Peptide Spectrum Matches (PSMs) refer to the number of times a spectrum matching the corresponding peptide is observed; the higher the number, the higher the likelihood. By applying a filter based on signal intensity, modification was identified only at the lysine residue at position 246, as shown in (2). The modification site identified only in (1) is likely to be noise. [Figure 20] Figure 20 shows the CID spectrum of a peptide containing the modification site of an azide-introduced form of trastuzumab, corresponding to the CID spectrum of the peptide in the boxed area in Figure 18. The spectrum shown has an m / z value that matches the theoretical value. [Figure 21] Figure 21 shows the b / y ions identified in the CID spectrum. The b / y ions identified in Figure 20 are shown. The identification of the b25 ion indicates that position 246 (EU numbering) is highly likely to be modified. [Figure 22] Figure 22 shows a comparison of the area values ​​of modified trastuzumab with unmodified trastuzumab. Based on the prediction that the area value of the unmodified peptide would be smaller at the site of modification, a comparison with the area value obtained with unmodified trastuzumab revealed a significant decrease only in the peptide containing lysine residues at positions 246 and 248 (EU numbering). [Figure 23] 23 shows the DAR analysis of modified trastuzumab by Q-TOFMS. From the peak observation results, the average DAR was 2. [Figure 24]Figure 24 shows the amino acid sequence data used in the search. Since it is known that deglycosylation changes N to D at the 300th residue in the heavy chain of trastuzumab, two types of heavy chain amino acid sequences were used for the analysis. [Figure 25] Figure 25 shows the MS spectrum (measured value: m / z 979.49982, theoretical value: 979.49975, tetravalent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 40), which contains a modification site (azide-introduced form (+254.102 Da)) on a lysine residue obtained by trypsin digestion of trastuzumab. [Figure 26] FIG. 26 shows a CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 40) containing a modification site (azide-introduced form (+254.102 Da)) in a lysine residue obtained by trypsin digestion of trastuzumab. [Figure 27] FIG. 27 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 16). [Figure 28] FIG. 28 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 17). [Figure 29] FIG. 29 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 18). [Figure 30] FIG. 30 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 19). [Figure 31] FIG. 31 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 20). [Figure 32]FIG. 32 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 21). [Figure 33] FIG. 33 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 22). [Figure 34] FIG. 34 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 23). [Figure 35] FIG. 35 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 24). [Figure 36] FIG. 36 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 25). [Figure 37] FIG. 37 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 26). [Figure 38] FIG. 38 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 27). [Figure 39] FIG. 39 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 28). [Figure 40] FIG. 40 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 29). [Figure 41] FIG. 41 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 225 nm or UV 280 nm) (Example 30). [Figure 42]42 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 31). AU on the vertical axis represents absorbance (the same applies to the subsequent figures). [Figure 43] FIG. 43 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 32). [Figure 44] FIG. 44 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 33). [Figure 45] FIG. 45 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 34). [Figure 46] FIG. 46 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 35). [Figure 47] FIG. 47 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 36). [Figure 48] FIG. 48 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 37). [Figure 49] FIG. 49 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 38). [Figure 50] FIG. 50 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 39). [Figure 51] Figure 51 shows the MS spectrum (m / z 791.74872, trivalent) of the peptide fragment of VVSVLTVLHQDWLNGKEYK (sequence number 66) containing the modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Example 39. [Figure 52] Figure 52 shows the CID spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (sequence number 66) containing a modification site (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) at a lysine residue obtained by trypsin digestion of trastuzumab modified with the binder peptide of Example 39. [Figure 53] Figure 53 shows the MS spectrum (m / z 886.93408, quadrivalent) of the peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (sequence number 67), which includes a modification site (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Example 39. [Figure 54] Figure 54 shows the CID spectrum of a peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (sequence number 67) containing a modification site (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) at a lysine residue obtained by trypsin digestion of trastuzumab modified with the binder peptide of Example 39. [Figure 55] FIG. 55 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 40). [Figure 56] Figure 56 shows the MS spectrum (m / z 769.04688, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 69) containing a modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Example 40. [Figure 57]Figure 57 shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 69) containing a modification site (a thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) at a lysine residue obtained by trypsin digestion of trastuzumab modified with the binder peptide of Example 40. [Figure 58] FIG. 58 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 41). [Figure 59] Figure 59 shows the MS spectrum (m / z 952.23145, tetravalent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 40), which contains a modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with a thiol-introduced product. [Figure 60] Figure 60 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 40), which contains a modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) at a lysine residue obtained by trypsin digestion of trastuzumab modified with a thiol-introduced product. [Figure 61] FIG. 61 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 42). [Figure 62] Figure 62 shows the MS spectrum (m / z 952.22968, tetravalent) of the peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 40), which contains the modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with a thiol-introduced product. [Figure 63] Figure 63 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 40), which contains a modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) at a lysine residue obtained by trypsin digestion of trastuzumab modified with a thiol-introduced product. [Figure 64] FIG. 64 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 43). [Figure 65] Figure 65 shows the MS spectrum (m / z 769.04529, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 69) containing a modification site (thiol-introduced compound (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with a thiol-introduced compound. [Figure 66] Figure 66 shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 69) containing a modification site (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide) at a lysine residue obtained by trypsin digestion of trastuzumab modified with a thiol-introduced product. [Figure 67] FIG. 67 shows the results of HIC-UPLC analysis of specific modifications of a human IgG2 antibody (detection wavelength: UV 280 nm) (Example 2-2). [Figure 68] FIG. 68 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modifications of a human IgG2 antibody (Example 43). [Figure 69] FIG. 69 shows the results of HIC-UPLC analysis of specific modifications of a human IgG4 antibody (detection wavelength: UV 280 nm) (Example 2-2). [Figure 70]FIG. 70 shows the results of HIC-UPLC analysis (detection wavelength: UV 280 nm) of specific modifications of a human IgG4 antibody (Example 43). DETAILED DESCRIPTION OF THE INVENTION

[0027] 1. A compound or its salt containing an affinity substance for soluble proteins, a cleavable moiety, and a reactive group Overview The present invention provides a compound or a salt thereof comprising an affinity substance for a soluble protein, a cleavable moiety, and a reactive group, represented by formula (I): ALBR (I) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R is a reactive group to the soluble protein.

[0028] In formula (I) and other formulas presented in connection with this invention, a hyphen (-) indicates that the two units on either side of it are covalently bonded. Thus, in formula (I), A is covalently bonded to L, L is covalently bonded to A and B, B is covalently bonded to L and R, and R is covalently bonded to B.

[0029] 1-2. Affinity substances for soluble proteins (A) In formula (I), A is an affinity substance for a soluble protein. An affinity substance is a substance that has the ability to bind to a target by non-covalent bonding.

[0030] The affinity substance used in the present invention targets soluble proteins, also known as secretory proteins, such as antibodies, soluble receptors, ligands, albumin, erythropoietin (EPO), vascular endothelial growth factor (anti-VEGF), bone morphogenetic proteins, follicle-stimulating hormone (FSH), glucagon, granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GCSF), chorionic gonadotropin (CG), insulin, and the like. Examples of soluble proteins include interleukins, interferons, platelet-derived growth factors (PDGF), and growth factors (TGF family, FGF family). Soluble proteins may be proteins (e.g., glycoproteins) modified with biomolecules (e.g., sugars) or proteins that are not modified with biomolecules.

[0031] The soluble protein that is the target of the affinity substance may be a naturally occurring protein or an artificial protein.

[0032] Examples of naturally occurring proteins include proteins derived from organisms and viruses. Examples of proteins derived from organisms include proteins derived from animals such as mammals and birds (e.g., chickens), insects, microorganisms, plants, fungi, and fish. Preferably, the naturally occurring protein is a protein derived from a mammal. Examples of mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, and rabbits), pet animals (e.g., dogs and cats), livestock (e.g., cows, pigs, and goats), and working animals (e.g., horses and sheep). More preferably, the naturally occurring protein is a protein derived from a primate or rodent, and even more preferably, from the perspective of clinical application of the present invention, a protein derived from a human. Examples of proteins derived from viruses include influenza viruses (e.g., avian influenza viruses and swine influenza viruses), AIDS viruses, Ebola viruses, and phage viruses.

[0033] Examples of artificial proteins include modified proteins of naturally occurring proteins (e.g., proteins in which one or more amino acid residue mutations selected from the group consisting of substitution, deletion, and insertion have been introduced into a naturally occurring protein), fusion proteins, and artificially designed monoclonal antibodies. Examples of artificially designed monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies with a specific glycosylation chain added (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated consensus sequence), bispecific antibodies, scFv antibodies, Fab antibodies, F(ab')2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins.

[0034] The soluble protein targeted by the affinity substance may further be a monomeric protein or a multimeric (e.g., dimeric, trimeric, or tetrameric) protein. When the protein targeted by the affinity substance is a multimeric protein, the multimeric protein may be a homomultimer or a heteromultimer. The multimeric protein may be a protein that forms a multimer via a covalent bond (e.g., a disulfide bond) (e.g., an antibody having a structure in which two units consisting of a light chain and a heavy chain are linked via a disulfide bond) or a protein that forms a multimer via a non-covalent bond (i.e., association), but a protein that forms a multimer via a covalent bond is preferred. Examples of multimeric proteins that are targets of affinity substances include bivalent antibodies (e.g., IgG, IgD, IgE), tetravalent or higher antibodies (e.g., IgA antibody, IgM antibody), and albumin.

[0035] Soluble proteins targeted by affinity substances may be composed of any amino acid residue, but are preferably composed of the 20 naturally occurring L-α-amino acid residues that normally constitute proteins, such as L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), L-lysine (K), and glycine (G) (hereinafter, the abbreviation "L" will be omitted). The soluble protein may be composed of, for example, 100 or more, preferably 120 or more, more preferably 150 or more, even more preferably 180 or more, and particularly preferably 200 or more amino acid residues. The soluble protein may also be composed of, for example, 1000 or less, preferably 900 or less, more preferably 800 or less, even more preferably 700 or less, and particularly preferably 600 or less amino acid residues. More specifically, the soluble protein may be composed of, for example, 100 to 1000, preferably 120 to 900, more preferably 150 to 800, even more preferably 180 to 700 or more, and particularly preferably 200 to 600 amino acid residues. When the soluble protein is an antibody (e.g., an artificially designed monoclonal antibody as described above), the above number of amino acid residues may correspond to the amino acid residues of the heavy chain of the antibody.

[0036] The soluble protein that is the target of the affinity substance may further comprise a specific amino acid having a side chain or terminus (N-terminus and / or C-terminus) capable of reacting with a reactive group as described below, preferably a side chain. The present invention relates to a protein containing amino acid residues at one or more positions (preferably at multiple positions). Examples of such specific amino acid residues include, for example, 14 types of amino acid residues as described below, preferably amino acid residues selected from the group consisting of lysine residues, tyrosine residues, tryptophan residues, and cysteine ​​residues. Considering that the compounds of the present invention can regioselectively modify soluble proteins, soluble proteins containing such specific amino acid residues at multiple positions are preferred. The multiple positions are not particularly limited as long as they are two or more positions, but may be, for example, three or more positions, preferably five or more positions, more preferably ten or more positions, even more preferably twenty or more positions, and particularly preferably thirty or more positions. The multiple positions may also be, for example, positions 200 or less, preferably positions 180 or less, more preferably positions 150 or less, even more preferably positions 120 or less, and particularly preferably positions 100 or less. 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 soluble proteins containing such specific amino acid residues at multiple positions, the compounds of the present invention can regioselectively modify a specific amino acid residue present at a specific position. For example, the number of lysine residues in human IgG1 is generally said to be approximately 70 to 90, although this varies depending on the amino acid composition in the variable region. The present invention has succeeded in regioselectively modifying such lysine residues present at a specific position in human IgG1.

[0037] More specifically, in the present invention, site-selective modification of amino acid residues exposed on the surface of a protein is preferred from the viewpoint of modifying amino acid residues present at specific positions in a protein while maintaining the function of the protein, such as an antibody (i.e., while maintaining native folding without denaturing the protein). For example, in human IgG, such as human IgG1, exposed lysine and tyrosine residues are present at the following positions (according to EU numbering; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html): (1) Exposed Lysine Residues CH2 domain (246, 248, 274, 288, 290, 317, 320, 322, 338) CH3 domain (360th, 414th, 439th) (2) Exposed tyrosine residues CH2 domain (ranks 278, 296, and 300) CH3 domain (rank 436) Therefore, when human IgG, such as human IgG1, is modified at a lysine or tyrosine residue, modification at the above positions is preferred.

[0038] Preferably, human IgG, such as human IgG1, is modified at a lysine or tyrosine residue. In this case, among the positions (1) and (2) above, lysine residues or tyrosine residues present at the following positions, which are highly exposed to the surface, may be modified: (1') exposed lysine residue CH2 domain (246, 248, 274, 288, 290, 317, 320, 322) CH3 domain (360th, 414th, 439th) (2') Exposed tyrosine residue CH2 domain (ranks 278, 296, and 300) CH3 domain (rank 436) Therefore, when human IgG, such as human IgG1, is modified at a lysine or tyrosine residue, modification at the above positions is more preferred.

[0039] More preferably, when human IgG such as human IgG1 is modified with lysine residues, the lysine residues present at specific positions in the CH2 domain that can be efficiently modified in the present invention (e.g., positions 246, 248, 288, 290, and 317) among the positions (1) above may be modified.

[0040] In certain embodiments, when a soluble protein targeted by an 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 may also contain 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, and 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 type of target protein and affinity substance, but may be, for example, specific positions in specific regions (e.g., CH1, CH2, or CH3) in the constant region of an antibody, preferably in CH2 of the antibody. More specifically, the target region may be the following residues according to EU numbering in human IgG Fc: (1) Lys248 residue (hereinafter also referred to simply as "Lys248" in this specification, corresponding to the 18th residue in the CH2 region of human IgG (SEQ ID NO: 1)) or Lys246 residue (hereinafter also referred to simply as "Lys246" in this specification, corresponding to the 16th residue in the CH2 region of human IgG (SEQ ID NO: 1)) : (2) Lys288 residue (hereinafter also referred to simply as "Lys288" in this specification, corresponding to the 58th residue in the CH2 region of human IgG (SEQ ID NO: 1)) or Lys290 residue (hereinafter also referred to simply as "Lys290" in this specification, corresponding to the 60th residue in the CH2 region of human IgG (SEQ ID NO: 1)); (3) Lys317 residue (hereinafter also referred to simply as "Lys317" in this specification, and corresponds to the 87th residue in the CH2 region of human IgG (SEQ ID NO: 1)).

[0041] 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% or more.

[0042] The target region also refers to a region in which a specific amino acid residue present at a specific position is present at the specific position in a region extending from the specific position to a distant position of a number of amino acid residues on the N-terminal side and the C-terminal side (where a is an integer of 1 to 10). In addition to the specific amino acid residues, the amino acid residues may not contain the same amino acid residues as the specific amino acid residues. a is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, even more preferably 1 or 2, and particularly preferably 1.

[0043] In a preferred embodiment, the affinity substance for a soluble protein is an affinity substance for an antibody. The antibody may be a polyclonal antibody or a monoclonal antibody. Examples of antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. The antibody may be a full-length antibody or an antibody fragment (e.g., F(ab')2, Fab', Fab, Fv, single-chain antibody), with full-length antibodies being preferred.

[0044] The antibody is an antibody against any antigen. For example, such antigens may be components found in the organisms or viruses described above. Such antigens also include, for example, proteins (including oligopeptides and polypeptides, and proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low-molecular-weight compounds.

[0045] Preferably, the antibody may be an antibody whose antigen is a protein, such as a cell membrane receptor, a cell membrane protein other than a cell membrane receptor (e.g., an extracellular matrix protein), a ligand, or a soluble receptor.

[0046] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0047] (1) Oncology PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1(CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfelin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Ruis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 inhibitory factor, TAG-72 (CA72-4), CD70,

[0048] (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

[0049] (3) Neurological disorders CGRP, CD20, β-amyloid, β-amyloid protofibrin, calcitonin gene-related peptide receptor, LINGO (Ig domain containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0050] (4) Infectious disease Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0051] (5) Genetic and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0052] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0053] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0054] (8) Hematological diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0055] (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

[0056] In a more preferred embodiment, the affinity substance for a soluble protein is an affinity substance for a monoclonal antibody. The isotype of the monoclonal antibody is the same as that described above for antibodies, with IgG (e.g., IgG1, IgG2, IgG3, IgG4) being preferred. Preferably, the monoclonal antibody is a full-length monoclonal antibody.

[0057] In an even more preferred embodiment, the affinity substance for a soluble protein is an affinity substance for a full-length monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody (eg, IgG such as IgG1, IgG2, IgG3, IgG4, etc.).

[0058] In a particularly preferred embodiment, the affinity substance for a soluble protein comprises any one Fc region protein selected from the group consisting of the following (A) to (C), and is an affinity substance for an antibody having antigen-binding ability: (A) an Fc region protein comprising the amino acid sequence of SEQ ID NO: 1; (B) an Fc region protein comprising an amino acid sequence in which one or several amino acid residues have been inserted, added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1; or (C) An Fc region protein comprising an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 1.

[0059] The amino acid sequence of SEQ ID NO: 1 is an Fc region protein. Such Fc region proteins are known to have secretory activity. Therefore, the Fc region proteins (A) to (C) above can have secretory activity. Furthermore, antibodies comprising such Fc region proteins can have antigen-binding activity. The amino acid residue at position 18 in SEQ ID NO: 1 can be any amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a nonpolar side chain as described below, even more preferably leucine, isoleucine, or alanine, and particularly preferably leucine or alanine. The amino acid residue at position 19 in SEQ ID NO: 1 can be 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, even more preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO: 1 can be 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 may be any amino acid residue but is preferably an acidic amino acid residue, more preferably glutamic acid or aspartic acid. The amino acid residue at position 142 in SEQ ID NO: 1 may be any amino acid residue but is preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, 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 may be any amino acid residue but is preferably a neutral amino acid residue, more preferably an amino acid residue having an uncharged polar side chain or an amino acid residue having a non-polar side chain as described below, even more preferably threonine, alanine, or glycine, and particularly preferably threonine or alanine.

[0060] In a preferred embodiment, the amino acid sequence of SEQ ID NO: 1 is SEQ ID NO: 2 or SEQ ID NO: 41 It may be an amino acid sequence consisting of amino acid residues at positions 220 to 449 in the amino acid sequence of above.

[0061] In another preferred embodiment, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence consisting of amino acid residues at positions 7 to 236 in the amino acid sequence of SEQ ID NO: 3.

[0062] In a specific embodiment, the antibody comprising an Fc region protein having the amino acid sequence described above may be an antibody comprising an Fc region protein having the amino acid sequence described above and an antibody constant region, which may be the constant region of a chimeric antibody, a humanized antibody, or a human antibody (e.g., IgG such as IgG1, IgG2, IgG3, or IgG4).

[0063] In the Fc region protein (B), one or several amino acid residues may be modified by one, two, three, or four types of mutation selected from the group consisting of deletion, substitution, addition, and insertion of amino acid residues. The amino acid mutations may be introduced into one region of the amino acid sequence, or into several different regions. The term "one or several" refers to the number of residues 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).

[0064] For Fc region proteins (C), the percent identity to the amino acid sequence of SEQ ID NO: 1 may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In the present invention, the percent identity of a peptide or polypeptide (protein) can be calculated using the blastp algorithm. More specifically, the percent identity of a polypeptide can be calculated using the blastp algorithm 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 percent identity of a polynucleotide (gene) can be calculated using the blastn algorithm. More specifically, the percent identity of polynucleotides can be calculated using the blastn algorithm provided by NCBI with default Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0065] The term "secretion" in secretory ability has the same meaning as the secretion of a secretory protein (so-called soluble). Therefore, "having secretory ability" means that the antibody functions as a soluble protein, similar to a normal antibody.

[0066] Mutations may be introduced into specific sites in antibodies containing the above-mentioned Fc region proteins, as long as the antibodies retain the desired properties (e.g., secretion ability, antigen-binding ability). The positions of amino acid residues into which mutations may be introduced that retain the desired properties will be clear to those skilled in the art. Specifically, those skilled in the art can 1) compare the amino acid sequences of multiple proteins with similar properties, 2) identify relatively conserved and relatively non-conserved regions, and then 3) predict regions that may play an important role in function and regions that may not play an important role in function from the relatively conserved and relatively non-conserved regions, respectively, thereby recognizing the correlation between structure and function. Therefore, those skilled in the art can easily recognize the correlation between structure and function by comparing the amino acid sequences of the above-mentioned Fc region. The positions of amino acid residues at which mutations may be introduced in the amino acid sequence of an antibody containing protein can be identified.

[0067] When an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution. As used herein, the term "conservative substitution" refers to replacing 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 (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, conservative amino acid substitutions may be 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.

[0068] Examples of antibodies comprising 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, and ortatoxacimab), humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), meclomid, and meloxicam). Antibodies include polizumab, elotuzumab, daratumumab, ikesekizumab (IgG4), reslizumab (IgG4), and atezolizumab; and human antibodies (e.g., adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), and olaratumab). (If no IgG subtype is specified, IgG1 is assumed.)

[0069] Examples of the affinity substance for the soluble protein as described above include peptides (including oligopeptides, polypeptides, and proteins), low molecular weight compounds, nucleic acids, nucleic acid- Examples include peptide complexes, peptide-low molecular weight compound complexes, and nucleic acid-low molecular weight compound complexes.

[0070] In a specific embodiment, the affinity substance for the soluble protein as described above may be a peptide (including an oligopeptide, a polypeptide, and a protein, which may be a glycoprotein). For example, the following peptides have been reported: (1) IgG-binding peptides having affinity for a specific region (CH2 region) of human IgG in general (i.e., human IgG1, IgG2, IgG3, and IgG4; the same applies below) (see, e.g., WO 2016 / 186206, WO 2013 / 027796, and WO 2008 / 054030); (2) Protein A mimetic (PAM) peptides that have affinity for a specific region (CH2 region) of human IgG in general (e.g., Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, VOL. see .6,564-569); (3) EPIHRSTLTALL (SEQ ID NO: 9) having 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, which has affinity for a specific region (Fc region) of human IgG in general (see, e.g., Ruvo M et al., ChemBioChem, 2005, VOL. 6, 1242-1253); (5) FARLVSSIRY (SEQ ID NO: 10), FGRLVSSIRY (SEQ ID NO: 11), and TWKTSRISIF (SEQ ID NO: 12) having affinity for a specific region (Fc region) of human IgG in general (see, e.g., Krook M et al., Journal of Immunological Methods, 1998, VOL. 221, 151-157); (6) QSYP (SEQ ID NO: 13) having affinity for a specific region of human IgG in general (see, for example, Jacobs JM et al., Bio. Techniques, 2003, VOL. 34, 132-141); (7) HWRGWV (SEQ ID NO: 14), HYFKFD (SEQ ID NO: 15), and HFRRHL (SEQ ID NO: 16) having affinity for a specific region (Fc region) of human IgG in general (see, e.g., Carbonell RG et al., Journal of Chromatography A, 2009, VOL. 1216, 910-918); (8) DAAG (SEQ ID NO: 17) having affinity for a specific region (Fc region) of human IgG in general (see, for example, Lund LN et al., Journal of Chromatography A, 2012, VOL. 1225, 158-167); (9) Fc-I, Fc-II, and Fc-III having affinity for specific regions (Fc regions) of human IgG in general (see, e.g., Warren L. Delano et al., Science, 2000, VOL. 287, 1279-1283; International Publication No. 2001 / 045746); and (10) NARKFYKG (SEQ ID NO: 18) and NKFRGKYK (SEQ ID NO: 19) (e.g., Biochemical (See Engineering Journal, 2013, VOL. 79, 33-40).

[0071] In another specific embodiment, the affinity substance for the soluble protein as described above may be a substance other than a peptide. Examples of such substances include aptamers (e.g., GGUG(C / A)(U / T) motif-containing aptamers such as GGUGCU and GGUGAU) that have affinity for a specific region (the CH2 region, particularly the side chain of Lys340) of human IgG (e.g., human IgG1 to IgG4) (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).

[0072] Affinity substances for soluble proteins as described above can be obtained by any method known in the art. For example, they can be obtained by producing antibodies (e.g., hybridoma method) using the entire soluble protein or a partial peptide of the soluble protein (e.g., a partial peptide present in a surface-exposed region of the protein, if the region is known) or by screening affinity substances from available libraries (e.g., peptide libraries, antibody libraries, antibody-producing cell libraries, aptamer libraries, phage libraries, mRNA libraries, cDNA libraries) (e.g., phage display, SELEX, mRNA display, ribosome display, cDNA display, yeast display). Furthermore, when the affinity substance for a soluble protein is an affinity substance for the Fc region (soluble region) of an antibody, partial peptides present in specific regions (e.g., CH1, CH2, CH3) of the Fc region of various antibodies (e.g., IgG, IgA, IgM, IgD, IgE) can be used to efficiently obtain affinity substances (e.g., antibodies, aptamers) that can selectively bind to any portion of the Fc region of an antibody. Among the affinity substances obtained in this manner, there will be a mixture of those with relatively strong and weak affinity binding ability. However, even affinity substances with weak affinity binding ability can be strengthened by using an excess amount.

[0073] The above-mentioned affinity substance for soluble proteins may be an IgG-binding peptide represented by the following formula (i) or a salt thereof (see, for example, the Examples and WO 2016 / 186206): (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 94) (i) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, and W is a tryptophan residue. and capable of binding to human IgG and / or rabbit IgG, or a salt thereof. Preferably, Xaa1 and Xaa2 are different amino acid residues.

[0074] As used herein, the N- or C-terminal X 1-3 The notation "X" means that there are 1 to 3 consecutive amino acid residues X other than cysteine ​​(C or Cys), which are independently any amino acid residue, and the amino acid residues constituting the X may be the same or different, but preferably, all three residues are not the same. Similarly, X2 means that there are two consecutive amino acid residues X other than cysteine ​​(C or Cys), which are independently any amino acid residue, and the amino acid residues constituting the X may be the same or different, but preferably, the two consecutive amino acid residues are not the same. X1, which will be described later, also means that there is one consecutive amino acid residue X other than cysteine ​​(C or Cys).

[0075] Also, herein, at least two cysteine ​​residues spaced apart in each amino acid sequence of a peptide can form a cyclic peptide through a disulfide bond. Typically, in a peptide of formula (i) or the like, the two outer cysteine ​​residues are disulfide bonded. Alternatively, in a peptide of formula (i) or the like, the sulfide groups in the two outer cysteine ​​residues may be linked by a carbonyl group-containing linker represented by the following formula:

[0076] [ka]

[0077] The dashed line portion of the carbonyl group-containing linker shown above represents the bond with the sulfide group. This linker is more stable against reduction reactions and the like than a typical disulfide bond. This peptide can be prepared, for example, by the method described in WO 2016 / 186206.

[0078] In certain embodiments, the affinity substance of formula (i) above may be an IgG-binding peptide represented by formula (i-1) below or a salt thereof (e.g., WO 2016 / 186206). (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 95) (i-1) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a glutamic acid or asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue. and capable of binding to human IgG and / or rabbit IgG, or a salt thereof. Preferably, Xaa1 and Xaa2 are different amino acid residues.

[0079] In another specific embodiment, the affinity substance for the soluble protein as described above may be an IgG-binding peptide represented by the following formula (i-2) or a salt thereof (see, for example, Examples): (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 96) (i-2) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is an arginine residue or a leucine residue, G is a glycine residue, Xaa2 is a lysine, glutamine, or aspartic acid residue; L is a leucine residue, V is a valine residue, and and W is a tryptophan residue.], and which is capable of binding to human IgG and / or rabbit IgG, or a salt thereof. Affinity not disclosed in WO 2016 / 186206 having such a specific structure The substance is useful for site-selective modification of the Lys248 or Lys246 residue according to Eu numbering in human IgG Fc, or other amino acid residues other than Lys248 or Lys246 (Examples).

[0080] Peptides represented by formula (i-1') and formula (i-1''), in which the amino acid residue X in the amino acid sequence of the peptide of formula (i-1) is further specified, are shown below.

[0081] That is, the peptide represented by formula (i-1') is (X 1-3 )-C-(X1)-YH-(Xaa1)-GNLVWC-(X 1-3 ) (SEQ ID NO: 97) (i-1') [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, Y is a tyrosine residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, N is an asparagine residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue. and is characterized by being capable of binding to human IgG and / or rabbit IgG.

[0082] The peptide represented by formula (i-1″) is (X 1-3 )-CA-(X1)-H-(Xaa1)-GELVWC-(X 1-3 ) (SEQ ID NO: 98) (i-1'') [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, A is an alanine residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue. and is characterized by being capable of binding to human IgG and / or rabbit IgG.

[0083] Furthermore, the peptide represented by formula (ii), in which the amino acid residue X in the amino acid sequence of the peptide of formula (i-1) is further specified, is shown below.

[0084] That is, the peptide represented by formula (ii) is (X 1-3 )-C-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (SEQ ID NO: 99) (ii) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a glutamic acid or asparagine residue, L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa3 is an alanine, serine, or threonine residue, and Xaa4 is a tyrosine residue or a tryptophan residue. and is characterized by being capable of binding to human IgG and / or rabbit IgG.

[0085] In the amino acid sequence of a peptide of a formula such as formula (i), if the peptide has 17 amino acid residues, the first and second amino acid residues, and the 16th and 17th amino acid residues X from the N-terminus may be deleted, resulting in a peptide having a length of 13 amino acids.

[0086] As used herein, the term "in the case of 17 amino acid residues" is a convenient expression for the purpose of numbering the amino acid residues of a peptide, such as that of formula (i), from 1 to 17 in order from the N-terminus of the 17 residues that are the longest amino acid length.

[0087] Furthermore, the peptide represented by formula (iii), in which the amino acid residue X in the amino acid sequence of the peptide of formula (i-1) is further specified, is shown below.

[0088] That is, the peptide represented by formula (iii) is (X 1-3 )-CAYH-(Xaa1)-GELVWC-(X 1-3 ) (SEQ ID NO: 100) (iii) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, A is an alanine residue, Y is a tyrosine residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, E is a glutamic acid residue, L is a leucine residue, V is a valine residue, and W is a tryptophan residue. and is characterized by being capable of binding to human IgG and / or rabbit IgG.

[0089] When the amino acid sequence of the peptide of formula (iii) is 17 amino acid residues, the first and second, and the 16th and 17th amino acid residues X from the N-terminus may be deleted, resulting in a peptide having a length of 13 amino acids.

[0090] Furthermore, in the case where the amino acid sequence of the peptide of each formula above is 17 amino acid residues, the amino acid residues at positions 1 to 3, 5, 6, and 15 to 17 from the N-terminus other than cysteine ​​(C) are preferably selected from the following, where each capital letter represents the single-letter code for an amino acid: 1st amino acid residue = S, G, F or none Second amino acid residue = D, G, A, S, P, homocysteine, or none 3rd amino acid residue = S, D, T, N, E or R, 15th amino acid residue = S, T or D, 16th amino acid residue = H, G, Y, T, N, D, F, homocysteine ​​or none, Amino acid residue 17 = Y, F, H, M or none. 5th amino acid residue = A or T, 6th amino acid residue = Y or W.

[0091] Furthermore, the peptide represented by formula (iv), in which the amino acid residue X in the amino acid sequence of the peptide of formula (i-1) is further specified, is shown below.

[0092] That is, the peptide represented by formula (iv) is DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 101) (iv) [During the ceremony, D is an aspartic acid residue, C is a cysteine ​​residue, H is a histidine residue, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a glutamic acid or asparagine residue, L is a leucine residue, V is a valine residue, W is a tryptophan residue, T is a threonine residue, Xaa3 is an alanine or threonine residue, and Xaa4 is a tyrosine residue or a tryptophan residue.] and is characterized by being capable of binding to human IgG and / or rabbit IgG.

[0093] Specific examples of the peptide of formula (i-1) are listed below in (1) to (19), but needless to say, the peptide is not limited to these: (1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 20); (2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 21); (3) RCAYH(Xaa1)GELVWCS (SEQ ID NO: 22); (4) GPRCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 23); (5) SPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 24); (6) GDDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 25); (7) GPSCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 26); (8) GPDCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 27); (9) GPDCAYH(Xaa1)GELVWCTHH (SEQ ID NO: 28); (10) GPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 29); (11) SPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 30); (12) SDDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 31); (13) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 32); (14) G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H (SEQ ID NO: 33); (15) RRGPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 34); (16) DCTYH(Xaa1)GNLVWCT (SEQ ID NO: 35); (17) DCAYH(Xaa1)GNLVWCT (SEQ ID NO: 36); (18) DCTYH(Xaa1)GELVWCT (SEQ ID NO: 37); and (19) DCAWH(Xaa1)GELVWCT (sequence number 38). [During the ceremony, Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; Xaa2 is homocysteine, Preferably, the homocysteines form disulfide bonds with each other.

[0094] Preferred specific examples of the peptide of formula (i-1) include the following: (1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 20); (2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 21); (13) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 22); (14) G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H (SEQ ID NO: 33); and (15) RRGPDCAYH(Xaa1)GELVWCTFH (sequence number 34). [During the ceremony, Xaa1 is a lysine residue, Xaa2 is homocysteine, Preferably, cysteines and / or homocysteines form disulfide bonds with each other.

[0095] The peptide (13) above may be RGNCAYHKGQLVWCTYH (SEQ ID NO: 39).

[0096] In another specific embodiment, the affinity substance of the above formula (i) may be an IgG-binding peptide represented by the following formula (v) or a salt thereof (see, for example, Examples): (X 1-3 )-C-(Xaa3)-(xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 102) (v) [During the ceremony, X may be the same or different and is any amino acid residue other than cysteine, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa5 is a threonine or lysine residue, Xaa6 is a tyrosine residue, a lysine residue, or none; and and Xaa7 is a histidine residue, a lysine residue, or is absent.], and is a peptide or a salt thereof that is capable of binding to human IgG.

[0097] Affinity substances having such specific structures and not disclosed in WO 2016 / 186206 are useful for site-selective modification of Lys248 or Lys246 residues in human IgG Fc according to EU numbering, or other amino acid residues other than Lys248 or Lys246 (see Examples). Preferably, any one of Xaa3, Xaa1, Xaa2, Xaa5, Xaa6, and Xaa7 is a lysine residue. Xaa1 may preferably be an arginine residue or a leucine residue. Alternatively, Xaa1 may preferably be a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue, more preferably a lysine residue, an aspartic acid residue, or a glutamic acid residue.

[0098] When the amino acid sequence of the peptide of formula (v) is 17 amino acid residues, the first and second, and the 16th and 17th amino acid residues X from the N-terminus may be deleted, resulting in a peptide having a length of 13 amino acids.

[0099] Furthermore, when the amino acid sequence of the peptide of formula (v) is 17 amino acid residues, the amino acid residues other than cysteine ​​(C) are preferably selected from the following, where each capital letter represents a single-letter code for an amino acid: 1st amino acid residue = R, S, G, F or none (preferably R or none) Second amino acid residue = D, G, A, S, P, homocysteine, or none (preferably G or none) 3rd amino acid residue = S, D, T, N, E or R (preferably N or D).

[0100] Some specific examples of the peptide of formula (v) are listed below in (16) to (34), but needless to say, the peptide is not limited to these: (16) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 73) (17) RGNCAWH(Xaa1)GQLVWCTYH (SEQ ID NO: 74) (18) RGNCAWH(Xaa1)GELVWCTYH (SEQ ID NO: 75) (19) RGNCKWH(Xaa1)GQLVWCTYH (SEQ ID NO: 76) (20) RGNCKYH(Xaa1)GELVWCTYH (SEQ ID NO: 77) (21) RGNCKYH(Xaa1)GQLVWCTYH (SEQ ID NO: 78) (22) DCKWH(Xaa1)GELVWCT (SEQ ID NO: 79) (23) DCKYH(Xaa1)GELVWCT (SEQ ID NO: 80) (24) DCKWH(Xaa1)GELVWCT (SEQ ID NO: 81) (25) DCKWH(Xaa1)GQLVWCT (SEQ ID NO: 82) (26) DCKYH(Xaa1)GELVWCT (SEQ ID NO: 83) (27) DCKYH(Xaa1)GQLVWCT (SEQ ID NO: 84) (28) DCKWH(Xaa1)GQLVWCT (SEQ ID NO: 85) (29) DCKYH(Xaa1)GQLVWCT (SEQ ID NO: 86) (30) RGNCAWH(Xaa1)GQLVWCKYH (SEQ ID NO: 87) (31) RGNCAWH(Xaa1)GELVWCKYH (SEQ ID NO: 88) (32) RGNCAYH(Xaa1)GQLVWCTKH (SEQ ID NO: 89) (33) RGNCAYH(Xaa1)GQLVWCTYK (SEQ ID NO: 90) (34) RGNCAYH(Xaa1)GQLVWCTKH (sequence number 91). [During the ceremony, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue. Preferably, Xaa1 is an arginine residue, a leucine residue, or a lysine residue, more preferably a lysine residue.

[0101] The IgG-binding peptide has a primary structure represented by the following formula (vi) in a broad sense: DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 103) (vi) D is an aspartic acid residue, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa5 is a threonine or lysine residue, Xaa6 is a tyrosine residue, a lysine residue, or none; and Xaa7 is a histidine residue, a lysine residue, or is absent.], and is a peptide characterized by comprising an amino acid sequence consisting of 13 to 15 amino acid residues represented by the following formula (I): and being capable of binding to human IgG and / or rabbit IgG (see, e.g., Examples and WO 2016 / 186206). Preferably, any one of Xaa3, Xaa1, Xaa2, Xaa5, Xaa6, and Xaa7 is a lysine residue. Xaa1 is preferably a lysine residue, an arginine residue, or a leucine residue, and Xaa2 is preferably a lysine residue, a glutamine residue, or a glutamic acid residue.

[0102] In certain embodiments, the IgG-binding peptide has the following formula (vii): DC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWCT (SEQ ID NO: 104) (vii) [During the ceremony, D is an aspartic acid residue, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, and and T is a threonine residue. The peptide is characterized by comprising an amino acid sequence consisting of 13 amino acid residues represented by the following formula (I): and being capable of binding to human IgG and / or rabbit IgG (e.g., International Publication No. 2016 / 186206). Preferably, any one of Xaa3, Xaa1, and Xaa2 is a lysine residue. Xaa1 is preferably a lysine residue, an arginine residue, or a leucine residue, and Xaa2 is preferably a lysine residue, a glutamine residue, or a glutamic acid residue.

[0103] In another particular embodiment, the IgG-binding peptide has the following formula (viii): RGNC-(Xaa3)-(Xaa4)-H-(Xaa1)-G-(Xaa2)-LVWC-(Xaa5)-(Xaa6)-(Xaa7) (SEQ ID NO: 105) (viii) [During the ceremony, R is an arginine residue, G is a glycine residue, N is an asparagine residue, C is a cysteine ​​residue, Xaa3 is an alanine residue or a lysine residue, Xaa4 is a tryptophan residue or a tyrosine residue, H is a histidine residue, Xaa1 is an arginine residue, a leucine residue, a lysine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid residue, or a diaminopropionic acid residue; G is a glycine residue, Xaa2 is a lysine residue, a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue; L is a leucine residue, V is a valine residue, W is a tryptophan residue, Xaa5 is a threonine or lysine residue, Xaa6 is a tyrosine residue, a lysine residue, or none; and Xaa7 is a histidine residue, a lysine residue, or is absent. A peptide (see, for example, Examples) is characterized by comprising an amino acid sequence consisting of 13 to 15 amino acid residues represented by the formula (I) and capable of binding to human IgG and / or rabbit IgG. Compounds having such a specific structure, not disclosed in WO 2016 / 186206, are useful for site-selective modification of Lys248 or Lys246 residues according to the EU numbering system in human IgG Fc, or amino acid residues other than Lys248 or Lys246 (see, for example, Examples). Preferably, any one of Xaa3, Xaa1, Xaa2, Xaa5, Xaa6, and Xaa7 is a lysine residue. Xaa1 may preferably be an arginine residue or a leucine residue. Alternatively, Xaa1 is preferably a lysine residue, an arginine residue, or a leucine residue, and Xaa2 is preferably a lysine residue, a glutamine residue, or a glutamic acid residue.

[0104] The peptide has at least two cysteine ​​(C) residues spaced apart in each amino acid sequence that are disulfide bonded to form a cyclic peptide, and may have one or two amino acid residues other than cysteine ​​at the N-terminal and C-terminal sides of each cysteine ​​residue. When each cysteine ​​residue has one or two amino acid residues at the N-terminal and C-terminal sides, and the total length is 17 amino acid residues, the amino acid residues 1-2 and 16-17 from the N-terminus are those exemplified above. The amino acids constituting the peptide may be in the L- or D-form. Either of these may be used, but the L-form is preferred (in the Examples, all of the amino acid residues constituting the peptide are in the L-form).

[0105] As described above, in the IgG-binding peptide, when the amino acid residue Xaa is an amino acid residue that can be easily modified with a cross-linking agent (a proteinogenic amino acid such as a lysine residue, cysteine ​​residue, aspartic acid residue, or glutamic acid residue, or a non-proteinogenic amino acid such as a diaminopropionic acid residue or 2-aminosuberic acid residue), a lysine residue is preferred among these amino acids. Examples of such crosslinkers include crosslinkers containing preferably two or more succinimidyl groups, such as DSG (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate); crosslinkers containing preferably two or more imidate moieties, such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2HCl), and DMS (dimethyl suberimidate·2HCl); and crosslinkers having an S-S bond, such as DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., WO 2016 / 186206). To enhance site specificity when modifying an IgG-binding peptide with a cross-linking agent, it is preferable that the IgG-binding peptide has no or few (e.g., only one or two) residues identical to Xaa1 in its sequence. For example, if Xaa1 is a lysine residue, it is preferable that the IgG-binding peptide has no or few lysine residues in positions other than Xaa1 in its sequence.

[0106] The IgG-binding peptide binds to the Fc domain of IgG. In the IgG-binding peptide, the amino acid residue of Xaa, such as Xaa1, is adjacent to a specific region of IgG Fc, i.e., Lys248 or Lys246 residue according to Eu numbering in human IgG Fc, preferably Lys248 (see Examples and WO 2016 / 186206). Alternatively, in the IgG-binding peptide, the amino acid residue of Xaa can be adjacent to an amino acid residue other than Lys248 or Lys246 residue according to Eu numbering in human IgG Fc.

[0107] More specifically, the peptides represented by the above formulas (i) to (viii) are as follows: (1')RGNCAYHKGQLVWCTYH (SEQ ID NO: 39) (2') RGNCKYHRGQLVWCTYH (SEQ ID NO: 42) (3') RGNCAWHRGKLVWCTYH (SEQ ID NO: 43) (4') RGNCKWHRGELVWCTYH (SEQ ID NO: 44) (5') RGNCKWHRGQLVWCTYH (SEQ ID NO: 45) (6') RGNCKYHLGELVWCTYH (SEQ ID NO: 46) (7') RGNCKYHLGQLVWCTYH (SEQ ID NO: 47) (8') DCKWHLGELVWCT (SEQ ID NO: 48) (9') DCKYHLGELVWCT (SEQ ID NO: 49) (10') DCKWHRGELVWCT (SEQ ID NO: 50) (11') DCKWHLGQLVWCT (SEQ ID NO: 51) (12') DCKYHRGELVWCT (SEQ ID NO: 52) (13') DCKYHLGQLVWCT (SEQ ID NO: 53) (14') DCKWHRGQLVWCT (SEQ ID NO: 54) (15') DCKYHRGQLVWCT (SEQ ID NO: 55) (16') RGNCAWHLGQLVWCKYH (SEQ ID NO: 56) (17') RGNCAWHLGELVWCKYH (SEQ ID NO: 57) (18')RGNCAYHLGQLVWCTKH (SEQ ID NO: 58) (19')RGNCAYHLGQLVWCTYK (SEQ ID NO: 59) (20') RGNCAYHRGQLVWCTKH (SEQ ID NO: 60)

[0108] In addition, the above-mentioned affinity substance for soluble proteins is (a) any amino acid residue in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 92) is substituted with one amino acid residue (preferably a lysine residue, an aspartic acid residue, or a glutamic acid residue, more preferably a lysine residue) selected from the group consisting of lysine residues, aspartic acid residues, glutamic acid residues, 2-aminosuberic acid residues, and diaminopropionic acid residues (amino acid residues that can be easily modified with a crosslinker); and (b) It may be an affinity peptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:92.

[0109] Preferably, the amino acid sequence having properties (a) and (b) is capable of binding to human IgG as described herein.

[0110] For convenience in peptide reagent synthesis, the peptide consisting of the amino acid sequence of SEQ ID NO: 92 is an affinity peptide known as Z34C in which the two K (lysine) residues at positions 26 and 28 from the N-terminus are replaced with R (arginine), and the N-terminus is further acetylated and the C-terminus is amidated. More specifically, an example of such an affinity peptide is Ac-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH2 (SEQ ID NO: 92). Affinity substances having such a specific structure are useful for site-selective modification of the Lys248 or Lys246 residue, Lys288 or Lys290, or Lys317 residue, or other amino acid residues other than these, according to Eu numbering, in human IgG Fc (Examples). The amino acid sequence of Z34C is FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO: 93) (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)).

[0111] The affinity peptide may have affinity for human IgG (e.g., the above-mentioned human IgG, preferably human IgG1). The affinity peptide may form a cyclic peptide via a disulfide bond between the cysteine ​​residues at positions 5 and 34.

[0112] The position at which an amino acid residue that can be easily modified with a cross-linking agent is introduced can be any position as long as it has affinity for human IgG, such as human IgG1. Such positions can be easily identified by those skilled in the art. Preferably, the position at which an amino acid residue that can be easily modified with a cross-linking agent is introduced is an amino acid residue other than the cysteine ​​residues at positions 5 and 34, which may form a disulfide bond. More preferably, the position at which an amino acid residue that can be easily modified with a cross-linking agent is introduced includes, for example, amino acid residues at positions 1, 3, 6, 7, 13, 20, 24, 31, and 32.

[0113] Preferably, the amino acid sequence having the properties (a) and (b) includes a lysine residue, an amino acid ... It is also preferred that the amino acid sequence has one specific amino acid residue (preferably lysine, aspartic acid, or glutamic acid, more preferably lysine) selected from the group consisting of aspartic acid, glutamic acid, 2-aminosuberic acid, and diaminopropionic acid (amino acid residues that can be easily modified with a crosslinker) at a predetermined position, and has mutations of one of the 20 amino acid residues commonly found in natural proteins (preferably 17 amino acid residues other than lysine, aspartic acid, and glutamic acid, more preferably 19 amino acid residues other than lysine) at a position other than the predetermined position. Such predetermined positions are not particularly limited, and examples include positions 1, 3, 6, 7, 13, 20, 24, 31, and 32. The amino acid sequence having properties (a) and (b) maintains two cysteine ​​residues at positions 5 and 34, and these two cysteine ​​residues may be linked by a disulfide bond. An amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 92 may be one in which one to three (preferably one or two, more preferably one) amino acid residues have been modified by one, two, three, or four types of mutations (preferably substitutions) selected from the group consisting of deletion, substitution, addition, and insertion of amino acid residues. The amino acid residue mutations may be introduced into one region or multiple different regions in the amino acid sequence.

[0114] More preferably, the amino acid sequence having the properties (a) and (b) may be the following (c) or (d): (c) an amino acid sequence selected from the group consisting of the following amino acid sequences (1) to (9): (1) KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 61); (2) FNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 62); (3) FNMQCQRRFYEAKHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 63); (4) FNMQCQRRFYEALHDPNLNEEQRKARIRSIRDDC (SEQ ID NO: 64); (5) FNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 65); (6) FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 68); (7) FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 70); (8) FNMQCKRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 71); and (9) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRKDC (SEQ ID NO: 72); or (d) An amino acid sequence having 90% or more identity to any of the amino acid sequences (1) to (9) above (which may include modifications of the number of amino acid residues described above), which has mutations of 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). The amino acid sequence (d) preferably maintains the two cysteine ​​residues at positions 5 and 34, and these two cysteine ​​residues may be linked by a disulfide bond. The affinity peptide having the amino acid sequence (d) above is preferably capable of binding to human IgG as described herein.

[0115] The affinity peptide may be the amino acid sequence of SEQ ID NO: 92 or any of the above (1) to (9). As long as the amino acid sequence has 90% or more identity, mutations of additional amino acid residues may be introduced in addition to the introduction of one amino acid residue that is easily modified by a cross-linking agent. Positions at which additional amino acid mutations can be introduced can be easily identified by those skilled in the art. For example, positions other than the cysteine ​​residues at positions 5 and 34 can be used. 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 at which an amino acid residue that can be easily modified by a cross-linking agent has already been introduced) can also be used. Amino acids that can be introduced by further amino acid mutation include, for example, alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Preferably, these 19 amino acids other than lysine may be used. Amino acids may be either L- or D-isomers, but L-isomers are preferred (in the examples, all amino acid residues constituting the peptide are L-isomers).

[0116] The percent identity to the amino acid sequence of SEQ ID NO: 92 or the amino acid sequences of (1) to (9) can be determined as described above. The percent identity may be preferably 92% or more, more preferably 94% or more, even more preferably 95% or more, and particularly preferably 97% or more (i.e., a sequence having only one amino acid residue mutation selected from the group consisting of lysine residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid residue, and diaminopropionic acid residue, relative to the amino acid sequence of SEQ ID NO: 92).

[0117] When the affinity substance is a peptide, the amino and carboxyl groups at the peptide termini may be protected. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). The acetyl group is preferred as a protecting group for the N-terminal amino group. Examples of protecting groups for the C-terminal carboxy 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. The protecting group for the C-terminal carboxyl group is preferably an amino group. When the affinity substance is a peptide containing two or more cysteine ​​residues, disulfide bonds may be formed via thiol groups in the side chains of the cysteine ​​residues.

[0118] 1-3. Linker (L) In formula (I), L is a cleavable linker, which is a divalent group that includes a cleavable moiety.

[0119] The cleavable linker represented by L is a divalent group containing a cleavable moiety. The cleavable moiety is a site that can be cleaved by a specific treatment under conditions (mild conditions) that cannot cause denaturation or decomposition of the protein (e.g., cleavage of an amide bond). Therefore, the cleavable moiety can be said to be a site (a bond other than an amide bond) that can be cleaved by a specific cleavage treatment under mild conditions. Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with a physicochemical stimulus selected from the group consisting of light, or (c) a cleavable moiety containing an autolytic cleavable moiety. For example, when using a cleavable linker, the cleavable linker and the cleavage conditions thereof are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry.20,571(2012); Feng P. et al., Journal of American Chemical Society.132,1500(2010); Bessodes M. et al., Journal of Controlled Release,99,423(2004); DeSimone, JM, Journal of American Chemical Society.132,17928(2010); Thompson, DH, Journal of Controlled Release,91,187(2003); Schoenmarks, RG, Journal of Controlled Release,95,291(2004)). Examples of such cleavable moieties include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues (e.g., tert-butyloxycarbamate residues), silane residues, hydrazone-containing residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0120] The aromatic ring group having an electron-withdrawing group is preferably one having an aromatic ring group selected from the group consisting of aryl, aralkyl, aromatic heterocyclic group, and alkyl having an aromatic heterocyclic group, and more preferably aralkyl or alkyl having an aromatic heterocyclic group. The electron-withdrawing group is preferably bonded to the 2-position of the ring. Even more preferably, the aromatic ring-containing residue having an electron-withdrawing group is, for example, an aralkyl (e.g., benzyl) having an electron-withdrawing group at the 2-position. Examples of the electron-withdrawing group include halogen atoms, alkyl substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl group, and keto group (e.g., acyl).

[0121] The definitions, examples, and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, aralkyl, etc. found as prefixes, suffixes, etc. in connection with the names of residues as cleavable moieties are the same as those described below.

[0122] Examples of the ester residue include ordinary ester residues composed of carbon atoms and oxygen atoms [e.g., alkyl esters (e.g., tertiary alkyloxycarbonyl such as tert-butyloxycarbonyl), aryl esters (e.g., phenacyl ester, 2-(diphenylphosphino)benzoate), glycosyl ester residues, orthoester residues, ester residues containing sulfur atoms and oxygen atoms (e.g., thioester residues such as α-thiophenyl ester residues and alkylthioester residues), ester residues containing phosphorus atoms and oxygen atoms (e.g., phosphodiester residues, phosphotriester residues), and activated ester residues (e.g., N-hydroxysuccinimide residues).

[0123] Examples of sulfone-containing residues include sulfone residues and quinolinylbenzenesulfonate residues.

[0124] The silane residue is preferably a silane residue having a group selected from the group consisting of alkyl, aryl, aralkyl, and alkoxy. Examples of such silane residues include dialkyldialkoxysilane residues (e.g., dimethyldialkoxysilane, diethyldialkoxysilane) and diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane, diphenyldialkoxysilane). Coxisilane).

[0125] The alkoxyalkyl (i.e., alkyloxyalkyl) residue is a group combining alkyloxy and alkyl as described below (the definitions, examples, and preferred examples of alkyloxy and alkyl are the same as those described below), and examples include, but are not limited to, methoxymethyl residue, ethoxymethyl residue, methoxyethyl residue, and ethoxyethyl residue.

[0126] The unsaturated bond-containing chain residue is a residue containing an unsaturated bond moiety consisting of only carbon atoms (e.g., vinyl (ethenyl), the smallest unit having a double bond between carbon atoms, or acetylenyl (ethynyl), the smallest unit having a triple bond between carbon atoms), or a residue containing an unsaturated bond moiety consisting of carbon atoms and heteroatoms (e.g., nitrogen atom, sulfur atom, oxygen atom) (e.g., aldehyde, cyano). Examples of the unsaturated bond-containing chain residue include vinyl ether residue, cyanoethyl residue, ethylene residue, and malondialdehyde residue.

[0127] Examples of acidic substances (also referred to as electrophiles) include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acidic substances such as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3-morpholinopropanesulfonic acid, sodium dihydrogenphosphate, citric acid, dodecylsulfuric acid, N-dodecanoylsarcosinic acid, and trifluoroacetic acid. Examples of sites that can be cleaved by acidic substances include alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, β-thiopropionate residues, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, and 2-(diphenylphosphino)benzoate residues.

[0128] Examples of basic substances (also called nucleophiles) include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, ammonium acetate, etc., and organic basic substances such as triethylamine, N,N'-diisopropylamine, etc. Examples of sites that can be cleaved by basic substances include silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyl disuccinate residues, α-thiophenyl ester residues, unsaturated vinyl sulfide residues, malondialdehyde residues, acylhydrazone residues, and alkylthioester residues.

[0129] Examples of reducing agents include cysteine, dithiothreitol, reduced glutathione, hydroxylamine, and β-mercaptoethanol. Examples of sites that can be cleaved by reducing agents include disulfide residues, alkoxyalkyl residues, and azo residues.

[0130] Examples of oxidizing agents include sodium periodate and oxidized glutathione. Examples of sites that can be cleaved by an oxidizing agent include vicinal diol residues and selenium residues.

[0131] Examples of enzymes include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloprotease, lipase, endoglycosidase, and PN gauze F. Examples of sites that can be cleaved by enzymes include ester residues, phosphodiester residues, and glycosyl residues.

[0132] Examples of the photocleavable moiety include a 2-nitrobenzyl residue, a phenacyl ester residue, an 8-quinolinebenzenesulfonate residue, a coumarin residue, and a phosphotriester. residues, bisarylhydrazone residues, and bimandithiopropionic acid residues.

[0133] Autolytic cleavable moieties include, for example, activated ester residues (eg, N-hydroxysuccinimide residues).

[0134] More specifically, the cleavable moiety may correspond to any one chemical structure selected from the group consisting of: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Multiple R 2a , multiple R 2b , and multiple R 2c are the same or different and are selected from the group consisting of a hydrogen atom or a substituent described below, J is -CH2-, -O-, or -S-; r is any integer from 1 to 4, A white circle (◯) indicates a bond to A (or La, which will be described later), and a black circle (●) indicates a bond to B (or Lb, which will be described later). When the chemical structure is asymmetric around the cleavage site, ● may represent a bond to A (or La, which will be described later), and ○ may represent a bond to B (or Lb, which will be described later).

[0135] J is -CH2-, -O-, or -S-. j is preferably -CH2- or -O-, more preferably -CH2-.

[0136] r is any integer from 1 to 4, preferably any integer from 1 to 3, and more preferably 1 or 2.

[0137] In one embodiment, the cleavable linker can be (i) a divalent group containing a cleavable moiety capable of cleaving to generate a bioorthogonal functional group on the reactive group side, or (ii) a divalent group containing a cleavable moiety that does not have the ability to cleave to generate a bioorthogonal functional group on the reactive group side.

[0138] Examples of the cleavable moiety (i) include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

[0139] More specifically, the cleavable moiety in (i) can be, for example, [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Multiple R 2a are the same or different and are selected from the group consisting of a hydrogen atom or a substituent described below, A white circle (◯) indicates a bond to A (or La, which will be described later), and a black circle (●) indicates a bond to B (or Lb, which will be described later). When the chemical structure is asymmetric around the cleavage site, the ● may represent a bond to A (or La described below), and the ○ may represent a bond to B (or Lb described below).

[0140] Examples of the cleavable moiety (ii) include ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues, silane residues, hydrazone-containing residues, phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0141] More specifically, the cleavable moiety in (ii) can be, for example, [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Multiple R 2b , multiple R 2c , J and r are selected from the group consisting of a hydrogen atom or a substituent described below; A white circle (◯) indicates a bond to A (or La, which will be described later), and a black circle (●) indicates a bond to B (or Lb, which will be described later). When the chemical structure is asymmetric around the cleavage site, the ● may represent a bond to A (or La described below), and the ○ may represent a bond to B (or Lb described below).

[0142] In certain embodiments, the cleavable linker (L) may be represented by any one of the following formulas (L1) to (L3): La-C-Lb (L1) La-C (L2) C-Lb (L3) [During the ceremony, La and Lb are each a divalent group; C is a cleavable moiety.

[0143] Examples of the divalent group include a divalent hydrocarbon group which may have a substituent, a divalent heterocyclic group which may have a substituent, -C(=O)-, and -NR a -(Ra represents a hydrogen atom or a substituent), -O-, -S-, -C(=S)-, and groups formed by combining two or more of these (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4).

[0144] The divalent hydrocarbon group is a straight-chain, branched-chain, or cyclic divalent hydrocarbon group, and is preferably Preferably, it is a straight-chain or branched-chain divalent hydrocarbon group. Examples of the divalent hydrocarbon group include alkylene, alkenylene, alkynylene, and arylene.

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

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

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

[0148] The arylene is preferably an arylene having 6 to 24 carbon atoms, more preferably an arylene having 6 to 18 carbon atoms, still more preferably an arylene having 6 to 14 carbon atoms, and even more preferably an arylene having 6 to 10 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of arylene include phenylene, naphthylene, and anthracenylene.

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

[0150] The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, even more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and even more preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of the substituents. More specific examples of the divalent aromatic heterocyclic group include pyrenediyl, pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrrolinediyl, piperidinediyl, triazolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, and isoquinolinediyl.

[0151] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, even more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 3 to 6 carbon atoms. The number of carbon atoms mentioned above does not include the number of carbon atoms of substituents. More specifically, examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0152] The divalent groups represented by La and Lb may have, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2 substituents. Such substituents are the same as those in the above R a and R b Examples of such substituents include the following: (i) halogen atoms; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v)R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or a monovalent hydrocarbon group; or (vi)NR d R e -, NR d Re -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; (vii) Nitro, sulfate, sulfonate, cyano, and carboxyl groups.

[0153] Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms.

[0154] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0155] A monovalent chain hydrocarbon group refers to a hydrocarbon group that is composed only of a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0156] The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0157] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0158] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. The number of carbon atoms in the substituent is not included in the number of carbon atoms. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0159] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0160] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not necessarily have to be composed only of alicyclic hydrocarbons, and may also contain a chain structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0161] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 or 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0162] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 or 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0163] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 or 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0164] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0165] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed of only aromatic rings, and it may contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, an aryl group having 6 to 12 carbon atoms is preferred, an aryl group having 6 to 10 carbon atoms is more preferred, and an aryl group having 6 carbon atoms is even more preferred. The number of carbon atoms mentioned above does not include the number of carbon atoms of the substituent. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0166] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0167] Among these, alkyl, cycloalkyl and aryl are preferred as the monovalent hydrocarbon group, and alkyl is more preferred.

[0168] Aralkyl refers to arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0169] A monovalent heterocyclic group refers to a group in which one hydrogen atom has been removed from the heterocyclic ring of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic group preferably contains one or more heteroatoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0170] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 3 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 3 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of monovalent aromatic heterocyclic groups include pyrenyl, pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolinyl, piperidinyl, triazonyl, purinyl, carbazonyl, fluorenyl, quinolinyl, and isoquinolinyl.

[0171] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 3 to 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0172] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0173] Preferably, the substituents may be: (i') a halogen atom; (ii') alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii') aralkyl having 3 to 15 carbon atoms; (iv') a 5- or 6-membered heterocycle; (v')R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; or (vi')NR d R e -, NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R eare the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; (vii') The same groups as those listed in (vii) above.

[0174] More preferably, the substituents may be: (i'') halogen atoms; (ii'') alkyl having 1 to 12 carbon atoms; (iii'')R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; or (iv'')NR d R e -, NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R e are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; (v'') The same groups as those listed in (vii) above.

[0175] Even more preferably, the substituents may be: (i''') halogen atoms; (ii''') alkyl having 1 to 6 carbon atoms; (iii''')R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; or (iv''')NR d R e -, NR d Re -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R e are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms; (v''') The same groups as those listed in (vii) above.

[0176] Particularly preferably, the substituents may be: (i'''') halogen atoms; (ii'''') alkyl having 1 to 4 carbon atoms; (iii'''')R c -O-, R c -C(=O)-, R c -OC(=O)- or R c -C(=O)-O-(R c represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; or (iv'''')NR d R e -, NR d R e -C(=O)-, NR d R e -C(=O)-O- or R d -C(=O)-NR e -(R d and R e are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms; (v'''') The same groups as those listed in (vii) above.

[0177] In certain embodiments, La and Lb are (La′) and (Lb′) below, respectively: [ka] [During the ceremony, p and p' are the same or different and are any integers from 0 to 10, q and q' are the same or different and each represents an integer of 0 to 10; X and X' may be the same or different and represent a carbon atom, a nitrogen atom, or a single bond (wherein when X is a nitrogen atom, R 1b is absent, and X' is a nitrogen atom, R 1b’ does not exist. If X is a single bond, R 1a and R 1b is absent and X' is a single bond, R 1a’ and R 1b’ does not exist), R 1a , R 1b , R 1a’ and R 1b’ are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents.

[0178] p and p' are the same or different and are any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, even more preferably an integer from 0 to 4, and particularly preferably 0, 1, or 2. Preferably, p and p' are the same.

[0179] q and q' may be the same or different and are any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, even more preferably an integer from 0 to 4, and particularly preferably 0, 1, or 2. Preferably, q and q' are the same.

[0180] X and X' are the same or different and each represent a carbon atom, a nitrogen atom, or a single bond, and preferably Preferably, it is a carbon atom or a single bond. Preferably, X and X' are the same.

[0181] R 1a , R 1b , R 1a’ and R 1b’are the same or different and are selected from the group consisting of a hydrogen atom or the substituents described below. The definitions, examples, and preferred examples of the substituents are as described above. Preferably, R 1a , R 1b , R 1a’ and R 1b’ is a hydrogen atom.

[0182] 1-4. (a) A divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group (B) In formula (I), B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group.

[0183] Bioorthogonal functional groups refer to groups that do not react with biological components (e.g., amino acids, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components.Bioorthogonal functional groups are well known in the art (e.g., see Sharpless KB et al., Angew.Chem.Int.Ed.40,2004(2015); Bertozzi CR et al., Science 291,2357(2001); Bertozzi CR et al., Nature Chemical Biology 1,13(2005)).

[0184] When the target of the affinity substance is a soluble protein, the bioorthogonal functional group is a protein-specific bioorthogonal functional group. A protein-specific bioorthogonal functional group is a group that reacts with a specific functional group without reacting with the side chains of the 20 naturally occurring amino acid residues that make up proteins. The 20 naturally occurring 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). Among these 20 naturally occurring amino acids, glycine, which has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, which have hydrocarbon side chains (i.e., do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inert to conventional reactions. Therefore, bioorthogonal functional groups for proteins are functional groups that cannot react with the side chains of these amino acids, which have side chains that are inert to conventional reactions, as well as with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0185] Examples of such 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, which is the smallest unit having a double bond between carbon atoms; the same applies below), alkyne residues (in other words, any residue that has an ethynylene moiety, which is the smallest unit having a triple bond between carbon atoms; the same applies below), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester groups, α-halocarbonyl residues (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position; the same applies below), isonitrile residues, sydnone residues, and selenium residues. Proteins include proteins that may contain free thiols (cysteine) (e.g., proteins other than antibodies) and proteins that may not contain free thiols (e.g., antibodies). In proteins that may not contain free thiols, the thiols function as bioorthogonal functional groups. Therefore, when the soluble protein targeted by the affinity substance is a protein that may not contain free thiols (e.g., an antibody), the bioorthogonal functional group preferably contains a thiol. Furthermore, when the soluble protein is a protein that may contain free thiols (e.g., a protein other than an antibody), the bioorthogonal functional group preferably does not contain a thiol. One or more (e.g., two, three, or four) types of bioorthogonal functional groups may be contained in the divalent group, but preferably, one type of bioorthogonal functional group may be contained in the divalent group.

[0186] In one embodiment, the divalent group containing a bioorthogonal functional group may be a divalent group containing a bioorthogonal functional group in the main chain selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene 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 groups, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[0187] In another embodiment, the divalent group containing a bioorthogonal functional group may be a divalent group containing a bioorthogonal functional group in a side chain 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, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[0188] More specifically, the bioorthogonal functional group may correspond to any one chemical structure selected from the group consisting of: [ka] [During the ceremony, R 1f , single or multiple R 1g and one or more R 1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups, · is a bond.)

[0189] Examples of the electron-withdrawing group include those mentioned above, with halogen atoms, boronic acid residues, mesyl, tosyl, and triflate being preferred.

[0190] In one embodiment, B may be (a) a divalent group containing a bioorthogonal functional group. The number of bioorthogonal functional groups contained in the divalent group may be single or multiple, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. When the divalent group contains multiple bioorthogonal functional groups, the multiple bioorthogonal functional groups may be the same or different, but from the perspective of adopting a simple structure, it is preferable that they are the same.

[0191] In certain embodiments, B may be (a1) a divalent group containing a bioorthogonal functional group in its main chain. The divalent group containing a bioorthogonal functional group in its main chain is selected from the group consisting of azide residues, aldehyde residues, thiol residues, alkyne residues, alkene 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 groups, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues, or a group in which the above-mentioned divalent group is linked to either one or both ends of such a divalent bioorthogonal functional group. The definition, examples, and preferred examples of the divalent group to be linked are the same as those of the divalent group described above.

[0192] In another specific embodiment, B may be (a2) a divalent group containing a bioorthogonal functional group in the side chain. The divalent group containing a bioorthogonal functional group in the side chain is a divalent group substituted with a bioorthogonal functional group or a group containing the same 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, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues. The definition, examples, and preferred examples of the divalent group to be substituted are the same as those of the divalent group described above.

[0193] In another embodiment, B may be (b) a divalent group that does not contain a bioorthogonal functional group. Such a divalent group may be an optionally substituted alkylene, an optionally substituted cycloalkylene, an optionally substituted aryl, an optionally substituted divalent heterocyclic group, or —NR a -(R a represents a hydrogen atom or a substituent), -O-, and a group consisting of a combination of two or more of these (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4). The substituents, which may be substituted, and R a The substituents are substituents other than bioorthogonal functional groups. Examples of such substituents include alkyl, cycloalkyl, aralkyl, monovalent heterocyclic groups, hydroxyl, amino, alkyloxy (alkoxy), cycloalkyloxy, and aralkyloxy. The number of such substituents is, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.

[0194] With respect to the substituents other than the bioorthogonal functional group, the definitions, examples and preferred examples of alkyl, cycloalkyl, aralkyl and monovalent heterocyclic group are as described above.

[0195] Regarding substituents other than the bioorthogonal functional group, alkyl in alkyloxy (alkoxy), cycloalkyl in cycloalkyloxy, and aralkyloxy The definition, examples, and preferred examples of aralkyl are as described above. More specifically, examples of alkyloxy include methyloxy, ethyloxy, propyloxy (e.g., n-propyloxy, iso-propyloxy), butyloxy (e.g., n-butyloxy, iso-butyloxy, sec-butyloxy, tert-butyloxy), pentyloxy (e.g., n-pentyloxy), and hexyloxy (e.g., n-hexyloxy). Examples of cycloalkyloxy include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. Examples of aralkyloxy include benzoyloxy, phenethyloxy, naphthylmethyloxy, and naphthylethyloxy.

[0196] A divalent group that does not contain a bioorthogonal functional group may also be a group that is highly inert to the reaction. Therefore, such a divalent group may be a group composed only of carbon and hydrogen atoms. Such a divalent group is alkylene, cycloalkylene, or aryl, and combinations of two or more of these (e.g., two or three). When a divalent group that does not contain a bioorthogonal functional group is a group that is highly inert to the reaction, such a divalent group may have a substituent selected from the group consisting of alkylene, cycloalkylene, and aryl as a highly inert substituent to the reaction. The number of highly inert substituents to the reaction is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

[0197] In certain embodiments, B is of the following formula (B-1): [ka] [During the ceremony, Y is -NH-, -O-, -CH2-, or the following formula (B-2): [ka] (In the formula, V and V' are the same or different and each represent -NH-, -O-, -CH2-, or a single bond; V1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; s is an integer between 0 and 10, The circle and the circle in formula (B-2) are in the same orientation as the circle and the circle in formula (B-1), respectively. Z is an oxygen atom, a sulfur atom, or a hydrogen atom (when Z is a hydrogen atom, -C(=Z)- represents -CH-), In formula (B-1), ◯ (white circle) indicates a bond to the L-side moiety, and ● (black circle) indicates a bond to the R-side moiety.

[0198] Y is -NH-, -O-, -CH2-, or a group represented by the above formula (B-2). From the viewpoint of simplifying the structure, Y may be -NH-, -O-, or -CH2-. Alternatively, from the viewpoint of designing a structure based on carbon atoms, Y may be -CH2-, or may be a group represented by the above formula (B-2).

[0199] Z is an oxygen atom, a sulfur atom, or a hydrogen atom, preferably an oxygen atom or a sulfur atom.

[0200] V and V' are -NH-, -O-, -CH2- or a single bond, preferably -CH2- or a single bond.

[0201] V1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group. Such divalent groups are the same as those described above.

[0202] Preferably, the divalent group in V1 is an optionally substituted divalent hydrocarbon group or an optionally substituted divalent heterocyclic group. The definition, examples, and preferred examples of the divalent hydrocarbon group are the same as those described above, except that in the case of V1, alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, and arylene are preferred. For example, when not substituted with a moiety containing a bioorthogonal functional group, alkenylene, alkynylene, cycloalkenylene, and cycloalkynylene are preferred. On the other hand, when substituted with a moiety containing a bioorthogonal functional group, alkylene, cycloalkylene, and arylene are preferred. Examples and preferred examples of these groups are as described above. The definition, examples, and preferred examples of the divalent heterocyclic group are the same as those described above, except that in the case of V1, a 5- or 6-membered heterocyclic group is preferred. Examples and preferred examples of the 5- or 6-membered heterocyclic group are the same as those described above. The definition, examples, and preferred examples of the substituent are as described above. V1 may have (a) 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1 bioorthogonal functional group(s). When the divalent group contains multiple bioorthogonal functional groups, the multiple bioorthogonal functional groups may be the same or different. From the viewpoint of adopting a simple structure and improving reactivity, the same type of bioorthogonal functional groups are preferred. From the viewpoint of ensuring differentiated reactions, the different types of bioorthogonal functional groups are preferred. V1 may also have (b) 1 to 5, preferably 1 to 3, more preferably 1 or 2 substituents.

[0203] s is any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, even more preferably an integer from 0 to 4, and particularly preferably 0, 1 or 2.

[0204] In a more specific embodiment, V1 is represented by the following formula (B-3): [ka] [During the ceremony, G and G′ are the same or different and each represent —NH—, —O—, —CH—, a single bond, or a group represented by the following formula (B-4): [ka] (In the formula, W and W' are the same or different and each represent -NH-, -O-, -CH2-, or a single bond; W1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; t is an arbitrary integer from 0 to 10. In formula (B-4), ○ (white circle) indicates a bond in the direction of the bond (·) in formula (B-3), and ● (black circle) indicates a bond in the direction of the b side. H is -CH2-, -C=O-, -C=S-, -NH-, or a single bond; I is a divalent hydrocarbon group, a divalent heterocycle, or a single bond; b is as follows: [ka] (In the formula, R 1f , single or multiple R 1g and one or more R 1h are the same or different and are atoms or groups selected from the group consisting of (i) to (vii), or electron-withdrawing groups, is a bond. ) is any one of the groups represented by the formula: ). It may also be a divalent group having a group represented by the formula: as a side chain. Such a divalent group is a divalent hydrocarbon group or a divalent heterocyclic group, preferably an optionally substituted divalent hydrocarbon group, more preferably alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, or arylene, even more preferably alkylene, cycloalkylene, or arylene, and particularly preferably alkylene. Examples and preferred examples of these groups are as described above. These groups may be substituted with a substituent other than the above-mentioned side chain. The number of such substituents is 1 to 5, preferably 1 to 3, and more preferably 1 or 2. Examples and preferred examples of the substituents are as described above.

[0205] G and G' are the same or different and each represent -NH-, -O-, -CH2-, a single bond, or a group represented by the above formula (B-4). From the viewpoint of simplifying the structure, G and G' may also represent -NH-, -O-, -CH2-, or a single bond. Alternatively, a carbon atom may be bonded to a group From the viewpoint of designing a balanced structure, G and G' may be -CH2-, a single bond, or a group represented by the above formula (B-4).

[0206] H is -CH2-, -C=O-, -C=S-, -NH-, or a single bond. Preferably, H is -CH2- or a single bond.

[0207] I is a divalent hydrocarbon group, a divalent heterocyclic ring, or a single bond. The divalent hydrocarbon group and the divalent heterocyclic ring may be substituted or unsubstituted. The definitions, examples, and preferred examples of the divalent hydrocarbon group, the divalent heterocyclic ring, and the substituent are the same as those described above for V1.

[0208] W and W' are the same or different and each represent -NH-, -O-, -CH2-, or a single bond, preferably -CH2- or a single bond.

[0209] W1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group. Such divalent groups are the same as those described above.

[0210] t is any integer from 0 to 10, preferably an integer from 0 to 8, more preferably an integer from 0 to 6, even more preferably an integer from 0 to 4, and particularly preferably 0, 1 or 2.

[0211] 1-5.Reactive group (R) In formula (I), R is a reactive group to soluble proteins. Such reactive groups are common knowledge in the art.

[0212] The reactive group may be the same or different from the bioorthogonal functional group.

[0213] For example, when B is (a) a divalent group containing a bioorthogonal functional group, the reactive group may be a group different from the bioorthogonal functional group. If the reactive group is the same type of group as the bioorthogonal functional group, the reaction specificity of the reactive group for soluble proteins will not be ensured. Furthermore, the bioorthogonal functional group is a group that cannot react with the side chains of the 20 naturally occurring amino acid residues that make up soluble proteins.

[0214] More specifically, of the 20 naturally occurring amino acids that constitute proteins, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, which have hydrocarbon side chains, are inactive in normal reactions. Therefore, the protein-reactive group is a group that can react with the side chains of one or more (e.g., two, three, or four) of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Depending on conditions such as the amino acid composition of the protein, one or more (e.g., two, three, or four) reactive groups may be contained in the compound represented by formula (I); however, preferably, one reactive group may be contained in the compound represented by formula (I).

[0215] Preferably, the reactive group is a group capable of reacting with the side chain of any one of the above-mentioned 14 amino acids that constitute proteins.

[0216] More preferably, the reactive group may be a reactive group specific for the side chain of any one of the amino acids lysine, tyrosine, tryptophan, or cysteine.

[0217] Even more preferably, the reactive group may be a reactive group specific for the side chain of any one of the amino acids lysine, tyrosine, or tryptophan.

[0218] Furthermore, when the protein is human IgG, such as human IgG1, the reactive group is preferably a reactive group specific for the side chain of lysine or tyrosine.

[0219] The reactive group specific to the side chain of a lysine residue is a group that can specifically react with the amino group (NH) present in the side chain of a lysine residue, and examples thereof include an activated ester residue (e.g., an N-hydroxysuccinimide residue), a vinyl sulfone residue, a sulfonyl chloride residue, an isocyanate residue, an isothiocyanate residue, an aldehyde residue, a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue, a 2-imino-2-methoxyethyl residue, and a diazonium terephthalate residue.

[0220] Examples of linking moieties formed by the reaction between the above-mentioned reactive group specific to the side chain of a lysine residue and the amino group (NH) present in the side chain of a lysine residue include amide residues, urea residues, pyridine residues, carbamate residues, and sulfonamide residues.

[0221] More specifically, the reactive group specific for the side chain of a lysine residue may correspond to any one chemical structure selected from the group consisting of: [ka] [Here, R 5a and R 5c is a hydrogen atom or a substituent as defined above, R 5b is an electron-withdrawing group, j is an integer from 1 to 5, and k is an integer of 1 to 4.

[0222] R 5a and R 5c is a hydrogen atom or a substituent as defined above. And preferred examples are the same as those mentioned above.

[0223] R 5b is an electron-withdrawing group. Examples of such an electron-withdrawing group include those mentioned above, with halogen atoms, boronic acid residues, mesyl, tosyl, and triflate being preferred.

[0224] j is an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably an integer of 1 Or 2.

[0225] k is any integer from 1 to 4, preferably an integer from 1 to 3, and more preferably 1 or 2.

[0226] The linking moiety formed by the reaction between the above chemical structure, which is a reactive group specific to the side chain of a lysine residue, and the amino group (NH) present in the side chain of a lysine residue may correspond to any one chemical structure selected from the group consisting of: [ka] (Here, ● (black circle) indicates a bond to the T side portion, and ○ (white circle) indicates a bond to the B side portion. The straight line perpendicular to the bond indicates the bond formed by the reaction.)

[0227] The reactive group specific to the side chain of a tyrosine residue is a group that can specifically react with the atom at the ortho position of the phenolic hydroxyl group (OH) present in the side chain of a tyrosine residue, and examples thereof include a diazonium residue, a diazodicarboxyl residue, and a 2,3-dihydro-1H-pyrazin-6-one residue.

[0228] More specifically, the reactive group specific for the side chain of a tyrosine residue may correspond to any one chemical structure selected from the group consisting of: [ka] [where R 4a represents a hydrogen atom or a substituent as described above, and ○ represents a bond to B.

[0229] R 4ais a hydrogen atom or the above-mentioned substituent. The definition, examples, and preferred examples of the substituent are the same as those described above.

[0230] The linking moiety formed by the reaction between the above chemical structure, which is a reactive group specific for the side chain of a tyrosine residue, and the atom ortho to the phenolic hydroxyl group (OH) present in the side chain of a tyrosine residue may correspond to any one chemical structure selected from the group consisting of: [ka] [where R 4a represents a hydrogen atom or a substituent as described above, ● represents a bond to T, and ○ represents a bond to B.

[0231] R 4a is a hydrogen atom or the above-mentioned substituent. The definition, examples, and preferred examples of the substituent are the same as those described above.

[0232] The reactive group specific to the side chain of a tryptophan residue is a group that can specifically react with the 3-ring atom of the indole group present in the side chain of a tryptophan residue, and an example of this is a 9-azabicyclo[3.3.1]nonan-3-one-N-oxyl residue.

[0233] More specifically, the reactive group specific for the side chain of a tryptophan residue may correspond to any one chemical structure selected from the group consisting of: [ka] (where ○ indicates a bond to B.)

[0234] The linking moiety formed by the reaction between the above chemical structure, which is a group specifically reactive with the side chain of a tryptophan residue, and the ring atom at position 3 of the indole group present in the side chain of a tryptophan residue, may correspond to any one chemical structure selected from the group consisting of: [ka] (wherein ● indicates a bond to T and ○ indicates a bond to B.)

[0235] Particularly preferably, the reactive group may be a reactive group specific to the side chain of lysine.

[0236] 1-6. Partial structure “LB” 1-6-1. Length of the main chain in the partial structure "LB" connecting A and R In formula (I), the length of the main chain (the linear portion in LB) connecting A (affinity substance) and R (reactive group) can be appropriately designed depending on various factors, such as the type of soluble protein and affinity substance, and the relationship between the target site of the affinity substance in the soluble protein and the position and number of specific amino acid residues in the target region (e.g., specific position) to which R should react and bind, as described above. The compound represented by formula (I) can covalently bind to a soluble protein by associating the affinity substance with the soluble protein, and then reacting the reactive group covalently bound to the affinity substance via LB with a group in the side chain of a specific amino acid residue (e.g., the amino group in the side chain of a lysine residue) present near the target site. In this case, if no other specific amino acid residues are present in the region near the specific amino acid residue to which R should react and bind, the region near the target site, or the region between the specific amino acid residue and the target site, the reactive group can bind regioselectively to the specific amino acid residue without strictly controlling the length of the main chain. Of course, even if the specific amino acid residue is present elsewhere in such a region, the reactive group can be site-selectively bound to the specific amino acid residue by controlling the length of the main chain.

[0237] The length of the main chain linking A and R can vary depending on factors such as the type of soluble protein and the affinity substance for it, as well as the position and number of specific amino acid residues in the target site in the soluble protein, but may be about 5 Å or more, preferably about 7.5 Å or more, and more preferably about 10.5 Å or more. The length of such a main chain may also be, for example, about 30 Å or less, preferably about 23 Å or less, and more preferably about 16.5 Å or less. More specifically, the length of such a main chain may be, for example, about 5.0 to 30 Å, preferably about 7.5 to 23 Å, and more preferably about 10.5 to 16.5 Å.

[0238] Incidentally, it is common general knowledge in the technical field that the relationship between the lengths (distances) of atoms is as shown in the table below. Therefore, a person skilled in the art can appropriately design a main chain having the number of atoms corresponding to the above-mentioned main chain length (Å) by referring to the interatomic lengths in the table below.

[0239] [Table 1]

[0240] More specifically, the length of the main chain connecting A and R can also be defined as the number of atoms constituting the main chain (excluding hydrogen atoms and substituents). The number of atoms constituting the main chain can be, for example, The number of atoms in the main chain may be, for example, 20 or less (about 30 Å), preferably 16 or less (about 23 Å), and more preferably 12 or less (about 16.5 Å). More specifically, the number of atoms in the main chain may be, for example, 4 to 20, preferably 6 to 16, and more preferably 8 to 12.

[0241] When the main chain does not contain a ring structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure.

[0242] On the other hand, when the main chain has a structure including a ring structure, the number of atoms in the main chain does not necessarily correspond to the above-mentioned length, and the length that can be determined by the number of atoms in the main chain tends to be shorter than the above-mentioned length. Even in such a case, the number of atoms in the main chain can be counted for convenience in terms of determining the length of the main chain. Specifically, the number of atoms in the main chain in such a case can be determined by counting the number of atoms in the chain structure that does not include a divalent ring structure in the main chain, as well as 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 bond. In the case of (a), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 2. In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 3. In the case of (c), both paths are the shortest paths (equidistant), so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4. In the case of (d), the path to the condensation site is the shortest path, so the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.

[0243] Preferably, the linking portion between A and R represented by LB (excluding the side chain) may be a chain structure that does not contain a divalent ring structure. In this case, L and B can be appropriately designed so that the linking chain portion between A and R represented by LB does not contain a divalent ring group.

[0244] 1-6-2. Specific structure of the partial structure "LB" In the above formula (I), L and B are structures that can be linked to each other. Therefore, in the above formula (I), L and B can be defined as a partial structure represented by "LB."

[0245] In one embodiment, the cleavable linker can be (i) a divalent group containing a cleavable moiety capable of cleaving to generate a bioorthogonal functional group on the reactive group side, or (ii) a divalent group containing a cleavable moiety that does not have the ability to cleave to generate a bioorthogonal functional group on the reactive group side.

[0246] In certain embodiments, when L is a cleavable linker as defined in (i) above, B is (a) a divalent group that includes a bioorthogonal functional group, or (b) a divalent group that does not include a bioorthogonal functional group.

[0247] Preferably, when L is the cleavable linker (i) above, B is (a) a divalent group containing a bioorthogonal functional group. In this case, the bioorthogonal functional group generated in (i) may be the same or different from the bioorthogonal functional group in (a). From the viewpoint of adopting a simpler structure and / or improving reactivity to a single functional substance, it is preferable to use (i). The bioorthogonal functional group generated in (i) may be the same as the bioorthogonal functional group in (a). On the other hand, in order to ensure differentiated reactivity with two or more functional substances and to avoid the use of some bioorthogonal functional groups in the reaction, the bioorthogonal functional group generated in (i) may be different from the bioorthogonal functional group in (a).

[0248] Alternatively, when L is the cleavable linker (i) above, B may be (b) a divalent group that does not contain a bioorthogonal functional group. In this case, the compound represented by formula (I) or a salt thereof has a simpler structure and is therefore easier to synthesize.

[0249] In another particular embodiment, when L is a cleavable linker as defined above in (ii), B is (a) a divalent group comprising a bioorthogonal functional group.

[0250] In a specific embodiment, the partial structure represented by LB preferably does not contain a peptide moiety. In this case, there is an advantage that a soluble protein (e.g., an antibody-drug conjugate) having the functional substance of the present invention obtained using the compound of the present invention does not contain a peptide moiety as a linker, which may have immunogenicity.

[0251] In certain embodiments, the partial structure represented by "LB" may have a symmetric structure (e.g., cis (i.e., Z) or trans (i.e., E)) based on the atom located at the center of the main chain (the linear portion of LB) connecting A and R (e.g., when the number of atoms constituting the main chain is odd) or the bonding site located at the center of the main chain (e.g., when the number of atoms constituting the main chain is even). For example, the bonding site located at the center can also be designed as a cleavable moiety in the cleavable linker as described above. When the partial structure represented by "LB" has a symmetric structure, the partial structure represented by "LB" can be easily synthesized. For example, a symmetric structure containing a cleavable moiety at the center of the main chain (a chain-like divalent group -SS-a chain-like divalent group) can be realized by reacting the same divalent groups having a functional group at one end that can react to form a cleavable moiety with each other (e.g., a chain-like divalent group having an SH group at one end).

[0252] Therefore, the partial structure represented by "LB" may be a partial structure represented by "B2-L'-B1" (i.e., L is a divalent group represented by B2-L', and B is B1). In this case, the compound represented by the above formula (I) can be defined as a compound represented by the following formula (I'): A-B2-L'-B1-R (I') [During the ceremony, A and R are the same as those in formula (I). L' is a cleavable linker, which is a divalent group that includes a cleavable moiety; B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; B1 and B2 may have a symmetrical structure with L' at the center.

[0253] B1 and B2 may be the same or different and have the same definition, examples and preferred examples as B. .

[0254] In certain embodiments, L' may be represented by any one of the following formulas (L1') to (L3'): La'-C'-Lb' (L1') La'-C' (L2') C'-Lb' (L3') [During the ceremony, La′ and Lb′ are each a divalent group, C' is a cleavable moiety.

[0255] The divalent groups represented by La' and Lb' have the same definitions, examples and preferred examples as those of the divalent groups represented by La' and Lb', respectively.

[0256] The cleavable moiety represented by C′ has the same definition, examples and preferred examples as the cleavable moiety represented by C.

[0257] In another particular embodiment, the structural unit represented by LB may be represented by the following formula (LB'): [ka] [During the ceremony, C, p, p', q, q', X, X', R 1a , R 1a’ , R 1b , R 1b’ The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above; ○ (open circle) indicates a bond to A, and ● (filled circle) indicates a bond to R.

[0258] Therefore, the above formula (I) can be defined as the following formula (I″). [ka] [During the ceremony, A, R, C, p, p', q, q', X, X', R 1a , R 1b , R 1a’ , R 1b’ The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above.

[0259] In the above formulas (LB') and (I''), the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' is the same as the length of the main chain connecting A and R. In these formulas, the length from C in C=Z to C in C=Z' can also be defined as the number of atoms constituting the connecting chain (excluding hydrogen atoms and substituents) of the partial structure connecting C in C=Z to C in C=Z'. Such number of atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain (excluding hydrogen atoms and substituents) of the partial structure connecting C in C=Z to C in C=Z' does not include a ring structure. The linking chain (excluding hydrogen atoms and substituents) may or may not contain a peptide moiety, but preferably does not contain a ring structure.

[0260] 1-8. Manufacturing method The compound or its salt containing an affinity substance for a soluble protein, a cleavable moiety, and a reactive group can be prepared appropriately. The compound containing an affinity substance for a soluble protein, a cleavable moiety, and a reactive group is represented by formula (I), preferably formula (I'), and more preferably formula (I'').

[0261] The affinity substance can be appropriately selected from those having any functional group. Therefore, by utilizing a reactive group capable of reacting with the functional group, the affinity substance can be reacted with a structural unit represented by LBR or a structural unit represented by RLBR (the two reactive groups may be the same or different) to prepare a structural unit represented by ALBR. For example, such a reaction can be carried out in an appropriate reaction system, such as an organic solvent system or an aqueous solution system, at an appropriate temperature (e.g., about 15 to 200°C). The reaction system may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0262] In the reaction system, the molar ratio (Y / X) of the structural unit represented by LBR or the structural unit represented by RLBR (Y) to the affinity substance (X) is not particularly limited, as it varies depending on the type of the 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.

[0263] The production of the affinity substance for the soluble protein and the soluble protein or its salt containing a cleavable moiety can be confirmed, depending on the specific raw material and the molecular weight of the product, by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry. The affinity substance for the soluble protein and the soluble protein or its salt containing a cleavable moiety can be appropriately purified by any method, such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0264] 1-9.Other In the inventions described below (e.g., inventions represented by formulas (II) to (v) and their subordinate formulas, and partial structural formulas (e.g., (L1) to (L3), (La'), (Lb'), (B-1) to (B-4))), any symbols (e.g., A, L, B, R), terms represented by such symbols, and details thereof (e.g., definitions, examples, and preferred examples) are common to the invention of the compound represented by formula (I) or a salt thereof. In addition, specific moieties (e.g., cleavable moieties, moieties capable of generating a bioorthogonal functional group on the reactive group side upon cleavage), specific groups (e.g., bioorthogonal functional groups, divalent groups, alkyl groups, substituents, electron-withdrawing groups), and specific numerical values, as well as optional technical elements such as salts (e.g., definitions, examples, and preferred examples) that can define the inventions described below can also be common to those described above. Therefore, these matters can be appropriately incorporated into the inventions described below without further reference. Similarly, technical elements of specific inventions described in the inventions described below can be appropriately incorporated as technical elements of the present invention and other inventions.

[0265] 2. Affinity substances for soluble proteins and soluble proteins or proteins containing cleavable moieties or its salt 2-1. Overview The present invention provides a soluble protein or a salt thereof comprising an affinity substance for a soluble protein and a cleavable moiety, represented by formula (II): ALB-R'-T (II) [During the ceremony, A is an affinity substance for soluble proteins, L is a cleavable linker that is a divalent group that includes a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; R' is a moiety generated by reaction between a soluble protein and a reactive group; T is a soluble protein.

[0266] 2-2. Moiety (R') generated by reaction between soluble protein and reactive group In the moiety generated by the reaction between a soluble protein and a reactive group, the definitions, examples, and preferred examples of the soluble protein and the reactive group are as described above. The moiety generated by the reaction between a soluble protein and a reactive group is common technical knowledge in the art and can be appropriately determined depending on the types of the soluble protein and the reactive group.

[0267] Preferably, the moiety generated by the reaction between a soluble protein and a reactive group is a moiety generated by the reaction between the side chain of any one of the 14 amino acids (asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine) that can be contained in a soluble protein and the reactive group therefor.

[0268] More preferably, the moiety formed by the reaction between the soluble protein and the reactive group may be a moiety formed by the reaction between the side chain of any one of the amino acids lysine, tyrosine, tryptophan, or cysteine ​​and a reactive group specific thereto.

[0269] Even more preferably, the moiety formed by the reaction between a soluble protein and a reactive group may be a moiety formed by the reaction between the side chain of any one of the amino acids lysine, tyrosine, or tryptophan and a reactive group specific thereto. Examples of the moiety formed by the reaction between the side chain of any one of the amino acids lysine, tyrosine, or tryptophan and a reactive group specific thereto include the linking moieties and / or chemical structures described above in "1-5. Reactive Group (R)."

[0270] Even more preferably, the moiety formed by the reaction between the soluble protein and the reactive group may be a moiety formed by the reaction between a lysine or tyrosine side chain and a reactive group specific thereto (particularly when the soluble protein is human IgG, such as human IgG1). Examples of the moiety formed by the reaction between a lysine or tyrosine side chain and a reactive group specific thereto include the linking moieties and / or chemical structures described above in "1-5. Reactive Group (R)."

[0271] Particularly preferably, the moiety formed by reaction between a soluble protein and a reactive group may be a moiety formed by reaction between a lysine side chain and a reactive group specific therefor.

[0272] 2-3. Partial structure “LB” Details of the partial structure "LB" are as described above in "1-6. Partial structure "LB"".

[0273] In a specific embodiment, the partial structure represented by "LB" may be a partial structure represented by "B2-L'-B1" (i.e., L is a divalent group represented by B2-L', and B is B1). In this case, the substance having affinity for soluble proteins represented by the above formula (II), and the soluble protein or a salt thereof comprising a cleavable moiety, can be represented by the following formula (II'): A-B2-L'-B1-R'-T (II') [During the ceremony, A, R' and T are the same as those in formula (II). L' is a cleavable linker, which is a divalent group that includes a cleavable moiety; B1 and B2 are the same or different and are (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; B1 and B2 may have a symmetrical structure with L' at the center.

[0274] In the above formula (II'), L' may be represented by any one of the above formulas (L1') to (L3').

[0275] In another particular embodiment, the structural unit represented by LB may be represented by the following formula (LB'): [ka] [During the ceremony, C, p, p', q, q', X, X', R 1a , R 1a’ , R 1b , R 1b’ The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above; A circle (○) indicates a bond to A, and a black circle (●) indicates a bond to R'.

[0276] Therefore, the above formula (II) can be defined as the following formula (II″). [ka] [During the ceremony, A, R', T, C, p, p', q, q', X, X', R 1a , R 1b , R 1a’ , R 1b’ The definitions, examples and preferred examples of Y, Y', Z and Z' are the same as those described above.

[0277] In the above formulas (LB') and (II''), the length from C (carbon atom) in C=Z to C (carbon atom) in C=Z' is the same as the length of the main chain connecting A and R as described above. In these formulas, the length from C in C=Z to C in C=Z' is also the length of the connecting chain of the partial structure connecting C in C=Z to C in C=Z'. It can also be defined as the number of atoms constituting the main chain (excluding hydrogen atoms and substituents) connecting A and R. This number of atoms is the same as the number of atoms constituting the main chain connecting A and R. The connecting chain (excluding hydrogen atoms and substituents) of the partial structure connecting C in C=Z to C in C=Z' may or may not contain a ring structure, but preferably does not contain a ring structure. The connecting chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide moiety.

[0278] 2-4. Binding sites of soluble proteins for substructures other than soluble proteins (regioselectivity) A partial structure other than the soluble protein (eg, ALB-R') can be site-selectively bound to the target region of the soluble protein (T) as described above.

[0279] As used herein, "regioselective" or "regioselectivity" refers to the fact that a specific structural unit capable of binding to a specific amino acid residue in a soluble protein is concentrated in a specific region of the soluble protein, even though the specific amino acid residue is not concentrated in a specific region of the soluble protein. Therefore, expressions related to regioselectivity, such as "regioselectively possessed," "regioselective binding," and "regioselective binding," mean that the retention or binding rate of a specific structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues homologous to the specific amino acid residues in the target region. Such regioselective binding or retention can be achieved by the present invention, which allows a specific structural unit to preferentially react with specific amino acid residues in a target region of a soluble protein, rather than randomly reacting the specific structural unit with specific amino acid residues in the soluble protein.

[0280] Specifically, when T contains one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues and contains five or more of the specific amino acid residues in a non-target region other than the target region, a partial structure other than a soluble protein can be bound to one or more specific amino acid residues contained in the target region with a regioselectivity of 30% or more. The definitions, examples, and preferred examples of the target region and regioselectivity are as described above.

[0281] 2-5. Number of substructures other than soluble proteins in a soluble protein The number of partial structures other than the soluble protein (e.g., ALB-R') possessed by a soluble protein (T) can vary. For example, when T is a multimeric protein containing multiple monomeric proteins, T can have partial structures other than T in multiple corresponding target regions in the multiple monomeric proteins. As a result, T can have multiple partial structures other than T. Therefore, the structure represented by formula (II), (II'), or (II'') indicates that T may have one or more partial structures other than T. The number of partial structures other than T possessed by T can be adjusted by appropriately setting conditions such as the type of soluble protein and the reaction ratio between the soluble protein and the structural unit to be introduced thereto. Such number varies depending on the type of soluble protein, but may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[0282] In certain embodiments, the number of partial structures other than soluble proteins possessed by a soluble protein may be more than one when the soluble protein is a multimeric protein composed of multiple monomeric proteins. According to the present invention, multiple partial structures other than soluble proteins can be introduced into the same target region of multiple monomeric proteins.

[0283] In a preferred embodiment, the soluble protein may be an antibody comprising multiple heavy chains. The definition, examples, and preferred examples of antibodies are as described above. The number of heavy chains varies depending on the type of antibody. For example, IgG, IgE, and IgD can have two heavy chains. IgA can have two or four heavy chains. IgM can have eight heavy chains. The number of partial structures other than antibodies possessed by an antibody (soluble protein) can be considered synonymous with DAR. In the present invention, the number of partial structures other than antibodies possessed by an antibody may be one or two (preferably two) for IgG, IgE, and IgD, one to four (preferably four) for IgA, and one to eight (preferably eight) for IgM.

[0284] 2-6. Manufacturing method The present invention provides a method for producing a soluble protein or a salt thereof comprising an affinity agent for a soluble protein and a cleavable moiety, the method comprising: (A1) Reacting a compound or its salt containing an affinity substance for a soluble protein, a cleavable moiety, and a reactive group with a soluble protein to produce a soluble protein or its salt containing an affinity substance for the soluble protein and a cleavable moiety.

[0285] The compound comprising an affinity substance for a soluble protein, a cleavable moiety, and a reactive group is represented by formula (I), preferably formula (I'), more preferably formula (I"). The affinity substance for a soluble protein and the soluble protein comprising a cleavable moiety are represented by formula (II), preferably formula (II'), more preferably formula (II").

[0286] The substance having affinity for soluble proteins, the compound containing a cleavable moiety and a reactive group, or its salt, can react with soluble proteins due to the reactive group. Such reactions can be carried out appropriately under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such reactions can be carried out in an appropriate reaction system, such as a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0287] In the reaction system, the molar ratio (Y / X) ...

Claims

1. The following formula (IV): L1-B-R'-T (IV) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; The partial structure represented by L1-B is a group that does not contain a peptide moiety, R' is a linking moiety formed by reaction with an amino group present in the side chain of a lysine residue, and is (1) an amide residue, or (2) The following: 【Chemical 1】 (wherein a black circle (●) represents a bond to the T side moiety, and a white circle (○) represents a bond to the B side moiety. A straight line perpendicular to the bond represents a bond formed by the reaction.) T is an IgG antibody comprising an Fc region of a human IgG antibody having a lysine residue at position 246, 248, 288, 290, or 317 according to EU numbering; The bioorthogonal functional group is selected from the group consisting of: 【Chemistry 2】 [During the ceremony, R 1f , one or more R 1g s and one or more R 1h s are the same or different; (i) a hydrogen atom or a halogen atom; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v) R c —O—, R c —C(═O)—, R c —O—C(═O)—, or R c —C(═O)—O— (R c represents a hydrogen atom or a monovalent hydrocarbon group); (vi) NR d R e —, NR d R e —C(═O)—, NR d R e —C(═O)—O—, or R d —C(═O)—NR e — (R d and R e are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); and (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group is an atom or group or an electron-withdrawing group selected from the group consisting of is a bond.], The number of atoms constituting the linking chain of the partial structure linking the L1 terminal portion and R' is 2 to 10.], and the structural unit represented by L1-B-R' is bonded to one or more lysine residues selected from positions 246, 248, 288, 290, and 317 of the EU numbering of the IgG antibody, or a salt thereof.

2. The following formula (V): F-(L1-B)'-R'-T (V) [During the ceremony, L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group; B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group not containing a bioorthogonal functional group; The structural unit represented by (L1-B)' is a functional substance and (i') and (a) a divalent structural unit that does not contain a peptide moiety and that includes a moiety generated by reaction with either or both of the bioorthogonal functional groups; F is a functional substance; R' is a linking moiety formed by reaction with an amino group present in the side chain of a lysine residue, and is (1) an amide residue, or (2) The following: 【Chemistry 3】 (wherein a black circle (●) represents a bond to the T side moiety, and a white circle (○) represents a bond to the B side moiety. A straight line perpendicular to the bond represents a bond formed by the reaction.) T is an IgG antibody comprising an Fc region of a human IgG antibody having a lysine residue at position 246, 248, 288, 290, or 317 according to EU numbering; The bioorthogonal functional group is selected from the group consisting of: 【Chemistry 4】 [During the ceremony, R 1f , one or more R 1g s and one or more R 1h s are the same or different; (i) a hydrogen atom or a halogen atom; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v) R c —O—, R c —C(═O)—, R c —O—C(═O)—, or R c —C(═O)—O— (R c represents a hydrogen atom or a monovalent hydrocarbon group); (vi) NR d R e —, NR d R e —C(═O)—, NR d R e —C(═O)—O—, or R d —C(═O)—NR e — (R d and R e are the same or different and represent a hydrogen atom). represents a monovalent hydrocarbon group; and (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, or a carboxyl group is an atom or group or an electron-withdrawing group selected from the group consisting of is a bond.], The number of atoms constituting the linking chain of the partial structure linking the L1 terminal portion and R′ is 2 to 10. An IgG antibody or a salt thereof, comprising a structural unit represented by F-(L1-B)'-R', which is bonded regioselectively to one or more lysine residues selected from positions 246, 248, 288, 290, and 317 of the IgG antibody according to EU numbering, and wherein the functional substance is a drug or a labeling substance.

3. The antibody or salt thereof according to any one of claims 1 to 2, wherein the antibody is a monoclonal antibody.

4. The antibody or salt thereof according to any one of claims 1 to 3, wherein the antibody is of human origin.

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