Compound or salt thereof comprising affinity substance to antibody, cleavable portion and reactive group

Abstract

To provide a technique enabling modification of an antibody, particularly regioselective modification of an antibody.SOLUTION: Disclosed is a compound or a salt thereof comprising a substance having affinity to an antibody, a cleavable portion and a reactive group or salt thereof, where the compound is represented by Formula (I): A-L-B-R, in which A is an affinity substance to an antibody, L is a cleavable linker being a divalent group comprising a cleavable group, B is: (a) a divalent group comprising a bioorthogonal functional group; or (b) a divalent group comprising no bioorthogonal functional group, R is a reactive group to the antibody, where the affinity substance to an antibody is a polypeptide having a glutamine residue (Q) at the N terminus thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an affinity substance for an antibody, 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 conducted. 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] Heterogeneity has been a problem for ADCs including T-DM1 since their development. That is, since low molecular weight drugs are randomly reacted with Lys residues, which are about 70 to 80 in an antibody, the drug antibody ratio (DAR) and conjugation position are not constant. Normally, when such random conjugation method is used, the DAR is in the range of 0 to 8, and it is known that multiple drugs with different numbers of drugs bound are generated. In recent years, it has been reported that changing the number and binding position of drugs bound to ADC changes the pharmacokinetics, drug release rate, and effect. For these reasons, it is required to control the number and position of drugs to be conjugated in next-generation ADCs. It is believed that if the number and position are constant, the expected efficacy, variation of conjugated drugs, lot differences, so-called regulation problems will be solved (Non-Patent Document 4).

[0004] Site-selective modification methods of antibodies have been researched around the world, but most of these methods use genetic engineering techniques or enzymes. Although the site selectivity and number selectivity can be controlled with genetic engineering modification methods, problems such as a decrease in the expression efficiency of the antibody itself (a decrease in the total yield when preparing ADC) have been pointed out. Another problem is that it takes a long time to establish an antibody expression system (Non-Patent Documents 5 to 7).

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

[0006] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed. The method of reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to a peptide (i.e., a method of producing an ADC via a linker containing a peptide portion) has succeeded in site-selective modification of an antibody. This method is the first in the world to successfully site-selectively modify an antibody Fc region with a drug by chemical synthesis, and has been confirmed to have good practical results (reaction time 30 minutes, yield 70% (in the case of DAR 1), site selectivity 100%). It has been demonstrated that the DAR can be controlled at 2 by adding about 5 equivalents of a peptide reagent, which is groundbreaking in that the modification site can also be controlled (Patent Document 1). [Prior art documents] [Patent documents]

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

[0008] [Non-Patent Document 1] Reichert JM et al., Nat Biotechnol 2005;23:1073-8 [Non-Patent Document 2] Kubota T et al., Cancer Sci 2009;100:1566-72 [Non-Patent Document 3] Wu AM et al., Nat Biotechnol 2005;23:1137-46 [Non-Patent Document 4] Junutula JR et al., Nat Biotechnol 2008;26:925-32 [Non-Patent Document 5] Shen BQ et al.,Nat Biotechnol 2012;30:184-9 [Non-Patent Document 6] Hofer T et al.,Biochemistry 2009;48:12047-57 [Non-Patent Document 7] Liu W et al., Nat Methods 2007;4:239-44 [Non-Patent Document 8] 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 allows for the modification of antibodies, in particular the site-selective modification of antibodies. [Means for solving the problem]

[0010] As a result of intensive research, the present inventors have found that a compound developed based on a novel and original design concept has structural features in which (1) an affinity substance for an antibody, (2) a reactive group for an amino acid residue constituting the antibody, and (3) a cleavable moiety between the affinity substance and the reactive group, and (4) a structural unit having 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, is useful for site-specific modification of an antibody (e.g., Figure 1-1, Figure 1-2, Figure 1-3, Figure 2).

[0011] The present inventors have also found that, by using such compounds, it is possible to prepare antibodies (e.g., antibody-drug conjugates (ADCs)) that do not contain a peptide moiety as a linker and that have a functional substance (e.g., a drug) in a regioselective manner. Avoiding the use of linkers that contain a peptide moiety, which has potential immunogenicity and is easily hydrolyzed in blood, is desirable in the clinical application of ADCs. In other words, the method developed by the present inventors is the first in the world to successfully modify an antibody Fc region with a drug in a regioselective manner by chemical synthesis without using a linker that contains a peptide moiety. Such a method makes it possible to develop various compounds that have the structural features (1) to (4) above (e.g., Figures 1-1, 1-2, 1-3, and 2).

[0012] The inventors have also found that by using a polypeptide containing a glutamine residue (Q) at its N-terminus as an affinity substance for an antibody, the amino group (-NH2) in the N-terminal glutamine residue (Q) can be protected by reacting (pyroglutamylation) with the amide group present in the side chain (e.g., -CONH2 in the glutamine residue (Q) when QET is contained at the N-terminus), and therefore a linker that mediates the binding of the affinity substance to the antibody can be specifically added to a specific amino acid residue (lysine residue having an amino group) in the affinity substance, making a polypeptide containing a glutamine residue (Q) at its N-terminus useful as an affinity substance for an antibody.

[0013] The present inventors also found that a method for secreting and producing a polypeptide using a coryneform bacterium as a host (WO 2013 / 062029) is useful for easily and mass-producing a polypeptide containing a glutamine residue (Q) at its N-terminus. This method allows the addition of the three N-terminal residues Gln-Glu-Thr (QET) of the mature Csp protein to the N-terminus of the target polypeptide, thereby enjoying the advantages of the method for secreting and producing the polypeptide, and is therefore suitable for preparing a polypeptide containing a glutamine residue (Q) at its N-terminus (an affinity substance for an antibody).

[0014] That is, the present invention is as follows.

[0015] In a first embodiment, the present invention provides a site-selective modification reagent for an antibody, comprising an affinity substance for an antibody, a compound or a salt thereof having a cleavable moiety and a reactive group, and the compound or a salt thereof. [1] A compound or a salt thereof having an affinity substance for an antibody, a cleavable moiety, and a reactive group, The compound has 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group to the antibody; A compound or a salt thereof, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus. [2] The compound or salt thereof according to [1], wherein the polypeptide is a polypeptide comprising a tripeptide (QET) consisting of a glutamine residue (Q)-glutamic acid (E)-threonine residue (T) at the N-terminus. [3] The compound according to [1] or [2], or a salt thereof, wherein the glutamine residue is pyroglutamylated. [4] The compound according to any one of [1] to [3] above, or a salt thereof, wherein the polypeptide has 16 to 123 amino acid residues. [5] The compound or salt thereof according to any one of [1] to [3], wherein the polypeptide has 20 to 70 amino acid residues. [6] The polypeptide is any one of the compounds according to [1] to [5] or a salt thereof, wherein the compound is selected from the group consisting of the following (1) to (4): [ka] [Q is an N-terminal glutamine residue, E is a glutamic acid residue, T is a threonine residue, the solid line is a bond, the spacer portion is a portion consisting of 1 to 50 amino acid residues, and the affinity substance for the antibody is a portion consisting of 15 to 70 amino acid residues.] [7] The compound or salt thereof according to [6], wherein the polypeptide portion having affinity for an antibody is selected from the group consisting of Protein A, Protein G, Protein L, Protein Z, fragments thereof having affinity for an antibody, and mutants thereof having affinity for an antibody. [8] The compound or salt of [6] or [7], wherein the antibody affinity polypeptide portion comprises an amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNAXIXSIRDDC (SEQ ID NO: 5) (each of the two Xs is independently an amino acid residue selected from the group consisting of arginine residues, glutamic acid residues, and glutamine residues), in which any one amino acid residue is substituted with a lysine residue, and which has an identity of 90% or more to the amino acid sequence of SEQ ID NO: 5. [9] A compound according to any one of [6] to [8], or a salt thereof, wherein the polypeptide portion having affinity for an antibody comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 6, in which any one of the amino acid residues in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 6) is replaced with a lysine residue.

[10] The compound or a salt thereof according to any one of [1] to [9], wherein the affinity substance for an antibody comprises an amino acid sequence selected from the group consisting of: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 8); (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 9); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (sequence number 25).

[11] Any of the compounds according to [1] to

[10] , 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.

[12] The compound or a salt thereof according to

[11] , wherein L is the cleavable linker (i).

[13] The compound or salt thereof according to

[11] or

[12] , wherein L is the cleavable linker of (i) above, and B is the divalent group of (b) above.

[14] The compound or a salt thereof according to

[11] , wherein L is the cleavable linker of (ii) above, and B is the divalent group of (a) above.

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

[14] , wherein the polypeptide is a binding peptide to the Fc region of a monoclonal antibody.

[16] The compound or salt thereof according to

[15] , wherein the polypeptide is a binding peptide to the Fc region of IgG.

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

[16] or a salt thereof, wherein the affinity substance comprises any one of the Fc domain proteins 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.

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

[17] , wherein the polypeptide is capable of binding to human IgG.

[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 one 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) standing when a cleavable linker containing a self-degrading cleavable moiety is used.

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

[19] , or a salt thereof, wherein the cleavable moiety is selected from the group consisting of a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a 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.

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

[11] to

[20] above, 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

[11] 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; A plurality of R2a, a plurality of R2b, and a plurality of R2c 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) Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc represents a hydrogen atom or a monovalent hydrocarbon group); (vi) NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and each represents 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 ● (closed circle) indicates a bond to B. When the chemical structure is asymmetric with respect to the cleavage site, a ● may represent a bond to A and a ○ may represent a bond to B.], the compound or salt thereof according to any one of [1] to

[22] ,

[24] The cleavable moiety of (i) is any one of the following: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; R2a is the same as

[23] , ○ (open circle) indicates a bond to A, and ● (closed 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.], the compound or salt thereof according to any one of

[11] to

[19] ,

[21] , and

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

[25] The cleavable portion of (ii) is [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; R2b, R2c, J, and r are the same as in

[23] . ○ (open circle) indicates a bond to A, and ● (closed 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.], the compound or salt thereof according to any one of

[11] to

[19] ,

[22] , and

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

[26] L is represented by 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, and C is a cleavable moiety.], wherein the compound or a salt thereof is 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 each is an integer of 0 to 10, q and q' are the same or different and each is an integer of 0 to 10, X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein, when X is a nitrogen atom, R1b does not exist, and when X' is a nitrogen atom, R1b' does not exist. When X is a single bond, R1a and R1b do not exist, and when X' is a single bond, R1a' and R1b' do not exist.); R1a, R1b, R1a' and R1b' are the same or different and are atoms or groups selected from the group consisting of (i) to (vii).

[28] Any of the compounds according to [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 residues, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[29] Any of the compounds according to [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 selected from the group consisting of the following: [ka] [During the ceremony, R1f, one or more R1g, and one or more R1h are the same or different and each is an atom or group selected from the group consisting of (i) to (vii), or an electron-withdrawing group; and · is a bond.], wherein the compound or a salt thereof is any one of the compounds represented by the following formula (1) to (29):

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

[30] , or a salt thereof, wherein the divalent group (b) is selected from the group consisting of an optionally substituted alkylene, an optionally substituted cycloalkylene, an optionally substituted aryl, an optionally substituted divalent heterocyclic group, -NRa- (Ra represents a hydrogen atom or a substituent), -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 is -NH-, -O-, -CH2-, or a single bond; V1 is a divalent group containing a bioorthogonal functional group; s is an integer from 0 to 10, The circles and circles in formula (B-2) are the same orientation as the circles and circles 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 moiety on the L side, and ● (black circle) indicates a bond to the moiety on the R side.], or a salt thereof, any one of compounds [1] to

[31] .

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

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

[34] The compound or a salt thereof according to

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

[35] The reactive group is one of the following: [ka] [Where: R5a and R5c each represent an atom or group selected from the group consisting of (i) to (vii). and R5b is an electron withdrawing group; j is an integer from 1 to 5; and k is an integer of 1 to 4.], wherein the compound or a salt thereof is any one of the compounds according to [1] to

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

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

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

[37] Any of the compounds according to [1] to

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

[38] Any of the compounds according to [1] to

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

[39] The compound represented by the 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 that is a divalent group that contains 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.] is a compound represented by the formula (1), or a salt thereof.

[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', R1a, R1a', R1b, and R1b' are the same as those in formulas (La') and (Lb') described in

[19] , 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 above formula (B-1).

[41] A regioselective modification reagent for an antibody, comprising: The compound has 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group to the antibody.], and A regioselective antibody modification reagent, in which the affinity substance for the antibody is a polypeptide comprising a glutamine residue (Q) at the N-terminus (preferably, a regioselective antibody modification reagent comprising any of the compounds or salts thereof described above in [2] to

[40] ).

[42] A compound or a salt thereof having an affinity substance for an antibody, a cleavable moiety, and a reactive group, 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group specific for the side chain of a lysine residue; A compound or a salt thereof, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[43] A regioselective modification reagent for an antibody, comprising: 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group specific to the side chain of a lysine residue.], and A reagent, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0016] Secondly, the present invention provides an affinity substance for an antibody, and an antibody or a salt thereof having a cleavable portion, and a method for producing the same. (Antibody having affinity for antibody, and cleavable moiety or its salt) [1] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion, 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody; An antibody or a salt thereof, wherein the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus. [2] The antibody or salt thereof according to [1], wherein the antibody is a monoclonal antibody. [3] The antibody or salt thereof according to [1] or [2], wherein the antibody is an IgG antibody. [4] The antibody or salt thereof according to any one of [1] to [3], wherein the antibody is of human origin. [5] The antibody according to any one of [1] to [4] or a salt thereof, wherein the antibody comprises any one of the Fc domain proteins selected from the group consisting of the following (A) to (C) and has 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. [6] 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; The antibody or salt thereof according to any one of [1] to [5], 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 site selectivity of 30% or more. [7] The antibody or salt thereof according to [6], wherein the target region is a region consisting of 1 to 10 consecutive amino acid residues. [8] The antibody or salt thereof according to [7], wherein the target region is a region consisting of 1 to 3 consecutive amino acid residues. [9] The antibody or salt thereof according to [8], wherein the target region is a region consisting of amino acid residues at positions 246 to 248 in the Fc region of human IgG.

[10] The antibody or salt thereof according to any one of [6] to [9], wherein the position selectivity is 50% or more.

[11] The antibody or salt thereof according to

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

[12] The antibody or salt thereof according to

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

[13] The antibody or salt thereof according to any of [6] to

[12] , wherein the specific amino acid residue does not contain any amino acid residue identical to the specific amino acid residue at the specific position, except for 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 of amino acid residues distant from the specific amino acid at the N-terminus and C-terminus (wherein a is any integer from 1 to 10).

[14] The antibody comprises multiple heavy chains, The antibody or salt thereof according to any one of [1] to

[13] , 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'.

[15] The antibody or salt thereof according to

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

[16] The antibody or salt thereof according to any one of [1] to

[15] , wherein the moiety generated by the reaction between the antibody and the reactive group is a moiety generated by the reaction of a reactive group specific for one of the side chains of a lysine residue, a tyrosine residue, or a tryptophan residue with a lysine residue, a tyrosine residue, or a tryptophan residue.

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

[16] , wherein the moiety generated by the reaction between the antibody 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 a lysine residue.

[18] The moiety produced by the reaction is as follows: [ka] [wherein ● (black circle) indicates a bond to the T side moiety, and ○ (white circle) indicates a bond to the B side moiety. A straight line perpendicular to the bond indicates a bond formed by the reaction.], the antibody or salt thereof according to any one of [1] to

[17] ,

[19] The antibody or salt thereof according to any one of [1] to

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

[20] The antibody or salt thereof according to any one of [1] to

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

[21] The antibody or salt thereof according to any one of [1] to

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

[22] The compound represented by the 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 that is a divalent group that contains 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 antibody or salt thereof according to any one of [1] to

[21] , wherein the compound is represented by the formula:

[23] 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) above; C is a cleavable moiety, p and p' are the same or different and each is an integer of 0 to 10, q and q' are the same or different and each is an integer of 0 to 10, X and X' are the same or different and each is a carbon atom, a nitrogen atom, or a single bond (wherein, when X is a nitrogen atom, R1b does not exist, and when X' is a nitrogen atom, R1b' does not exist. When X is a single bond, R1a and R1b do not exist, and when X' is a single bond, R1a' and R1b' do not exist); R1a, R1b, R1a' and R1b' 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) Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc represents a hydrogen atom or a monovalent hydrocarbon group); (vi) NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and each represents 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) above, Z and Z' are the same or different and are the same as Z in the formula (B-1).

[24] An antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion, 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain 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. An antibody or a salt thereof, wherein the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0017] (Method for Producing an Affinity Substance for an Antibody, and an Antibody Having a Cleavable Moiety or a Salt thereof)

[25] A method for producing an antibody or a salt thereof having an affinity substance for an antibody and a cleavable portion, comprising the steps of: 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; and R is a reactive group to the antibody.], by reacting a compound or a salt thereof having an affinity substance for the antibody, a cleavable moiety and a reactive group, with the antibody, 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 the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.] and producing an antibody or a salt thereof having an affinity substance for the antibody, and a cleavable moiety, A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

[26] The method according to

[25] , wherein the reactive group is a reactive group specific for the side chain of a lysine residue.

[0018] Third, the present invention provides a complex or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety, a functional substance and an antibody, and a method for producing the same. (Conjugates or salts thereof having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody) [1] A complex or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, 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 contains a cleavable moiety; B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody; A conjugate or a salt thereof, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus. [2] The conjugate or salt thereof according to [1], wherein the antibody is a monoclonal antibody. [3] The conjugate or salt thereof of [1] or [2], wherein the antibody is an IgG antibody. [4] The conjugate or salt thereof according to any one of [1] to [3], wherein the antibody is of human origin. [5] A conjugate or a salt thereof according to any one of [1] to [4], wherein the antibody comprises any one Fc domain protein selected from the group consisting of the following (A) to (C) and has 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. [6] 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; The complex or salt thereof according to any one of [1] to [5], 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 site selectivity of 30% or more. [7] The complex or salt thereof according to [6], 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 present at the specific position, in a region extending to a number (wherein a is any integer from 1 to 10) of amino acid residues away from the specific amino acid present at the specific position on the N-terminal and C-terminal sides, respectively. [8] The antibody comprises multiple heavy chains, The conjugate or salt thereof according to any one of [1] to [7], 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, so that the antibody has a plurality of structural units represented by AL-B'(-F)-R'. [9] When the functional substance has a functional group that easily reacts with a bioorthogonal functional group, or when the functional substance has been derivatized to have a functional group that easily reacts with a bioorthogonal functional group, the functional group that easily reacts with the 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, a thioester residue, an α-halocarbonyl residue, an isonitrile residue, a sydnone residue, or a selenium residue. A complex or a salt thereof according to any one of [1] to [8].

[10] When the functional substance has a functional group that easily reacts with a bioorthogonal functional group, or is derivatized to have a functional group that easily reacts with a bioorthogonal functional group, the functional group that easily reacts with a bioorthogonal functional group is one of the following: [ka] [During the ceremony, R1f, one or more R1g, and one or more R1h are the same or different and each represents an atom or group selected from the group consisting of (i) to (vii), or an electron is a bonding hand to the functional substance.]. The complex or salt thereof according to any one of [1] to [9],

[11] The conjugate according to any one of [1] to

[10] or a salt thereof, wherein the moiety generated by the reaction between the antibody and the 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 one of the side chains of a lysine residue, a tyrosine residue, or a tryptophan residue.

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

[11] , wherein the moiety generated by the reaction between the antibody 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 a lysine residue.

[13] The moiety produced by the reaction is as follows: [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.], wherein the complex or salt thereof is any one of [1] to

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

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

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

[15] A complex or a salt thereof according to any one of [1] to

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

[16] The conjugate or salt thereof of

[14] or

[15] , wherein the drug is an anticancer drug.

[17] A complex or a salt thereof according to any one of [1] to

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

[18] Any of the complexes according to [1] to

[17] or salts thereof, wherein the main chain connecting A and R does not contain a ring structure.

[19] A complex or a salt thereof according to any one of [1] to

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

[20] 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 that is a divalent group that contains a cleavable moiety; B1' and B2' are the same or different and are trivalent 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 and are functional substances; B1' (-F1) and B2' (-F2) may have a symmetric structure with L' at the center.], The complex or salt thereof according to any one of [1] to

[19] ,

[21] The compound represented by the formula (III') is the following (III''): [ka] [During the ceremony, A, R' and T are the same as those in formula (III) above; C is a cleavable moiety, p and p' are the same or different and each is an integer of 0 to 10, q and q' are the same or different and each is an integer of 0 to 10, X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond (wherein, when X is a nitrogen atom, R1b does not exist, and when X' is a nitrogen atom, R1b' does not exist. When X is a single bond, R1a and R1b do not exist, and when X' is a single bond, R1a' and R1b' do not exist.); R1a, R1b, R1a' and R1b' 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 each is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1), Z and Z' are the same or different and are the same as Z in formula (B-1), F and F' are the same or different and each is a functional substance.

[22] A complex or a salt thereof comprising an affinity substance, a functional substance, and an antibody, 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 contains a cleavable moiety; B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; 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; A conjugate or a salt thereof, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

[0019] (Method of producing a complex having an affinity substance for an antibody, a cleavable moiety, a functional substance and an antibody, or a salt thereof)

[23] A method for producing a complex or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, comprising the steps of: 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 contains a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; R' is the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.], and an antibody or a salt thereof having a cleavable portion is reacted with a functional substance to form the antibody. 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 trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional substance; and A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

[24] The method according to

[23] , wherein the reactive group is a reactive group specific for the side chain of a lysine residue.

[25] A method for producing a complex or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, comprising the steps of: (A) 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 contains a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; and R is a group reactive to the antibody. 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 the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.] and producing an affinity substance for the antibody, and an antibody or a salt thereof having a cleavable moiety, (B) reacting an affinity substance for the antibody and an antibody or a salt thereof 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 trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional substance; and A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

[0020] Fourth, the present invention provides a method for producing an antibody having a bioorthogonal functional group. [1] A method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising the steps of: 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.], and the cleavable portion of the antibody or its salt having the cleavable portion is cleaved to obtain 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 in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus. [2] The method of [1], 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] L is the cleavable linker of (i), L1 is (i') a monovalent group containing a bioorthogonal functional group; The method according to [2], wherein B is the divalent group represented by (a) or (b). [4] L is the cleavable linker of (i), L1 is (i') a monovalent group containing a bioorthogonal functional group; The method according to [2] or [3], wherein B is the divalent group of (b). [5] L is the cleavable linker of (ii) above, L1 is (i') a monovalent group that does not contain a bioorthogonal functional group; The method according to [2], wherein B is the divalent group represented by (a). [6] The method according to any one of [1] to [5], wherein the reactive group is a group that is specifically reactive with a side chain of a lysine residue. [7] A method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising the steps of: (A) 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; and R is a group reactive to the antibody. 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 the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.] and producing an affinity substance for the antibody, and an antibody or a salt thereof having a cleavable moiety, (B) cleaving the cleavable portion of the antibody or its salt having an affinity substance for the antibody 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 in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

[0021] Fifth, the present invention provides a method for producing an antibody or a salt thereof having a functional substance. [1] A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: 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 contains a cleavable moiety; B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; R' is the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.], by cleaving the cleavable portion of a complex or a salt thereof having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody, 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; B', F, R' and T are the same as those of the formula (III), A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus. [2] The method according to [1], wherein the reactive group is a group that is specifically reactive with a side chain of a lysine residue. [3] A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: (A) 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 contains a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; R' is the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.], and an antibody or a salt thereof having a cleavable portion is reacted with a functional substance to form the antibody. 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 trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional substance. forming a conjugate or a salt thereof having the active substance and the antibody; and (B) cleaving the cleavable portion of a complex or a salt thereof having an affinity substance for the antibody, a cleavable portion, a functional substance, and an antibody, 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; B', F, R' and T are the same as those of the formula (III), A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus. [4] A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: (A) 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 contains a cleavable moiety; B is (a) a divalent group containing a bioorthogonal functional group; and R is a reactive group to the antibody.], by reacting a compound or a salt thereof having an affinity substance for the antibody, a cleavable moiety and a reactive group, with the antibody, 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 the moiety generated by reaction between the antibody and the reactive group, and T is an antibody. (B) reacting an affinity substance for the antibody and an antibody or a salt thereof 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 trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; and F is a functional substance.], and (C) cleaving the cleavable portion of a complex or a salt thereof having an affinity substance for the antibody, a cleavable portion, a functional substance, and an antibody, 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; B', F, R' and T are the same as those of the formula (III), A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus. [5] A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: (A) 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.], and the cleavable portion of the antibody or its salt having the cleavable portion is cleaved to obtain 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 antibody 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 reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; F is a functional material; 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), B' is a divalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; Fa and Fb are the same or different functional substances, A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus. [6] A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: (A) 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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; and R is a group reactive to the antibody. 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 the moiety generated by reaction between the antibody and the reactive group, and T is an antibody. (B) cleaving the cleavable portion of the antibody or its salt having an affinity substance for the antibody 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 antibody 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 reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; F is a functional material; 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), B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; Fa and Fb are the same or different functional substances, A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus. Effect of the Invention

[0022] (I) The compounds of the present invention having an affinity substance for an antibody, a cleavable moiety and a reactive group, or salts thereof, are useful, for example, for site-selective modification of an antibody. (I) the compound of the present invention or a salt thereof, (II) the antibody of the present invention having an affinity substance for an antibody and a cleavable portion (regioselectively) or a salt thereof, (III) the complex of the present invention having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody (regioselectively) or a salt thereof are useful, for example, as intermediates for the preparation of an antibody having a bioorthogonal functional group (regioselectively) or a salt thereof, and an antibody having a functional substance (regioselectively) or a salt thereof. An antibody having a functional substance (regioselectively) or a salt thereof is useful, for example, as a medicine or a reagent (e.g., a diagnostic agent, a research reagent). An antibody having a bioorthogonal functional group (regioselectively) or a salt thereof is useful as an intermediate for the preparation of an antibody having a functional substance (regioselectively) or a salt thereof. Thus, (I) the compound of the present invention or a salt thereof, (II) the antibody of the present invention or a salt thereof, and (III) the complex of the present invention or a salt thereof are useful, for example, as intermediates for the synthesis of medicines or reagents. In addition, a polypeptide containing a glutamine residue (Q) at the N-terminus is used as an affinity substance for antibodies. By using this pyroglutamyl group, the amino group (-NH2) in the N-terminal glutamine residue (Q) can be protected by reacting it with the amide group present in its side chain (e.g., -CONH2 in the glutamine residue (Q) when QET is contained at the N-terminus) (pyroglutamylation), thereby making it possible to more specifically attach a linker that mediates the binding of the affinity substance to the antibody to a specific amino acid residue in the affinity substance. Furthermore, according to a method for secreting and producing a polypeptide using a coryneform bacterium as a host (CspB fusion method), the advantages of the polypeptide secretion production method can be enjoyed by adding the N-terminal three residues of the mature CspB protein, Gln-Glu-Thr (QET), to the N-terminus of the target polypeptide, and this method is suitable for preparing polypeptides (substances with affinity for antibodies) that contain a glutamine residue (Q) at the N-terminus. [Brief description of the drawings]

[0023] [Figure 1-1] FIG. 1-1 is a schematic diagram (part 1) showing the concept of site-selective modification of an antibody by a compound of the present invention [a compound having an affinity substance for an antibody, a cleavable portion, and a reactive group: ALBR (I)]. First, the compound of the present invention associates with an antibody (T) via an affinity substance for the antibody (A). Next, the compound of the present invention reacts with a side chain of a specific amino acid residue (a side chain of a lysine residue in the figure) in a target region present in the vicinity of the association site between the affinity substance and the antibody via a reactive group (R) (an activated ester in the figure), to generate a conjugate of the compound of the present invention and the antibody [an antibody having a structural unit containing an affinity substance for the antibody and a cleavable portion in a site-selective manner: ALB-R'-T (II)]. [Figure 1-2] Figure 1-2 is a schematic diagram (part 2) showing the concept of site-selective modification of an antibody with the compounds of the present invention. Cleavage of the cleavable moiety in the linker (L) generates an antibody site-specifically modified with a bioorthogonal functional group. [Figure 1-3] Figure 1-3 is a schematic diagram (part 3) showing the concept of site-selective modification of an antibody with the compound of the present invention. The reaction of a bioorthogonal functional group with a functional substance (e.g., a drug) produces an antibody site-specifically modified with the functional substance. [Diagram 2]FIG. 2 is a diagram showing the relationship between each invention of the present invention (salts are omitted). In reaction (1), a compound having an affinity substance for an antibody, a cleavable portion, and a reactive group is reacted with an antibody to produce an affinity substance for the antibody and an antibody having a cleavable portion. In reaction (2), an affinity substance for an antibody and an antibody having a cleavable portion are reacted with a functional substance to produce a complex having an affinity substance for the antibody, a cleavable portion, a functional substance, and an antibody. In reaction (3), the cleavable portion of the complex having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody is cleaved to produce an antibody having a functional substance (at this time, the portion containing the affinity substance is produced as a by-product). In reaction (4), an affinity substance for an antibody and the cleavable portion of the antibody having a cleavable portion are cleaved to produce an antibody having a bioorthogonal functional group (at this time, the portion containing the affinity substance is produced as a by-product). In reaction (5), an antibody having a bioorthogonal functional group is reacted with a functional substance to produce an antibody having a functional substance. Reactions (2) and (5) can be carried out in a similar manner. Reactions (3) and (5) can also be carried out similarly. [Diagram 3] FIG. 3 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 4] FIG. 4 shows (1) the amino acid sequence of the heavy chain of trastuzumab (sequence number 2), (2) the amino acid sequence of the IgG1 Fc region whose glycans have been cleaved with PNGase (sequence number 3), and (3) the amino acid sequence of the light chain of trastuzumab (sequence number 4). [Diagram 5] 5 is a diagram showing secretion expression of each modified protein A in C. glutamicum. Lane 1: molecular weight marker; lane 2: protein band estimated to be CspB6Tev-QZ34C; lane 3: protein band estimated to be QZ34C-60; lane 4: protein band estimated to be QET-Z34C1; lane 5: protein band estimated to be QET-Z34C2. [Figure 6]6 is a diagram showing the results of HIC (Hydrophobic Interaction Chromatography)-UPLC analysis of specific modifications of trastuzumab (Example 3) (detection wavelength: UV280 nm). The AU on the vertical axis indicates absorbance (the same applies to the following figures). [Figure 7] FIG. 7 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 4). [Figure 8] FIG. 8 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 5). [Figure 9] FIG. 9 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 6). [Figure 10] FIG. 10 shows the MS spectrum (measured value: m / z 952.23196, theoretical value: 952.22900, 4-valent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 26), a peptide consisting of 33 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab (Example 7). [Figure 11-1] FIG. 11-1 shows the CID spectrum of the product ion at m / z 764.68 (theoretical value: 764.40) corresponding to divalent y12, indicating the modification of the lysine residue at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 7). [Figure 11-2]FIG. 11-2 shows the results of searching for peptide fragments containing lysine residue modifications (thiol-introduced form (+145.019 Da) that have been carbamidomethylated with iodoacetamide) in a tryptic digest of trastuzumab using Proteome Discoverer (Thermo Fisher Scientific) (Example 7). The horizontal axis shows the identified lysine residues, and the vertical axis shows Peptide Spectrum Matches (PSMs). Note that the residue numbers of lysine residues are indicated by the numbers in the sequence (i.e., the N-terminal amino acid is number 1; the same applies below) in the heavy chain VH domain and light chain, and by EU numbering in the heavy chain CH1, CH2, and CH3 domains. [Figure 12] FIG. 12 shows the MS spectrum (measured value: m / z 952.23227, theoretical value: 952.22900, 4-valent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 26), a peptide consisting of 33 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab (Example 8). [Figure 13-1] FIG. 13-1 shows the CID spectrum of the product ion at m / z 838.23 (theoretical value: 837.94), which corresponds to doubly charged y13 and indicates the modification of the lysine residue at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 8). [Figure 13-2]FIG. 13-2 shows the results of searching for peptide fragments containing lysine residue modifications (thiol-introduced derivatives (+145.019 Da) that have been carbamidomethylated with iodoacetamide) in tryptic digests of trastuzumab using Proteome Discoverer (Thermo Fisher Scientific) (Example 8). The horizontal axis shows the identified lysine residues, and the vertical axis shows Peptide Spectrum Matches (PSMs). Note that the residue numbers of lysine residues are indicated by the numbers in the sequence for the heavy chain VH domain and light chain, and by EU numbering for the heavy chain CH1, CH2, and CH3 domains. [Figure 14] FIG. 14 shows the MS spectrum (measured value: m / z 952.23032, theoretical value: 952.22900, 4-valent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 26), a peptide consisting of 33 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab (Example 9). [Figure 15-1] FIG. 15-1 shows the CID spectrum of the product ion at m / z 838.29 (theoretical value: 837.94), which corresponds to doubly charged y13 and indicates the modification of the lysine residue at positions 246 / 248 of the human IgG heavy chain in the EU numbering system (Example 9). [Figure 15-2]FIG. 15-2 shows the results of searching for peptide fragments containing lysine residue modifications (thiol-introduced derivatives (+145.019 Da) that have been carbamidomethylated with iodoacetamide) in tryptic digests of trastuzumab using Proteome Discoverer (Thermo Fisher Scientific) (Example 9). The horizontal axis shows the identified lysine residues, and the vertical axis shows Peptide Spectrum Matches (PSMs). Note that the residue numbers of lysine residues are indicated by the numbers in the sequence for the heavy chain VH domain and light chain, and by EU numbering for the heavy chain CH1, CH2, and CH3 domains. [Figure 16] FIG. 16 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm). [Figure 17] FIG. 17 shows the MS spectrum (m / z 791.74872, trivalent) of a peptide fragment of VVSVLTVLHQDWLNGKEYK (sequence number 47) including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Reference Example 1. [Figure 18] FIG. 18 shows the CID spectrum of a peptide fragment of VVSVLTVLHQDWLNGKEYK (sequence number 47) including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Reference Example 1. [Figure 19]FIG. 19 shows the MS spectrum (m / z 886.93408, tetravalent) of a peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (sequence number 48) including 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 Reference Example 1. [Figure 20] FIG. 20 shows the CID spectrum of a peptide fragment of EEQYDSTYRVVSVLTVLHQDWLNGKEYK (sequence number 48) including 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 Reference Example 1. [Figure 21] FIG. 21 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Reference Example 3). [Figure 22] FIG. 22 shows the MS spectrum (m / z 769.04688, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 50) including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab modified with the binder peptide of Reference Example 3. [Diagram 23] FIG. 23 shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 50), which includes 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 Reference Example 3. [Figure 24] FIG. 24 shows SDS-PAGE results of the secretion and expression of each modified protein A (QET-Z34C3 and QET-Z34C4) in C. glutamicum. [Diagram 25] FIG. 25 shows SDS-PAGE results of the secretion and expression of each modified Fc-III (CspBss-QET-LV and CspBss-QET-II) in C. glutamicum. [Figure 26] FIG. 26 shows the MS spectrum (measured value: m / z 769.04353, theoretical value: 769.04482, trivalent) of a peptide fragment of FNWYVDGVEVHNAKTKPR (sequence number 50), a peptide consisting of 18 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab. [Figure 27-1] Figure 27-1 shows the CID spectrum of the product ion at m / z 1022.73 (theoretical value: 1022.51), which corresponds to divalent y16, indicating modification of the lysine residues at positions 288 / 290 of the human IgG heavy chain in EU numbering (Example 12). [Figure 27-2] Figure 27-2 shows the results of searching for peptide fragments containing a thiol-introduced form (+145.019 Da) that has been modified at a lysine residue (carbamidomethylated with iodoacetamide) in a trypsin digest of trastuzumab using BioPharma Finder (Example 12). The horizontal axis shows the identified lysine residues, and the vertical axis shows the intensity. Note that the residue numbers of the lysine residues are indicated by the numbers in the sequence (i.e., the N-terminal amino acid is number 1; the same applies below) in the heavy chain VH domain and light chain, and by EU numbering in the heavy chain CH1, CH2, and CH3 domains. [Figure 28] FIG. 28 shows confirmation of specific modified forms of trastuzumab by ESI-TOFMS analysis (Example 13). [Figure 29] FIG. 29 shows the results of HIC-UPLC analysis of specific modifications of trastuzumab (detection wavelength: UV 280 nm) (Example 13). [Diagram 30]FIG. 30 shows the MS spectrum (measured value: m / z 952.23425, theoretical value: 952.22942, 4-valent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 26), a peptide consisting of 33 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab (Example 14). [Diagram 31] FIG. 31 shows the MS spectrum (measured value: m / z 952.23425, theoretical value: 952.22942, 4-valent) of a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPKDTLMISR (sequence number 26), a peptide consisting of 33 amino acid residues including a modification site (thiol-introduced form (+145.019 Da) that has been carbamidomethylated with iodoacetamide) on a lysine residue by trypsin digestion of trastuzumab (Example 14). [Figure 32-1] Figure 32-1 shows the CID spectrum of the product ion at m / z 1166.99 (theoretical value: 1166.61), which corresponds to doubly charged y20, indicating modification of the lysine residue at positions 246 / 248 of the human IgG heavy chain in EU numbering (Example 14). [Figure 32-2] Figure 32-2 shows the results of searching for peptide fragments containing a thiol-introduced form (+145.019 Da) that has been modified at a lysine residue (carbamidomethylated with iodoacetamide) in a tryptic digest of trastuzumab, using BioPharma Finder (Example 14). The horizontal axis shows the identified lysine residues, and the vertical axis shows the intensity. Note that the residue numbers of the lysine residues are indicated by the numbers in the sequence (i.e., the N-terminal amino acid is number 1; the same applies below) in the heavy chain VH domain and light chain, and by EU numbering in the heavy chain CH1, CH2, and CH3 domains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] 1. A compound or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety and a reactive group 1-1. Overview The present invention provides a compound comprising an affinity substance for an antibody, a cleavable moiety and a reactive group, represented by formula (I) or a salt thereof: ALBR (I) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group to the antibody.

[0025] In formula (I) and other formulae 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.

[0026] 1-2. Substances with affinity for antibodies (A) In formula (I), A is an affinity substance for an antibody. The affinity substance is a substance that has the ability to bind to an antibody via a non-covalent bond.

[0027] The affinity substance used in the present invention targets an antibody. The antibody may be a protein (e.g., glycoprotein) modified with a biomolecule (e.g., sugar) or a protein unmodified with a biomolecule. The antibody may be any antibody against any component, such as a biological component, a viral component, or a component found in the environment, but an antibody against a biological component or a viral component is preferred. The biological component may be, for example, a component (e.g., protein) derived from an animal, such as a mammal or bird (e.g., chicken), an insect, a microorganism, a plant, a fungus, or a fish. Preferably, the biological component is a component derived from a mammal. The mammal may be, for example, a primate (e.g., human, monkey, chimpanzee), a rodent (e.g., mouse, rat, guinea pig, hamster, rabbit), a pet animal (e.g., dog, cat), a livestock animal (e.g., cow, pig, goat), or a working animal (e.g., horse, sheep). The biological component is more preferably a component (e.g., protein) derived from a primate or rodent, and even more preferably a component (e.g., protein) derived from a human from the viewpoint of clinical application of the present invention. Examples of the virus-derived component include components (e.g., proteins) derived from influenza viruses (e.g., avian influenza viruses, swine influenza viruses), AIDS viruses, Ebola viruses, and phage viruses.

[0028] The antibody may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a specific sugar chain has been added (e.g., antibodies modified to have a sugar chain consensus sequence such as an N-type sugar chain consensus sequence), bispecific antibodies, scFv antibodies, Fab antibodies, F(ab')2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE), or an antibody having 4 or more valencies (e.g., IgA antibody, IgM antibody).

[0029] The antibody that is the target of the affinity substance may be composed of any amino acid residues, but is preferably composed of the 20 naturally occurring L-α-amino acid residues that normally constitute proteins. Examples of such amino acid residues include L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (hereinafter, the notation of L is omitted). The antibody may be composed of, for example, 100 or more amino acid residues, preferably 120 or more, more preferably 150 or more, even more preferably 180 or more, and particularly preferably 200 or more. The antibody may also be, 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. More specifically, the antibody may be composed of, for example, 100 to 1000 amino acid residues, preferably 120 to 900, more preferably 150 to 800, even more preferably 180 to 700 or more, and particularly preferably 200 to 600. When the antibody is an antibody (e.g., a monoclonal antibody as described above), the above number of amino acid residues may correspond to the amino acid residues of the heavy chain of the antibody.

[0030] The antibody which is the target of the affinity substance further has a specific amino acid residue having a side chain or terminus (N-terminus and / or C-terminus), preferably a side chain, to which a reactive group as described below can react. The protein contains the specific amino acid residue at one position or multiple positions (preferably multiple positions). Examples of such specific amino acid residues include 14 types of amino acid residues as described below, and preferably, the amino acid residue is selected from the group consisting of lysine residues, tyrosine residues, tryptophan residues, and cysteine ​​residues. More preferably, such specific amino acid residues are selected from the group consisting of lysine residues, tyrosine residues, and tryptophan residues. In view of the fact that the compound of the present invention can modify an antibody site-selectively, an antibody containing such specific amino acid residues at multiple positions is 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, 200 or less positions, preferably 180 or less positions, more preferably 150 or less positions, even more preferably 120 or less positions, and particularly preferably 100 or less positions. More specifically, the multiple positions may be, for example, positions 3 to 200, preferably positions 5 to 180, more preferably positions 10 to 150, even more preferably positions 20 to 120, and particularly preferably positions 30 to 100. Even in an antibody containing such a specific amino acid residue at multiple positions, the compound 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 about 70 to 90, depending on the amino acid composition in the variable region. In the present invention, the lysine residues present at a specific position of such human IgG1 have been successfully regioselectively modified.

[0031] More specifically, in the present invention, from the viewpoint of modifying amino acid residues present at specific positions in an antibody while maintaining the antibody function (i.e., while maintaining the native folding without denaturing the antibody), site-selective modification of amino acid residues exposed on the antibody surface is preferred. For example, in human IgG such as human IgG1, exposed lysine residues and exposed tyrosine residues are present at the following positions (according to EU numbering; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For convenience of explanation, in the present application, EU numbering is used for the heavy chain CH1, CH2, and CH3 domains, and numbers in the sequence (i.e., the N-terminal amino acid is numbered 1) are used for the heavy chain VH domain and light chain. (1) Exposed Lysine Residues CH2 domain (246, 248, 274, 288, 290, 317, 320, 322, 338) CH3 domain (ranks 360, 414, and 439) (2) Exposed tyrosine residues CH2 domain (278th, 296th, 300th) CH3 domain (436th position) Therefore, when human IgG, such as human IgG1, is modified at lysine or tyrosine residues, modification at the above positions is preferred.

[0032] Preferably, when human IgG, such as human IgG1, is modified with lysine or tyrosine residues, the lysine or tyrosine residues at the following positions, which are highly surface-exposed, among the above positions (1) and (2) may be modified: (1') Exposed Lysine Residue CH2 domain (246, 248, 274, 288, 290, 317, 320, 322) CH3 domain (ranks 360, 414, and 439) (2') Exposed tyrosine residue CH2 domain (278th, 296th, 300th) CH3 domain (436th position) Therefore, when human IgG, such as human IgG1, is modified at lysine or tyrosine residues, modification at the above positions is more preferred.

[0033] More preferably, when human IgG, such as human IgG1, is modified with lysine residues, among the positions (1) above, 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) may be modified.

[0034] In a specific embodiment, when the antibody that is the target of the affinity substance contains specific amino acid residues at multiple positions as described above, it may contain one or more specific amino acid residues in a target region consisting of 1 to 50 consecutive amino acid residues, and may 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 present at a specific position. Such a specific position may vary depending on the type of the target protein and the affinity substance, and may be, for example, a specific position in a specific region (e.g., CH1, CH2, CH3) in the constant region of the antibody, and may preferably be a position 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, which corresponds 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, which corresponds 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, which corresponds 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, which corresponds 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, human I (corresponding to residue 87 of the gG CH2 region (SEQ ID NO:1)).

[0035] According to the present invention, specific amino acid residues in the target region can be modified with high position selectivity. Such position selectivity 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.

[0036] The target region may also be one in which a specific amino acid residue at a specific position does not contain any amino acid residue of the same type as the specific amino acid residue, except for the specific amino acid residue at the specific position, in a region that is a number (here, a is any integer from 1 to 10) of amino acid residues away from the specific position on the N-terminal and C-terminal sides. 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.

[0037] In a preferred embodiment, the antibody is a monoclonal antibody. Examples of isotypes of antibodies such as monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. The monoclonal antibody may be a full-length antibody or an antibody fragment (e.g., F(ab')2, Fab', Fab, Fv, single-chain antibody), with full-length antibodies being preferred.

[0038] The antibody is an antibody against any antigen. For example, such an antigen may be a component found in an organism or virus as described above. Such antigens may also be, for example, proteins (including oligopeptides and polypeptides, or proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low molecular weight compounds.

[0039] Preferably, the antibody may be an antibody against a protein as an antigen, for example, 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.

[0040] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of the disease target protein include the following:

[0041] (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 receptor), factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, Frizzled 7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL -8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfelin receptor, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Luis blood group B antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 inhibitory protein, TAG-72 (CA72-4), CD70

[0042] (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

[0043] (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

[0044] (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

[0045] (5) Genetic and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0046] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0047] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0048] (8) Hematological diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0049] (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

[0050] In a more preferred embodiment, the affinity substance for an antibody is an affinity substance for a monoclonal antibody. The isotype of the monoclonal antibody is the same as that described above for the antibody, with IgG (e.g., IgG1, IgG2, IgG3, IgG4) being preferred. Preferably, the monoclonal antibody is a full-length monoclonal antibody.

[0051] In an even more preferred embodiment, the affinity substance for an antibody 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.).

[0052] In a particularly preferred embodiment, the affinity substance for an antibody comprises any one of the Fc region proteins 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.

[0053] The amino acid sequence of SEQ ID NO: 1 is an Fc region protein. Such Fc region proteins are known to have secretory ability. Therefore, the Fc region proteins of (A) to (C) above can have secretory ability. Furthermore, an antibody containing such an Fc region protein can have antigen binding ability. 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 non-polar 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 non-polar side chain or an acidic amino acid residue, and even more preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO: 1 can 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, 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 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.

[0054] In a preferred embodiment, the amino acid sequence of SEQ ID NO:1 may be an amino acid sequence consisting of amino acid residues at positions 220 to 449 in the amino acid sequence of SEQ ID NO:2.

[0055] 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.

[0056] In a specific embodiment, the antibody comprising the Fc region protein comprising the amino acid sequence as described above may be an antibody comprising the Fc region protein comprising the amino acid sequence as described above and an antibody constant region, which may be a chimeric antibody, a humanized antibody, or a human antibody (e.g., IgG such as IgG1, IgG2, IgG3, IgG4, etc.).

[0057] In the Fc region protein (B), one or several amino acid residues may be modified by one, two, three or four types of mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. Mutations of amino acid residues may be introduced into one region in the amino acid sequence, or into several different regions. The term "one or several" refers to the number that does not significantly impair the activity of the protein. The number referred to 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).

[0058] In the case of the Fc region protein (C), the percent identity with the amino acid sequence of SEQ ID NO: 1 may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In the present invention, the percent identity of a peptide or polypeptide (protein) can be calculated by the algorithm blastp. More specifically, the percent identity of a polypeptide can be calculated by the algorithm blastp provided by the National Center for Biotechnology Information (NCBI) using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11 Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment). The percent identity of a polynucleotide (gene) can be calculated by the algorithm blastn. More specifically, the percent identity of polynucleotides can be calculated using the algorithm blastn provided by NCBI with default Scoring Parameters (Match / Mismatch Scores=1, -2; Gap Costs=Linear).

[0059] The term "secretion" in the secretory ability has the same meaning as the secretion of secretory proteins (so-called soluble). Therefore, "having secretory ability" means that it functions as an antibody, like a normal antibody.

[0060] The antibody containing the Fc region protein may have a mutation introduced at a specific site as long as it retains the desired properties (e.g., secretion ability, antigen binding ability). The position of the amino acid residue into which the mutation may be introduced, which can retain the desired properties, is clear to a person skilled in the art. Specifically, a person skilled in the art can 1) compare the amino acid sequences of multiple proteins having similar properties, 2) clarify the relatively conserved regions and the relatively non-conserved regions, and then 3) predict the regions that may play an important role in function and the regions that may not play an important role in function from the relatively conserved regions and the relatively non-conserved regions, respectively, and thus recognize the correlation between structure and function. Thus, a person skilled in the art can identify the position of the amino acid residue into which the mutation may be introduced in the amino acid sequence of the antibody containing the Fc region protein.

[0061] 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 beta-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.

[0062] Examples of antibodies comprising any one of the Fc regions selected from the group consisting of (A) to (C) above include chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab), humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, efalizumab (IgG2), efalizumab (IgG4), ... rotuzumab, daratumumab, ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab), human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (if no IgG subtype is mentioned, it refers to IgG1).

[0063] In the present invention, the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus. By using a polypeptide containing a glutamine residue (Q) at the N-terminus as an affinity substance for an antibody, the amino group (-NH2) in the N-terminal glutamine residue (Q) can be protected by reacting with the amide group present in the side chain (pyroglutamylation). Therefore, a linker that mediates the binding of an affinity substance to an antibody can be used. The carboxyl group can be specifically added to a specific amino acid residue (a lysine residue having an amino group) in the affinity substance.

[0064] Examples of polypeptides containing a glutamine residue (Q) at the N-terminus include polypeptides in which a glutamic acid residue or a peptide containing a glutamic acid residue at the N-terminus has been added to the N-terminus of an affinity polypeptide for an antibody. Various affinity polypeptides for antibodies have been reported (e.g., International Publication No. 2016 / 186206; International Publication No. 2013 / 027796; International Publication No. 2008 / 054030; International Publication No. 2001 / 045746; Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, VOL. 6, 564-569; Ehrlich GK et al., J. Biochem. Biophys. Methods, 2001, VOL. 49, 443-454; Ruvo M et al., ChemBioChem, 2005, VOL. 6, 1242-1253; Krook M et al., Journal of Immunological Methods, 1998, VOL. 221, 151-157; Jacobs JM et al., J. Biochem. Biophys. Methods, 2001, VOL. 49, 443-454; al.,Bio.Techniques,2003,VOL.34,132-141;Carbonell RG et al.,Journal of Chromatography A,2009,VOL.1216,910-918;Lund LN et al., Journal of Chromatography A, 2012, VOL. 1225, 158-167; Warren L. Delano et al., Science, 2000, VOL. 287, 1279-1283; Biochemical Engineering Journal, 2013, VOL. 79, 33-40).

[0065] As the polypeptide containing a glutamine residue (Q) at the N-terminus, it is preferable to use a polypeptide that can be prepared by a method for secreting and producing a polypeptide using a coryneform bacterium as a host (International Publication No. 2013 / 062029). This method can add the N-terminal three residues Gln-Glu-Thr (QET) of a Csp mature protein to the N-terminus of a target polypeptide, and can easily prepare a large amount of a polypeptide containing a glutamine residue (Q) at the N-terminus, so it is suitable for preparing an affinity substance for an antibody. Examples of coryneform bacteria that can be used in this method include bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum, Corynebacterium stathionis) and bacteria of the genus Brevibacterium.

[0066] The number of amino acid residues of a polypeptide containing a glutamine residue (Q) at the N-terminus is not particularly limited, but may be 16 to 123 in terms of secretory production efficiency in a method for secretory production of a polypeptide using a coryneform bacterium as a host. Preferably, such number of amino acid residues may be 20 or more, 25 or more, 30 or more, 35 or more, 37 or more, or 40 or more. Such number of amino acid residues may also be 73 or less, 68 or less, 64 or less, 61 or less, 58 or less, or 53 or less.

[0067] In certain embodiments, the polypeptide comprising a glutamine residue (Q) at the N-terminus may be selected from the group consisting of (1) to (4) below: [ka] [Q is an N-terminal glutamine residue, E is a glutamic acid residue, T is a threonine residue, the solid line is a bond, the spacer portion is a portion consisting of 1 to 50 amino acid residues, and the affinity substance for the antibody is a portion consisting of 15 to 70 amino acid residues.]

[0068] In the above polypeptides (1) to (4), the number of amino acid residues in the spacer portion is preferably from 1 to 40, more preferably from 1 to 30, even more preferably from 1 to 20, and particularly preferably from 1 to 10. Any amino acid residue can be used as the amino acid residue, but the 20 standard L-α-amino acids constituting natural proteins are preferred.

[0069] In the above polypeptides (1) to (4), the number of amino acid residues in the affinity polypeptide portion for an antibody is 15 to 70, and may be preferably 15 or more, 17 or more, 20 or more, 25 or more, 30 or more, 34 or more, 35 or more, or 40 or more, and may be preferably 65 or less, 61 or less, 60 or less, 55 or less, or 50 or less. In the above polypeptides (1) to (4), the affinity polypeptide portion for an antibody may be any polypeptide having affinity for an antibody (e.g., the above-mentioned previously reported polypeptides), and is preferably one selected from the group consisting of Protein A, Protein G, Protein L, Protein Z, fragments thereof having affinity for an antibody, and mutants thereof having affinity for an antibody.

[0070] Protein A is a non-glycosylated protein (Staphylococcus aureus Protein A: SpA) present in the cell wall of Staphylococcus aureus. It is known that multiple domains of Protein A (domains A, B, C, D, and E) each have affinity for antibodies (e.g., Moks T et al., Eur J Biochem. 1986 May 2; 156(3): 637-43). Therefore, a polypeptide corresponding to such a domain can be used as a fragment of Protein A. More specifically, a polypeptide corresponding to such a domain has the following amino acid sequence (58 to 61 amino acid residues): (1) Domain A: ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNESQAPK (SEQ ID NO: 27) (2) Domain B: ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 28) (3) Domain C: ADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 29) (4) Domain D: ADAQQNKFNKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQAPK (SEQ ID NO: 30) (5) Domain E: AANAAQHDEAQQNAFYQVLNMPNLNADQRNG FIQSLKDDPSQSANVLGEAQKLNDSQAPK (SEQ ID NO: 31)

[0071] Protein G is a protein present in the cell wall of group G streptococci. It is known that multiple domains of protein G (domains C2 and C3) each have affinity for antibodies (e.g., Sjobring UJ et al., Biol Chem. 1991 Jan 5; 266(1): 399-405). Therefore, a polypeptide corresponding to such a domain can be used as a fragment of protein G. More specifically, the polypeptide corresponding to such a domain has the following amino acid sequence (60 amino acid residues): (1) Domain C2: TPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 32) (2) Domain C3: TPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE (SEQ ID NO: 33)

[0072] Protein L is a protein derived from Peptostreptococcus magnus. It is known that several domains of protein L (domains C, C1-C4, B1-B5) have affinity for antibodies (e.g., Graille M el al., Structure. 2001 Aug;9(8):679-87). Therefore, a polypeptide corresponding to such a domain can be used as a fragment of protein L. More specifically, a polypeptide corresponding to such a domain has the following amino acid sequence (61 amino acid residues): (1) Domain C: EVTIKVNLIFADGKIQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGNHMNIKFA (SEQ ID NO: 34) (2) Domain C1: EVTIKANLIFADGSTQNAEFKGTFAKAVSDAYAYADALKKDNGEYTVDVADKGLTLNIKFA (SEQ ID NO: 35) (3) Domain C2: EVTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFA (SEQ ID NO: 36) (4) Domain C3: EVTIKVNLIFADGKIQTAEFKGTFEEATAKAYAYANLLAKENGEYTADLEDGGNTINIKFA (SEQ ID NO: 37) (5) Domain C4: EVTIKVNLIFADGKTQTAEFKGTFEEATAEAYRYADLLAKVNGEYTADLEDGGYTINIKFA (SEQ ID NO: 38) (6) Domain B1: EVTIKANLIFANGSTQTAEFKGTFEKATSEAYAYADTLKKDNGEYTVDVADKGYTLNIKFA (SEQ ID NO: 39) (7) Domain B2: EVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADALKKDNGEYTVDVADKGYTLNIKFA (SEQ ID NO: 40) (8) Domain B3: EVTIKANLIYADGKTQTAEFKGTFEEATAEAYRYADLLAKENGKYTVDVADKGYTLNIKFA (SEQ ID NO: 41) (9) Domain B4: EVTIKANLIYADGKTQTAEFKGTFAEATAEAYRYADLLAKENGKYTADLEDGGYTINIRFA (SEQ ID NO: 42) (10) Domain B5: QVTIKENIYFEDGTVQTATFKGTFAEATAEAYRYADLLSKEHGKYTADLEDGGYTINIRFA (SEQ ID NO: 43)

[0073] Protein Z is an artificial IgG-binding polypeptide designed based on the above Protein A (e.g., Nilsson B et al., Protein Eng. 1987 Feb-Mar;1(2):107-13). Protein Z has the following amino acid sequence (58 amino acid residues): Z34C, like Protein Z, is an artificial IgG-binding polypeptide designed based on the above Protein A and consisting of a total of 34 amino acid residues (e.g., Starovasnik MA et al., Proc Natl Acad Sci (2012) 143:1111-1123). cad Sci US A. 1997 Sep 16;94(19):10080-5). Although Z34C has multiple amino acid residue substitutions introduced into it to increase the affinity to antibodies, it has a shorter total amino acid length than protein Z, and therefore can be said to correspond to a fragment of protein Z. Therefore, a truncated polypeptide of protein Z (e.g., an N-terminally and / or C-terminally truncated polypeptide consisting of at least 34 amino acid residues) and Z34C can be used as a fragment of protein Z. (1) Protein Z: VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 44) (2) Z34C:FNMQCQRRFYEALHDPNLNEEQRNAKIKSIRDDC (SEQ ID NO: 45)

[0074] A fragment of Protein A, Protein G, Protein L, or Protein Z having affinity for an antibody is a truncated polypeptide in which the N-terminal and / or C-terminal amino acid residues of Protein A, G, L, or Z are deleted. It is known that Protein A, G, L, or Z does not require all of its constituent amino acid residues for affinity to an antibody, and that it has affinity for an antibody even when the N-terminal and / or C-terminal amino acid residues are deleted (e.g., those having the above-mentioned domains). Therefore, a person skilled in the art can appropriately prepare a fragment of Protein A, G, L, or Z having affinity for an antibody by preparing a truncated polypeptide in which the terminal and / or C-terminal amino acid residues of Protein A, G, L, or Z are deleted, and then evaluating whether the prepared truncated polypeptide has affinity for an antibody.

[0075] Examples of mutants of Protein A, Protein G, Protein L, or Protein Z, or fragments thereof, that have affinity for antibodies include homologous polypeptides that have 60% or more amino acid sequence identity to Protein A, Protein G, Protein L, or Protein Z, or fragments thereof, and have affinity for antibodies. It is known that even homologous polypeptides that have such a degree of amino acid sequence identity have affinity for antibodies (see, for example, Starovasnik MA et al., Proc Natl Acad Sci US A. 1997 Sep 2001). 16;94(19):10080-5). The amino acid sequence identity may be preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more). The amino acid sequence identity can be calculated in the same manner as the % identity of the polypeptide described above.

[0076] In a specific embodiment, the antibody affinity polypeptide portion is a fragment of protein A having affinity for an antibody or a variant thereof. Preferably, the fragment of protein A having affinity for an antibody or a variant thereof may include an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5, in which any one amino acid residue in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNAXIXSIRDDC (SEQ ID NO: 5) (each of the two Xs is independently an amino acid residue selected from the group consisting of an arginine residue, a glutamic acid residue, and a glutamine residue) is replaced with a lysine residue (the replacement with a lysine residue is maintained in the amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 5). In other words, such a polypeptide portion can be said to be (i) a polypeptide portion in which any one amino acid residue in the amino acid sequence of SEQ ID NO: 5 is replaced by a lysine residue, or (ii) a polypeptide portion in which any one amino acid residue in the amino acid sequence of SEQ ID NO: 5 is replaced by a lysine residue and 1 to 3 amino acid residues other than the lysine residue are replaced by other amino acid residues or are inserted or added. Preferably, the amino acid sequence of SEQ ID NO: 5 is It may also be the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO:6).

[0077] In a preferred embodiment, the affinity polypeptide for an antibody is a mutant of a fragment of Protein A having affinity for an antibody, to which a glutamine residue (Q) or QET has been added at the N-terminus. Such mutants include, for example, the following: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 8); (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 9); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 52); and (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (sequence number 53).

[0078] In another specific embodiment, the affinity polypeptide for the antibody may be a modified peptide (modified Fc-III) of peptide Fc-III (WO2001 / 045746) consisting of the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 54), which has been reported to have binding ability to human IgG. Such modified Fc-III may be, for example, the following: (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (sequence number 25).

[0079] At least two cysteine ​​residues spaced apart in each amino acid sequence of the affinity polypeptide can form a cyclic peptide through a disulfide bond. Alternatively, the sulfide groups in the two cysteine ​​residues in the peptide can be linked by a carbonyl group-containing linker as shown below.

[0080] [ka]

[0081] The dashed line portion of the carbonyl group-containing linker shown above means the bond portion with the sulfide group. The linker is more stable against reduction reactions and the like than a normal disulfide bond. Such a peptide can be prepared, for example, by the method described in WO 2016 / 186206.

[0082] Such novel peptides having a specific structure are useful for site-selective modification of Lys248 or Lys246 residues according to Eu numbering in human IgG Fc, or other amino acid residues other than Lys248 or Lys246 residues (Examples). The amino acids constituting the above peptides may be in either L- or D-form, but L-form is preferred (in the Examples, all amino acid residues constituting the peptides are in L-form).

[0083] The peptide may be modified at specific amino acid residues with a crosslinking agent. Examples of such specific amino acid residues include lysine residues, aspartic acid residues, and glutamic acid residues, and preferably lysine residues. Examples of the crosslinking agent include crosslinking agents containing preferably two or more succinimidyl groups, such as DSG (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate), crosslinking agents containing preferably two or more imide acid moieties, such as DMA (dimethyl adipimidate·2HCl), DMP (dimethyl pimelimidate·2 HCl), and DMS (dimethyl suberimidate·2 HCl), as well as crosslinking agents having an SS bond, such as DTBP (dimethyl 3,3'-dithiobispropionimidate·2HCl) and DSP (dithiobis(succinimidyl propionate)) (e.g., WO 2016 / 186206).

[0084] The peptide may have terminal amino and carboxy groups protected. Examples of the protecting group 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, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). The protecting group for the N-terminal amino group is preferably an acetyl group. Examples of the protecting group for the C-terminal carboxy group include groups capable of forming esters or amides. Examples of the groups capable of forming esters or amides include alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, and hexyloxy), aryloxy groups (e.g., phenyloxy and naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups. The protecting group for the C-terminal carboxy group is preferably an amino group.

[0085] 1-3. Linker (L) In formula (I), L is a cleavable linker, which is a divalent group that includes a cleavable moiety.

[0086] 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 do not 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 physicochemical stimuli selected from the group consisting of light, or (c) leaving the cleavable linker containing a self-degrading cleavable moiety. Such cleavable linkers and the cleavage conditions thereof are common technical knowledge in the field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Jounal of American Chemical Society. 132, 1500). (2010).;Bessodes M. et al.,Journal of Controlled Release, 99,423(2004).;DeSimone,JM,Journal of American Chemical Society.132,17928(2010);Thompson,DH,Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)). 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 electron-withdrawing groups, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0087] 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 and 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).

[0088] 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.

[0089] Examples of the ester residue include normal 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 esters, 2-(diphenylphosphino)benzoate), glycosyl ester residues, orthoester residues], ester residues containing a sulfur atom and an oxygen atom (e.g., thioester residues such as α-thiophenyl ester residues and alkylthioester residues), ester residues containing a phosphorus atom and an oxygen atom (e.g., phosphodiester residues, phosphotriester residues), and activated ester residues (e.g., N-hydroxysuccinimide residues).

[0090] Examples of the sulfone-containing residue include a sulfone residue and a quinolinylbenzenesulfonate residue.

[0091] 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).

[0092] 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 thereof include methoxymethyl residue, ethoxy ... Examples of aryl groups include, but are not limited to, oxymethyl, methoxyethyl, and ethoxyethyl residues.

[0093] The unsaturated bond-containing chain residue is a residue containing an unsaturated bond portion 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 portion consisting of carbon atoms and heteroatoms (e.g., nitrogen atoms, sulfur atoms, oxygen atoms) (e.g., aldehyde, cyano). Examples of the unsaturated bond-containing chain residue include vinyl ether residues, cyanoethyl residues, ethylene residues, and malondialdehyde residues.

[0094] Examples of acidic substances (also called 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 dihydrogen phosphate, 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.

[0095] 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 hydroxylamine, 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.

[0096] Examples of reducing agents include cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Examples of sites that can be cleaved by a reducing agent include disulfide residues, alkoxyalkyl residues, and azo residues.

[0097] Examples of the oxidizing agent include sodium periodate and oxidized glutathione. Examples of the site that can be cleaved by the oxidizing agent include vicinal diol residues and selenium residues.

[0098] Examples of the enzyme include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloprotease, lipase, endoglycosidase, and PNase F. Examples of the site cleavable by the enzyme include ester residues, phosphodiester residues, and glycosyl residues.

[0099] Examples of the site cleavable by light include a 2-nitrobenzyl residue, a phenacyl ester residue, an 8-quinolinebenzenesulfonate residue, a coumarin residue, a phosphotriester residue, a bisarylhydrazone residue, and a bimandithiopropionic acid residue.

[0100] Autolytic cleavable moieties include, for example, activated ester residues (eg, N-hydroxysuccinimide residues).

[0101] More specifically, the cleavable moiety has any one of the following chemical structures: It may correspond to: [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; A plurality of R2a, a plurality of R2b, and a plurality of R2c are the same or different and are selected from the group consisting of a hydrogen atom or a substituent described later; J is -CH2-, -O-, or -S-; r is any integer from 1 to 4; A white circle (◯) indicates a bond to A (or La described below), and a black circle (●) indicates a bond to B (or Lb described below). When the chemical structure is asymmetric about the cleavage site, the black circle may represent a bond to A (or La, described below) and the open circle may represent a bond to B (or Lb, described below).

[0102] J is -CH2-, -O-, or -S-. j is preferably -CH2- or -O-, more preferably -CH2-.

[0103] r is any integer from 1 to 4, preferably any integer from 1 to 3, and more preferably 1 or 2.

[0104] In one embodiment, the cleavable linker can be (i) a divalent group that includes a cleavable moiety that has the ability to generate a bioorthogonal functional group on a reactive group upon cleavage, or (ii) a divalent group that includes a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on a reactive group upon cleavage.

[0105] The cleavable moiety of (i) can be, for example, a disulfide residue, an ester residue, or an acetal. residues, ketal residues, imine residues, and vicinal diol residues.

[0106] More specifically, the cleavable moiety of (i) can be, for example, [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; Each of R2a is the same or different and is selected from the group consisting of a hydrogen atom or a substituent described later, A white circle (◯) indicates a bond to A (or La described below), and a black circle (●) indicates a bond to B (or Lb described below). When the chemical structure is asymmetric with respect to 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).

[0107] 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.

[0108] More specifically, the cleavable moiety in (ii) can be, for example, [ka] [wherein the wavy lines perpendicular to the bonds indicate the cleavage sites; A plurality of R2b, a plurality of R2c, J, and r are selected from the group consisting of a hydrogen atom or a substituent described later, A white circle (◯) indicates a bond to A (or La described below), and a black circle (●) indicates a bond to B (or Lb described below). When the chemical structure is asymmetric with respect to 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).

[0109] In certain embodiments, the cleavable linker (L) may be represented by any one of the following formulas (L1)-(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.

[0110] 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)-, -NRa- (Ra represents a hydrogen atom or a substituent), -O-, -S-, -C(=S)-, and a group consisting of a combination of two or more of these (for example, 2 to 8, preferably 2 to 6, and more preferably 2 to 4).

[0111] The divalent hydrocarbon group is a straight-chain, branched-chain, or cyclic divalent hydrocarbon group, and is preferably a straight-chain or branched-chain divalent hydrocarbon group. Examples of the divalent hydrocarbon group include alkylene, alkenylene, alkynylene, and arylene.

[0112] 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.

[0113] 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 of the substituent is not included in the number of carbon atoms. The alkenylene may be linear, branched, or cyclic, but linear alkenylene is preferred. Examples of such alkenylene include ethylenylene, propynylene, butenylene, pentenylene, and hexenylene.

[0114] 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 of the substituent is not included in the number of carbon atoms. The alkynylene may be linear, branched, or cyclic, but linear alkynylene is preferred. Examples of such alkynylene include ethynylene, propynylene, butynylene, pentynylene, and hexynylene.

[0115] 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 the substituent. Examples of the arylene include phenylene, naphthylene, and anthracenylene.

[0116] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocycle, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom and a nitrogen atom.

[0117] The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 1 to 21 carbon atoms, more preferably a divalent aromatic heterocyclic group having 1 to 15 carbon atoms, even more preferably a divalent aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably a divalent aromatic heterocyclic group having 1 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. More specifically, examples of the divalent aromatic heterocyclic group include pyrrolidinediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolidinediyl, thiophenediyl, pyridinedi ... Examples of the aryl group include aryldiyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl.

[0118] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 21 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, even more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The above number of carbon atoms does not include the number of carbon atoms of the substituent. More specifically, examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolindiyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl.

[0119] 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 represented by Ra and Rb above. Examples of such substituents include the following: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) aralkyl; (iv) a monovalent heterocyclic group; (v) Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc represents a hydrogen atom or a monovalent hydrocarbon group); or (vi) NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group); (vii) Nitro, sulfate, sulfonate, cyano, and carboxyl groups.

[0120] Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0121] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0122] The monovalent chain-like hydrocarbon group means a hydrocarbon group that is composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain-like hydrocarbon group include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0123] The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of the substituent. Examples of the alkyl group 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. Can be obtained.

[0124] 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 the substituent. Examples of the alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0125] 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 does not include the number of carbon atoms of the substituent. Examples of the alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0126] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0127] The monovalent alicyclic hydrocarbon group means a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it is not necessary to be composed only of alicyclic hydrocarbons, and it may contain a chain structure as a part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0128] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0129] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0130] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 to 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of the cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0131] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0132] The monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it is not necessary to be composed of only an aromatic ring, and it may contain a chain structure or an alicyclic hydrocarbon as a part thereof, and the aromatic ring may be either a monocyclic or polycyclic ring. As the monovalent aromatic hydrocarbon group, an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is even more preferable. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

[0133] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0134] Among these, the monovalent hydrocarbon group is preferably alkyl, cycloalkyl, or aryl, and more preferably alkyl.

[0135] The aralkyl refers to an arylalkyl. The definitions, examples and preferred examples of aryl and alkyl in the arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such an aralkyl include benzoyl, phenethyl, naphthylmethyl and naphthylethyl.

[0136] The monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. As the heteroatom constituting the heterocyclic group, it is preferable to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably to include at least one selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom.

[0137] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0138] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. The number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0139] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0140] 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') Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc is a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (vi') NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); (vii') The same groups as those enumerated in (vii) above.

[0141] More preferably, the substituents may be: (i'') a halogen atom; (ii'') alkyl having 1 to 12 carbon atoms; (iii'') Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc is a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or (iv'') NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); (v'') The same groups as those listed in (vii) above.

[0142] Even more preferably, the substituents may be: (i'') halogen atoms; (ii''') alkyl having 1 to 6 carbon atoms; (iii''') Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); or (iv''') NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms); (v''') The same groups as those listed in (vii) above.

[0143] Particularly preferably, the substituents may be: (i'''') halogen atoms; (ii'''') alkyl having 1 to 4 carbon atoms; (iii'''') Rc-O-, Rc-C(=O)-, Rc-OC(=O)-, or Rc-C(=O)-O- (Rc is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); or (iv'''') NRdRe-, NRdRe-C(=O)-, NRdRe-C(=O)-O-, or Rd-C(=O)-NRe- (Rd and Re are the same or different and each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms); (v'''') The same groups as those listed in (vii) above.

[0144] In certain embodiments, La and Lb are, respectively, (La') and (Lb') as follows: [ka] [During the ceremony, p and p' are the same or different and each is an integer of 0 to 10, q and q' are the same or different and each is an integer of 0 to 10, X and X' are the same or different and each is a carbon atom, a nitrogen atom, or a single bond (wherein, when X is a nitrogen atom, R1b does not exist, and when X' is a nitrogen atom, R1b' does not exist; when X is a single bond, R1a and R1b do not exist, and when X' is a single bond, R1a' and R1b' do not exist); R1a, R1b, R1a' and R1b' may be the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents.

[0145] 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.

[0146] q and q' 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, q and q' are the same.

[0147] X and X' are the same or different and are a carbon atom, a nitrogen atom, or a single bond, preferably a carbon atom or a single bond. Preferably, X and X' are the same.

[0148] R1a, R1b, R1a' and R1b' are the same or different and are selected from the group consisting of a hydrogen atom or a substituent described below. The definition, examples and preferred examples of the substituent are as described above. Preferably, R1a, R1b, R1a' and R1b' are hydrogen atoms.

[0149] 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 that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group.

[0150] A bioorthogonal functional group refers to a group that does not react with biological components (e.g., amino acids, nucleic acids, lipids, sugars, phosphates) or reacts slowly with biological components, but selectively reacts with components other than biological components. Bioorthogonal functional groups are well known in the art (e.g., see Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

[0151] When the target of the affinity substance is an antibody, the bioorthogonal functional group is a bioorthogonal functional group for a protein. A bioorthogonal functional group for a protein is a group that reacts with a predetermined functional group without reacting with the side chains of the 20 natural amino acid residues that make up proteins. The 20 natural amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Among these 20 naturally occurring amino acids, glycine, which has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, which have a hydrocarbon side chain (i.e., do not contain a heteroatom selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inactive to normal reactions. Therefore, the bioorthogonal functional group for proteins is a functional group that cannot react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine in addition to the side chains of these amino acids that have side chains that are inactive to normal reactions.

[0152] 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 residues, α-halocarbonyl residues (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α position; the same applies below), isonitrile residues, sydnone residues, and selenium residues. 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, thiols function as bioorthogonal functional groups. Therefore, since the target of the affinity substance is an antibody that may not contain free thiols, the bioorthogonal functional group contains a thiol. One or more (e.g., two, three, or four) types of bioorthogonal functional groups may be included in the bivalent group, but preferably one type of bioorthogonal functional group may be included in the bivalent group.

[0153] 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 groups, 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 residues, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues.

[0154] 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.

[0155] More specifically, the bioorthogonal functional group may correspond to any one of the chemical structures selected from the group consisting of: [ka] [During the ceremony, R1f, one or more R1g, and one or more R1h are the same or different and each is an atom or group selected from the group consisting of (i) to (vii), or an electron-withdrawing group; · is a bond.

[0156] Examples of the electron-withdrawing group include those mentioned above, with halogen atoms, boronic acid residues, mesyl, tosyl and triflate being preferred.

[0157] 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 is single or multiple, for example, 1 to 5, preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. When the number of bioorthogonal functional groups contained in the divalent group is multiple, the multiple bioorthogonal functional groups may be the same or different, but from the viewpoint of adopting a simple structure, it is preferable that they are the same.

[0158] In certain embodiments, B may be (a1) a divalent group containing a bioorthogonal functional group in the main chain. The divalent group containing a bioorthogonal functional group in the 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 residues, α-halocarbonyl residues, isonitrile residues, sydnone residues, and selenium residues, and is a group in which the above-mentioned divalent group is linked to either one end 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 above-mentioned divalent group.

[0159] In another specific embodiment, B may be (a2) a divalent group having a bioorthogonal functional group in a side chain. The divalent group having a bioorthogonal functional group in a side chain is an azide residue, an aldehyde residue, A divalent group substituted with a bioorthogonal functional group selected from the group consisting of 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, or a group containing the same. The definition, examples, and preferred examples of the divalent group to be substituted are the same as those of the divalent group described above.

[0160] In another embodiment, B may be a divalent group not including (b) 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, -NRa- (Ra 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 in the optionally substituted cases and the substituents of Ra are substituents other than the bioorthogonal functional group. Examples of such substituents include alkyl, cycloalkyl, aralkyl, monovalent heterocyclic group, 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.

[0161] Regarding 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.

[0162] Regarding the substituents other than the bioorthogonal functional group, the definition, examples and preferred examples of the alkyl in the alkyloxy (alkoxy), the cycloalkyl in the cycloalkyloxy, and the aralkyl in the aralkyloxy are as described above. More specifically, the alkyloxy includes, for example, 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). The cycloalkyloxy includes, for example, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. The aralkyloxy includes, for example, benzoyloxy, phenethyloxy, naphthylmethyloxy, and naphthylethyloxy.

[0163] A divalent group that does not contain a bioorthogonal functional group may also be a group that is highly inert to the reaction. Thus, such a divalent group may be a group composed only of carbon atoms and hydrogen atoms. Such a divalent group is an alkylene, a cycloalkylene, or an aryl, and a combination of two or more of these (e.g., 2 or 3). 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 an alkylene, a cycloalkylene, and an aryl as a substituent that is highly inert to the reaction. The number of substituents that are highly inert to the reaction is, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

[0164] In certain embodiments, 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 is -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 from 0 to 10, The circles and circles in formula (B-2) are the same orientation as the circles and circles 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 moiety on the L side, and ● (black circle) indicates a bond to the moiety on the R side.

[0165] 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 a group represented by the above formula (B-2).

[0166] Z is an oxygen atom, a sulfur atom, or a hydrogen atom, and is preferably an oxygen atom or a sulfur atom.

[0167] V and V' are -NH-, -O-, -CH2- or a single bond, preferably -CH2- or a single bond.

[0168] 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.

[0169] 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, but 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, but 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 bioorthogonal functional groups, for example, 1 to 3, more preferably 1 or 2, and even more preferably 1. When the number of bioorthogonal functional groups contained in the divalent group is more than one, the multiple bioorthogonal functional groups may be the same or different. From the viewpoint of adopting a simple structure and improving reactivity, it is preferable that they are the same. From the viewpoint of ensuring differentiated reactions, it is preferable that they are different. V1 may also have (b) 1 to 5, preferably 1 to 3, and more preferably 1 or 2 substituents.

[0170] 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.

[0171] 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—, —CH2-, 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 is -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, R1f, one or more R1g, and one or more R1h are the same or different and each is an atom or group selected from the group consisting of (i) to (vii), or an electron-withdrawing group; · is a bond. ) is any one of the groups represented by. ) may be a divalent group having a group represented by as a side chain. Such a divalent group is a divalent hydrocarbon group or a divalent heterocyclic group, preferably a divalent hydrocarbon group which may be substituted, more preferably an alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, or arylene, even more preferably an alkylene, cycloalkylene, or arylene, and particularly preferably an 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, more preferably 1 or 2. Examples and preferred examples of the substituents are as described above.

[0172] G and G' are the same or different and are -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 be -NH-, -O-, -CH2-, or a single bond. Alternatively, from the viewpoint of designing a structure based on carbon atoms, G and G' may be -CH2-, a single bond, or a group represented by the above formula (B-4).

[0173] H is -CH2-, -C=O-, -C=S-, -NH-, or a single bond. Preferably, H is -CH2- or a single bond.

[0174] I is a divalent hydrocarbon group, a divalent heterocycle, or a single bond. The divalent hydrocarbon group and the divalent heterocycle may be substituted or unsubstituted with a substituent. The definitions, examples, and preferred examples of the divalent hydrocarbon group, the divalent heterocycle, and the substituent are the same as those described above in V1.

[0175] W and W' are the same or different and each is -NH-, -O-, -CH2-, or a single bond, preferably -CH2- or a single bond.

[0176] 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.

[0177] 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.

[0178] 1-5.Reactive group (R) In formula (I), R is a reactive group to an antibody. Such reactive groups are common knowledge in the art.

[0179] The reactive group may be the same or different from the bioorthogonal functional group.

[0180] 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 to the antibody is not ensured. In addition, the bioorthogonal functional group is a group that cannot react with the side chains of the 20 natural amino acid residues that constitute the antibody.

[0181] More specifically, among the 20 naturally occurring amino acids constituting proteins as described above, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, which have a hydrocarbon side chain, are inactive in normal reactions. Therefore, the reactive group for proteins is a group that can react with one or more (e.g., two, three, or four) side chains of 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), but preferably, one reactive group may be contained in the compound represented by formula (I).

[0182] Preferably, the reactive group is a group capable of reacting with the side chain of any one of the above-mentioned 14 types of amino acids that constitute proteins.

[0183] 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.

[0184] 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.

[0185] Also, 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.

[0186] The reactive group specific to the side chain of a lysine residue is a group capable of specifically reacting with an amino group (NH2) present in the side chain of a lysine residue, and examples thereof include activated ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethane residues, and the like. Examples of the terephthalic acid residue include a terephthalic acid residue and a diazonium terephthalic acid residue.

[0187] Linking moieties formed by the reaction between the above-mentioned reactive group specific for the side chain of a lysine residue and the amino group (NH2) present in the side chain of a lysine residue include, for example, amide residues, urea residues, pyridine residues, carbamate residues, and sulfonamide residues.

[0188] More specifically, the reactive group specific for the side chain of a lysine residue may correspond to any one of the chemical structures selected from the group consisting of: [ka] [Where: R5a and R5c are hydrogen atoms or the above-mentioned substituents; R5b is an electron withdrawing group; j is an integer from 1 to 5; and k is an integer of 1 to 4.

[0189] R5a and R5c are hydrogen atoms or the above-mentioned substituents. Definitions of the substituents, e.g. And preferred examples are the same as those mentioned above.

[0190] R5b is an electron-withdrawing group. Examples of such an electron-withdrawing group include those mentioned above, and are preferably a halogen atom, a boronic acid residue, mesyl, tosyl, or triflate.

[0191] j is an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 1 or 2.

[0192] k is an integer of 1 to 4, preferably an integer of 1 to 3, and more preferably 1 or 2.

[0193] 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 (NH2) present in the side chain of a lysine residue may correspond to any one of the chemical structures selected from the group consisting of: [ka] (Here, ● (black circle) indicates a bond to the part on the T side, and ○ (white circle) indicates a bond to the part on the B side. The straight line perpendicular to the bond indicates a bond formed by the reaction.)

[0194] The reactive group specific to the side chain of a tyrosine residue is a group that can specifically react with an 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 diazodicarboxylate residue, and a 2,3-dihydro-1H-pyrazin-6-one residue.

[0195] More specifically, the reactive group specific for the side chain of a tyrosine residue may correspond to any one of the chemical structures selected from the group consisting of: [ka] [wherein R4a is a hydrogen atom or a substituent as described above, and ○ indicates a bond to B.]

[0196] R4a 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.

[0197] 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 ortho atom of the phenolic hydroxyl group (OH) present in the side chain of a tyrosine residue, may correspond to any one of the chemical structures selected from the group consisting of: [ka] [wherein R4a is a hydrogen atom or a substituent as described above, ● indicates a bond to T, and ○ indicates a bond to B.]

[0198] R4a 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.

[0199] The reactive group specific to the side chain of a tryptophan residue is a group capable of specifically reacting 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]nonane-3-one-N-oxyl residue.

[0200] More specifically, the reactive group specific for the side chain of a tryptophan residue may correspond to any one of the chemical structures selected from the group consisting of: [ka] (wherein ○ indicates a bond to B.)

[0201] The linking moiety formed by the reaction between the above chemical structure, which is a group specifically reactive to 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 of the chemical structures selected from the group consisting of: [ka] (wherein ● indicates a bond to T and ○ indicates a bond to B.)

[0202] Particularly preferably, the reactive group may be a reactive group specific for the side chain of lysine.

[0203] 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) linking A (affinity substance) and R (reactive group) can be appropriately designed depending on various factors such as the types of antibody and affinity substance, and the relationship between the target site of the affinity substance in the antibody and the position and number of specific amino acid residues in the target region (e.g., specific position) as described above to which R should react and bind. In the compound represented by formula (I), the affinity substance is associated with the antibody, and then the reactive group covalently bonded to the affinity substance via LB reacts 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 in the vicinity of the target site. By this, it can be covalently bonded to the antibody. At this time, if there is no other specific amino acid residue in the vicinity of the specific amino acid residue to which R should react and bind, in the vicinity of the target site, and in the region between the specific amino acid residue and the target site, the reactive group can be site-selectively bonded to the specific amino acid residue without strictly controlling the length of the main chain. Of course, even if there is another specific amino acid residue in such a region, the reactive group can be site-selectively bonded to the specific amino acid residue by controlling the length of the main chain.

[0204] The length of the main chain linking A and R may vary depending on factors such as the type of antibody and the affinity substance therefor, and the relationship between the position and number of specific amino acid residues in the target site in the antibody, but may be about 5 Å or more, preferably about 7.5 Å, 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.

[0205] Incidentally, it is common 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 lengths between atoms in the table below.

[0206] [Table 1]

[0207] 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 may be, for example, 4 (about 5.0 Å) or more, preferably 6 (about 7.5 Å), and more preferably 8 (about 10.5 Å) or more. The number of atoms in the main chain may also be, for example, 20 (about 30 Å) or less, preferably 16 (about 23 Å) or less, and more preferably 12 (about 16.5 Å) or less. 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.

[0208] 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.

[0209] On the other hand, when the main chain is a structure containing 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 contain 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 (for example, see the bold 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), since both paths are the shortest paths (equidistant), 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), since the path to the condensation site is the shortest path, the number of atoms in the divalent ring structure counted as the number of atoms in the main chain is 4.

[0210] Preferably, the linking portion between A and R represented by LB (excluding the side chain) may be a chain structure that does not include 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 include a divalent ring group.

[0211] 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".

[0212] In one embodiment, the cleavable linker can be (i) a divalent group that includes a cleavable moiety that has the ability to generate a bioorthogonal functional group on a reactive group upon cleavage, or (ii) a divalent group that includes a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on a reactive group upon cleavage.

[0213] 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.

[0214] 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, etc., in (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 using some bioorthogonal functional groups in the reaction, the bioorthogonal functional group generated in (i) may be different from the bioorthogonal functional group in (a).

[0215] 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.

[0216] In another particular embodiment, when L is a cleavable linker as defined above in (ii), B is (a) a divalent group that includes a bioorthogonal functional group.

[0217] 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 the antibody having the functional substance of the present invention (e.g., antibody-drug conjugate) obtained by using the compound of the present invention does not contain a peptide moiety that may have immunogenicity as a linker.

[0218] In a particular embodiment, the partial structure represented by "LB" may have a symmetric structure (e.g., cis type (i.e., Z) or trans type (i.e., E)) based on the atom (e.g., when the number of atoms constituting the main chain is odd) at the center of the main chain (straight-chain portion in LB) connecting A and R, or the binding site (e.g., when the number of atoms constituting the main chain is even) at the center of the main chain. For example, the binding site 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 (e.g., a chain-like divalent group having an SH group at one end) having a functional group at one end that can react to form a cleavable moiety with each other.

[0219] 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 specified 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 that is a divalent group that contains 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.

[0220] B1 and B2 may be the same or different and have the same definition, examples and preferred examples as B.

[0221] 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.

[0222] 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.

[0223] The cleavable moiety represented by C' has the same definition, examples and preferred examples as the cleavable moiety represented by C.

[0224] In another specific embodiment, the structural unit represented by LB may be represented by the following formula (LB'): [ka] [During the ceremony, The definitions, examples and preferred examples of C, p, p', q, q', X, X', R1a, R1a', R1b, R1b', 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.

[0225] Therefore, the above formula (I) can be defined as the following formula (I″): [ka] [During the ceremony, The definitions, examples and preferred examples of A, R, C, p, p', q, q', X, X', R1a, R1b, R1a', R1b', Y, Y', Z and Z' are the same as those described above.

[0226] 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' 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.

[0227] 1-7. Manufacturing method The compound or salt thereof comprising an affinity agent for the antibody, a cleavable moiety and a reactive group is suitably The compound comprising an affinity substance for an antibody, a cleavable moiety and a reactive group is represented by formula (I), preferably formula (I'), more preferably formula (I'').

[0228] As the affinity substance, one having any functional group can be appropriately selected. 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 are the same or different) to prepare a structural unit represented by ALBR. For example, such a reaction can be carried out in a suitable reaction system, such as an organic solvent system or an aqueous solution system, at a suitable temperature (for example, about 15 to 200°C). The reaction system may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0229] 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 types 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.

[0230] The production of the affinity substance for the antibody, and the antibody or its salt containing a cleavable moiety can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw material and the molecular weight of the product. The affinity substance for the antibody, and the antibody 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).

[0231] 1-8.Other In the inventions described below [examples, inventions represented by formulas (II) to (v) and their subordinate formulas, and partial structural formulas (examples, (L1) to (L3), (La'), (Lb'), (B-1) to (B-4)], any symbols (e.g., A, L, B, R), terms represented by the 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 by cleavage), specific groups (e.g., bioorthogonal functional groups, divalent groups, alkyl groups, substituents, electron-withdrawing groups), and specific numerical values, as well as any technical elements such as salts (e.g., definitions, examples, and preferred examples) that can define the inventions described below can be common to those described above. Therefore, these matters can be appropriately incorporated in the inventions described below without special 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.

[0232] 2. An antibody or a salt thereof comprising an affinity substance for the antibody and a cleavable moiety 2-1. Overview The present invention provides an antibody or a salt thereof comprising an affinity substance for an antibody, and a cleavable moiety, represented by 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 contains a cleavable moiety; 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. is a divalent group not R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody.

[0233] 2-2. Moiety (R') generated by reaction between antibody and reactive group In the moiety generated by the reaction between an antibody and a reactive group, the definitions, examples, and preferred examples of the antibody and the reactive group are as described above. The moiety generated by the reaction between an antibody and a reactive group is common technical knowledge in the art, and can be appropriately determined depending on the types of the antibody and the reactive group.

[0234] Preferably, the moiety generated by the reaction between an antibody 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 an antibody and a reactive group therefor.

[0235] More preferably, the moiety formed by the reaction between the antibody 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.

[0236] Even more preferably, the moiety generated by the reaction between the antibody and the reactive group may be a moiety generated 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 generated 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)".

[0237] Even more preferably, the moiety generated by the reaction between the antibody and the reactive group may be a moiety generated by the reaction between a lysine or tyrosine side chain and a specific reactive group therefor (particularly when the antibody is a human IgG, such as human IgG1). Examples of the moiety generated by the reaction between a lysine or tyrosine side chain and a specific reactive group therefor include the linking moieties and / or chemical structures described above in "1-5. Reactive group (R)".

[0238] Particularly preferably, the moiety resulting from the reaction between the antibody and the reactive group may be a moiety resulting from the reaction between a lysine side chain and a reactive group specific therefor.

[0239] 2-3. Partial structure “LB” The details of the partial structure "LB" are as described above in "1-6. Partial structure "LB"".

[0240] 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 affinity substance for the antibody represented by the above formula (II) and the antibody or a salt thereof containing 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 that is a divalent group that contains 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.

[0241] In the above formula (II'), L' may be represented by any one of the above formulas (L1') to (L3').

[0242] In another specific embodiment, the structural unit represented by LB may be represented by the following formula (LB'): [ka] [During the ceremony, The definitions, examples and preferred examples of C, p, p', q, q', X, X', R1a, R1a', R1b, R1b', 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'.

[0243] Therefore, the above formula (II) can be defined as the following formula (II″). [ka] [During the ceremony, The definitions, examples and preferred examples of A, R', T, C, p, p', q, q', X, X', R1a, R1b, R1a', R1b', Y, Y', Z and Z' are the same as those described above.

[0244] 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' 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' 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.

[0245] 2-4. Binding sites of substructures other than antibodies that an antibody has (position selectivity) A partial structure other than an antibody (eg, ALB-R') can be site-selectively bound to a target region of the antibody (T) as described above.

[0246] As used herein, "position-selective" or "position selectivity" refers to a situation in which a specific structural unit capable of binding to a specific amino acid residue in an antibody is distributed in a specific region of the antibody, even though a specific amino acid residue in the antibody is not distributed in a specific region. Thus, expressions related to position selectivity, such as "position-selectively possessed," "position-selective binding," and "position-selective 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 that are the same type as the specific amino acid residue in the target region. Such position-selective binding or retention can be achieved by the present invention, which can preferentially react a specific structural unit with a specific amino acid residue in a target region of an antibody, rather than randomly reacting a specific structural unit with a specific amino acid residue in the antibody.

[0247] 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 an antibody can be bound to one or more specific amino acid residues contained in the target region with a site selectivity of 30% or more. The definitions, examples, and preferred examples of the target region and site selectivity are as described above.

[0248] 2-5. Number of substructures other than antibodies contained in an antibody The number of partial structures other than the antibody (e.g., ALB-R') possessed by the antibody (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 target regions corresponding to 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 antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number varies depending on the type of antibody, but may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[0249] In a specific embodiment, the number of partial structures other than an antibody possessed by an antibody may be more than one when the antibody is a multimeric protein composed of multiple monomeric proteins. According to the present invention, multiple partial structures other than an antibody can be introduced into the same target region of multiple monomeric proteins.

[0250] In a preferred embodiment, the antibody may be an antibody containing multiple heavy chains. The definition, examples, and preferred examples of the antibody 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 the antibody possessed by an antibody (antibody) can be considered as synonymous with DAR. In the present invention, the number of partial structures other than the antibody possessed by an antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[0251] 2-6. Manufacturing method The present invention provides a method for producing an affinity agent for an antibody, and an antibody or salt thereof comprising a cleavable moiety, comprising: (A1) Reacting a compound or a salt thereof containing an affinity substance for the antibody, a cleavable moiety and a reactive group with the antibody to produce an antibody or a salt thereof containing an affinity substance for the antibody and a cleavable moiety.

[0252] The compound comprising an affinity substance for an antibody, a cleavable moiety and a reactive group is represented by formula (I), preferably formula (I'), more preferably formula (I"). The affinity substance for an antibody, and an antibody comprising a cleavable moiety are represented by formula (II), preferably formula (II'), more preferably formula (II").

[0253] The compound or its salt containing an affinity substance for an antibody, a cleavable moiety and a reactive group has a reactive group and can react with the antibody. Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction can be carried out in an appropriate reaction system, for example, a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain an appropriate catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such a reaction, see, for example, GJL Bernardes et al. 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).

[0254] In the reaction system, the molar ratio (Y / X) of the compound containing an affinity substance for the antibody, a cleavable moiety and a reactive group, or a salt thereof (Y) to the antibody (X) varies depending on the types of the affinity substance for the antibody, the compound containing a cleavable moiety and a reactive group, and the antibody, the number of sites in the antibody to be modified by the affinity substance for the antibody, the compound containing a cleavable moiety and a reactive group (e.g., DAR), and the like, and is not particularly limited, but is, for example, 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

[0255] The production of the antibody or its salt containing an affinity substance for the antibody and a cleavable portion can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw material and the molecular weight of the product. The position selectivity can be confirmed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (e.g., trypsin, chymotrypsin) treatment and mass spectrometry. The protease is preferably an endoprotease. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of partial structures other than the antibody that the antibody has can be confirmed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. The antibody or its salt containing an affinity substance for the antibody and a cleavable portion can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0256] 3. A complex having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof 3-1. Overview The present invention provides a complex having an affinity substance for an antibody, a cleavable moiety, a functional substance and an antibody, represented by formula (III), or a salt thereof. 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 contains a cleavable moiety; B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; R' is the moiety generated by reaction between the antibody and the reactive group, and T is an antibody.] A complex or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, the complex being represented by the following formula:

[0257] In formula (III) or other formulae, R' is covalently bonded to B', not to F.

[0258] 3-2.Functional substances (F) The functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and examples thereof include drugs, labeling substances, and stabilizers, with drugs or labeling substances being preferred. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked together.

[0259] The drug may be a drug for any disease. Examples of such diseases include cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), genetic and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). When the antibody is an antibody against a target protein of a given disease, the drug may be a drug related to the given disease (e.g., a drug that treats the given disease, or a drug that alleviates side effects associated with the use of an antibody against a target protein of the given disease).

[0260] More specifically, the drug is an anticancer drug. Examples of the anticancer drug include chemotherapeutic agents, toxins, radioisotopes, or substances containing the same. Examples of the chemotherapeutic agent include DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of the toxin include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioisotopes of hydrogen atoms (e.g., 3H), radioisotopes of carbon atoms (e.g., 14C), radioisotopes of phosphorus atoms (e.g., 32P), radioisotopes of sulfur atoms (e.g., 35S), radioisotopes of yttrium (e.g., 90Y), radioisotopes of technetium (e.g., 99mTc), radioisotopes of indium (e.g., 111In), radioisotopes of iodine atoms (e.g., 123I, 125I, 129I, 131I), radioisotopes of samarium (e.g., 153Sm), and radioisotopes of rhenium. Radioisotopes of bismuth (e.g., 186Re), astatine (e.g., 211At), and bismuth (e.g., 212Bi). More specifically, drugs include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBDs (pyrrolobenzodiazepines), IGN, camptothecin analogues, calicheamicin, and duocarmycin. , eribulin, anthracyclines, dmDNA31, and tubulysin.

[0261] Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), or substances containing the same.

[0262] The functional substance may also be a polymeric compound, a medium molecular compound, or a low molecular compound, preferably a low molecular compound. A low molecular compound refers to a compound with a molecular weight of 1500 or less. A low molecular compound may be a natural compound or a synthetic compound. The molecular weight of a low molecular compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of a low molecular compound may also be 30 or more, 40 or more, or 50 or more. A low molecular compound may be a drug or a labeling substance as described above. Examples of low molecular compounds include amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids, and salts thereof.

[0263] 3-3. Trivalent group (B') containing a moiety generated by reaction between a functional substance and a bioorthogonal functional group B' is the same as the divalent group containing the bioorthogonal functional group (a) in B described above, except that the bioorthogonal functional group (a) is reacted with a functional substance. Therefore, the definitions, examples and preferred examples of the trivalent group and orthogonal functional group in B' are the same as those in B, except that the bioorthogonal functional group (a) is reacted with a functional substance.

[0264] The functional substance has various functional groups according to its structure. When the functional substance has a functional group that easily reacts with the bioorthogonal functional group, the functional group of the functional substance can be appropriately reacted with the bioorthogonal functional group. The functional group that easily reacts with the bioorthogonal functional group can also differ depending on the specific type of the bioorthogonal functional group. Those skilled in the art can appropriately select an appropriate functional group as a functional group that easily reacts with the bioorthogonal functional group (e.g., Boutureira et al., Chem. Rev.,2015,115,2174-2195). Examples of functional groups that are easily reactive with bioorthogonal functional groups include, but are not limited to, maleimide and disulfide residues when the bioorthogonal functional group is a thiol residue, azide residues when the bioorthogonal functional group is an alkyne residue, and hydrazine residues when the bioorthogonal functional group is an aldehyde or ketone residue. For example, when the bioorthogonal functional group is a thiol residue and the functional group that is easily reactive with the bioorthogonal functional group is a maleimide residue or a disulfide residue (or vice versa), the trivalent group containing the moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be a trivalent group containing a thiosuccinimide residue, or a trivalent group containing a disulfide residue; when the bioorthogonal functional group is an alkyne residue and the functional group that is easily reactive with the bioorthogonal functional group is an azide residue (or vice versa), the functional The trivalent group containing a moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be a trivalent group containing a triazole residue (which may or may not be condensed with another ring); if the bioorthogonal functional group is an aldehyde or ketone residue and the functional group reactive with the bioorthogonal functional group is a hydrazine residue (or vice versa), the trivalent group containing a moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be a trivalent group containing a hydrazone residue (e.g., Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). Trivalent groups containing thiosuccinimide residues, trivalent groups containing disulfide residues A trivalent group, a trivalent group containing a triazole residue (which may or may not be fused with other rings), or a trivalent group containing a hydrazone residue are preferred examples of trivalent groups that contain a moiety formed by reaction between a functional agent and a bioorthogonal functional group.

[0265] On the other hand, when the functional substance does not have a functional group that is easily reactive with the bioorthogonal functional group, a functional substance that has been derivatized to have a desired functional group can be used. For example, when the functional substance is an antibody, a functional substance that has been derivatized to have a functional group that the antibody does not naturally have can be used. In this case, the antibody may be derivatized by the method of the present invention. In this case, an antibody that has been derivatized to have a desired bioorthogonal functional group regioselectively can be used as the functional substance.

[0266] Derivatization is common knowledge in the art (e.g., WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649). For example, derivatization may be performed using a crosslinker as described above. Alternatively, derivatization may be performed using a specific linker having a desired functional group. For example, such a linker may be one that allows the functional substance and the antibody to be separated by cleavage of the linker in an appropriate environment (e.g., inside or outside a cell). Examples of such linkers include peptidyl linkers that are degraded by specific proteases (e.g., intracellular proteases (e.g., proteases present in lysosomes or endosomes), extracellular proteases (e.g., secretory proteases)) (e.g., U.S. Pat. No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83:67-123 (1999)), and linkers that can be cleaved at local acidic sites present in vivo (e.g., U.S. Pat. Nos. 5,622,929, 5,122,368, and 5,824,805). The linker may be self-immolative (e.g., WO 02 / 083180, WO 04 / 043493, and WO 05 / 112919). In the present invention, a derivatized functional substance may also be simply referred to as a "functional substance."

[0267] In the trivalent group containing the part generated by the reaction between the functional substance and the bioorthogonal functional group, the definition, example and preferred example of the functional substance and the bioorthogonal functional group are as described above.The part generated by the reaction between the functional substance and the bioorthogonal functional group is common technical knowledge in the technical field, and can be appropriately determined according to the type of the functional substance (if the functional substance is derivatized, its derivatized part) and the bioorthogonal functional group.

[0268] The trivalent group containing a moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be (1) a trivalent group containing a thiosuccinimide residue, a triazole residue, or a hydrazone residue, which are the residues mentioned in the above-mentioned preferred examples, or (2) a trivalent group containing a residue selected from the group consisting of, but not limited to, a disulfide residue (which is the residue mentioned in the above-mentioned preferred examples), an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a 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.

[0269] The trivalent group containing a moiety generated by the reaction between the functional substance and the bioorthogonal functional group also includes, but is not limited to, for example, the following: [ka] [Where: The black squares indicate bonds to the functional substance side moiety. A white circle (◯) indicates a bond to the L-side moiety, and a black circle (●) indicates a bond to the R'-side moiety. When the chemical structure is asymmetric with respect to the cleavable moiety, a ● may represent a bond to the moiety on the L side, and a ○ may represent a bond to the moiety on the R' side.

[0270] 3-4. Partial structure "L-B'(-F)" The details of the partial structure "L-B'(-F)" are as described above in "1-6. Partial structure "LB"", except that B is changed to B'(-F).

[0271] In one embodiment, the cleavable linker can be (i) a divalent group that includes a cleavable moiety that has the ability to generate a bioorthogonal functional group on a reactive group upon cleavage, or (ii) a divalent group that includes a cleavable moiety that does not have the ability to generate a bioorthogonal functional group on a reactive group upon cleavage.

[0272] In certain embodiments, the cleavable linker may be the cleavable linker (ii) above, since B' is already bound to the functional substance (F), there is no need to generate a bioorthogonal functional group on the reactive group side.

[0273] In another specific embodiment, the partial structure represented by L-B'(-F) may be a partial structure represented by B2'(-F2)-L'-B1'(-F1) (i.e., L is B2'(-F2)-L', and B' is B1'.) In this case, the complex or salt thereof having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, represented by the above formula (III), can be 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 (III), L' is a cleavable linker that is a divalent group that contains a cleavable moiety; B1' and B2' are the same or different and are trivalent 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 and are functional substances; B1' (-F1) and B2' (-F2) may have a symmetrical structure with L' at the center.

[0274] B1' and B2' may be the same or different and have the same definition, examples and preferred examples as those of B'.

[0275] In the above formula (III'), L' may be represented by any one of the above formulas (L1') to (L3').

[0276] In another particular embodiment, the structural unit represented by L-B'(-F) may be represented by the following formula (LB'(F)'): [ka] [During the ceremony, The definitions, examples and preferred examples of C, F1, F2, p, p', q, q', X, X', R1a, R1a', R1b, R1b', Z and Z' are the same as those described above; Y and Y' are the same or different and each is a residue obtained by removing one hydrogen atom from Y in formula (B-1), A circle (○) indicates a bond to A, and a black circle (●) indicates a bond to R'.

[0277] More specifically, in Y and Y', the residue obtained by removing one hydrogen atom from Y in the above formula (B-1) is -N(-)-, -CH(-)-, or a group obtained by removing one hydrogen atom from the above formula (B-2). From the viewpoint of simplifying the structure, Y may be -N(-)- or -CH(-)-. Alternatively, from the viewpoint of designing a structure based on carbon atoms, Y may be -CH(-)- or a group obtained by removing one hydrogen atom from the above formula (B-2).

[0278] The group obtained by removing one hydrogen atom from the above formula (B-2) is represented by the following formula (B-2'): [ka] (In the formula, V and V' are the same or different and each is -NH-, -O-, -CH2-, or a single bond; V1 is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group containing a bioorthogonal functional group. is a divalent group that does not s is an integer from 0 to 10, The ○ and ● in formula (B-2') are in the same orientation as the ○ and ● in formula (B-1), respectively. ) is a group obtained by removing one hydrogen atom from ). The definition, examples and preferred examples of s in formula (B-2') are the same as those in formula (B-2). Thus, in formula (B-2'), one of V and V' may be -N(-)- or -CH(-)-. Alternatively, in formula (B-2'), V1 may be a trivalent group obtained by removing one hydrogen atom from the divalent group of (a) or (b) above. Alternatively, in formula (B-2'), one of the s -CH2- may be -CH(-)-.

[0279] In this case, the above formula (III) can be defined as the following formula (III″). [ka] [During the ceremony, The definitions, examples and preferred examples of A, R, C, F1, F2, p, p', q, q', X, X', R1a, R1b, R1a', R1b', Y, Y', Z and Z' are the same as those described above.

[0280] In the above formulas (LB'(F)') and (III''), 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' 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' 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.

[0281] 3-5. Binding sites of substructures other than antibodies that an antibody has (regioselectivity) A partial structure other than an antibody (e.g., AL-B'(-F)-R') can be regioselectively bound to the above-mentioned target region of an antibody (T). 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, the partial structure other than an antibody 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.

[0282] 3-6. Number of substructures other than antibodies contained in an antibody The number of partial structures other than an antibody (e.g., AL-B'(-F)-R') possessed by an antibody (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. Thus, the formula (III), (III'), or (III'' ) indicates that T may have one or more partial structures other than T. The number of partial structures other than T that T has can be adjusted by appropriately setting conditions such as the type of antibody and the reaction ratio between the antibody and the structural unit to be introduced therein. The number varies depending on the type of antibody, and may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[0283] In a specific embodiment, the number of partial structures other than an antibody possessed by an antibody may be more than one when the antibody is a multimeric protein composed of multiple monomeric proteins. According to the present invention, multiple partial structures other than an antibody can be introduced into the same target region of multiple monomeric proteins.

[0284] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples, and preferred examples of the antibody are as described above. In the present invention, the number of partial structures other than the antibody possessed by the antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[0285] 3-7. Manufacturing method The present invention provides a method for producing a conjugate having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, comprising: (B1) Reacting an affinity substance for an antibody, and an antibody or a salt thereof containing a cleavable moiety with a functional substance to produce a complex or a salt thereof having an affinity substance for the antibody, the cleavable moiety, the functional substance, and the antibody.

[0286] The affinity substance for the antibody, and the antibody comprising a cleavable moiety are represented by formula (II), preferably formula (II'), more preferably formula (II"). The complex comprising an affinity substance for the antibody, a cleavable moiety, a functional substance, and an antibody is represented by formula (III), preferably formula (III'), more preferably formula (III").

[0287] The affinity substance for the antibody, and the antibody or its salt containing a cleavable portion have a bioorthogonal functional group that can react with the functional substance, and therefore can react with the functional substance. Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds), as described above in "2-6. Production method."

[0288] In the reaction system, the molar ratio (Y / X) of the functional substance (Y) to the affinity substance for the antibody, and the antibody or its salt containing a cleavable moiety (X) varies depending on the types of antibody and functional substance, the number of sites in the antibody to be modified by the functional substance (e.g., DAR), etc., and is not particularly limited; it is, for example, 0.1 to 100, preferably 0.5 to 80, more preferably 1 to 70, even more preferably 2 to 50, and particularly preferably 3 to 30.

[0289] The production of a complex or a salt thereof having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Confirmation of regioselectivity, confirmation of the number of partial structures other than the antibody, and purification can be appropriately performed by the methods described above in "2-6. Production method."

[0290] The present invention also provides a method for producing a conjugate having an affinity agent for an antibody, a cleavable moiety, a functional agent, and an antibody, or a salt thereof, comprising: (B2) reacting a compound or a salt thereof containing an affinity substance for the antibody, a cleavable moiety and a reactive group with the antibody to produce an antibody or a salt thereof containing an affinity substance for the antibody and a cleavable moiety; and (B3) Reacting an affinity substance for the antibody and an antibody or a salt thereof containing a cleavable moiety with a functional substance to produce a complex or a salt thereof having an affinity substance for the antibody, the cleavable moiety, the functional substance and the antibody.

[0291] The above step (B2) can be carried out in the same manner as the above step (A1) described in "2-6. Manufacturing method". The above step (B3) can be carried out in the same manner as the above step (B1). The above steps (B2) and (B3) can be carried out separately. Alternatively, the above steps (B2) and (B3) can be carried out simultaneously depending on the combination of the reaction pair between the specific amino acid residue that is the reaction target in the antibody and the reactive group, and the reaction pair between the functional group in the functional substance and the bioorthogonal functional group. That is, when the above steps (B2) and (B3) are carried out simultaneously, the product-containing reaction solution obtained in the above step (B2) may be subjected to the above step (B3) without isolating the product obtained in the above step (B2).

[0292] 4. Antibody having a bioorthogonal functional group or its salt 4-1. Overview of manufacturing method The present invention provides a method for producing an antibody having a bioorthogonal functional group represented by formula (IV) or a salt thereof. 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 that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody.

[0293] 4-2. (i') A monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group (L1) In formula (IV), L1 is (i') a monovalent group containing a bioorthogonal functional group, or (ii') a monovalent group not containing a bioorthogonal functional group. Such a monovalent group is a monovalent group that can be generated by cleavage of a cleavable linker that is a divalent group containing a cleavable moiety as described above. Thus, L1 may be a monovalent group that can be generated by cleavage of a residue or chemical structure of a cleavable moiety as described above. More specifically, (i') the monovalent group containing a bioorthogonal functional group may be a monovalent group that can be generated by cleavage of a cleavable linker that is a divalent group containing a cleavable moiety having the ability to generate a bioorthogonal functional group on the reactive group side by cleavage as described above (i), and (ii') the monovalent group not containing a bioorthogonal functional group may be a monovalent group that can be generated by cleavage of 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 by cleavage as described above (ii).

[0294] In certain embodiments, L1 may be represented by any one of the following formulas (L1-1) or (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.

[0295] 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)-, -NRa- (Ra represents a hydrogen atom or a substituent), -O-, -S-, -C(=S)-, and a group consisting of a combination of two or more of these (for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4). The definitions, examples, and preferred examples of the divalent hydrocarbon group, divalent heterocyclic group, and substituent are as described above.

[0296] The definition, examples, and preferred examples of bioorthogonal functional groups are as described above.

[0297] Examples of groups other than bioorthogonal functional groups include, for example, those of the above-mentioned substituents other than bioorthogonal functional groups. Examples of such groups other than bioorthogonal functional groups include, for example, alkyl, cycloalkyl, aralkyl, monovalent heterocyclic groups, hydroxyl, amino, alkyloxy (alkoxy), cycloalkyloxy, and aralkyloxy. The definitions, examples, and preferred examples of these groups and the components of these groups (e.g., alkyl in alkyloxy (alkoxy), cycloalkyl in cycloalkyloxy, and aralkyl in aralkyloxy) are the same as those described above.

[0298] In certain embodiments, Lb is as follows (Lb'): [ka] [During the ceremony, p is an integer from 0 to 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, R1b is absent; when X is a single bond, R1a and R1b are absent); R1a and R1b are the same or different and are selected from the group consisting of a hydrogen atom or the above-mentioned substituents. ○ (open circle) indicates a bond to C1, and ● (filled circle) indicates a bond to B.].

[0299] The definitions, examples, and preferred examples of p, q, and X, as well as R1a and R1b (substituents) are as described above.

[0300] 4-3. Partial structure “L1-B” 4-3-1. Length of the partial structure "L1-B" connecting the L1 terminal portion and R' In formula (IV), the length of the partial structure (the linear portion in "L1-B") linking the L1 terminal portion (or the C1 terminal portion) and R' produced by cleavage (or the number of atoms in the main chain linking the bioorthogonal functional group and the side chain of a specific amino acid residue in an antibody or a salt thereof having a bioorthogonal functional group regioselectively, not limited to a specific formula such as formula (IV) (the same applies below)) can be appropriately designed depending on various factors such as those described above in "1-6-1. Length of the main chain in the partial structure "LB" linking A and R".

[0301] A partial structure that links the L1 terminal portion (or C1 terminal portion) generated by cleavage and R' The length of the partial structure may be, for example, about 2.5 Å or more, preferably about 4 Å or more, more preferably about 5 Å or more. The length of the partial structure may also be, for example, about 15 Å or less, preferably about 12 Å or less, more preferably about 8 Å or less. More specifically, the length of the partial structure may be, for example, about 2.5 to 15 Å, preferably about 4 to 12 Å, more preferably about 5 to 8 Å.

[0302] More specifically, the length of the partial structure linking the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage can also be defined as the number of atoms constituting the linking chain of the partial structure (excluding hydrogen atoms and substituents). The number of atoms constituting the linking chain may be, for example, 2 (about 2.5 Å) or more, preferably 3 (about 4 Å) or more, more preferably 4 (about 5 Å) or more. The number of atoms constituting the linking chain may also be, for example, 10 (about 15 Å) or less, preferably 8 (about 12 Å) or less, more preferably 6 (about 8 Å) or less. More specifically, the number of atoms constituting the linking chain may be, for example, 2 to 10, preferably 3 to 8, more preferably 4 to 6.

[0303] When the linking chain of the partial structure linking the L1 terminal portion (or the C1 terminal portion) and R' produced by cleavage is a chain structure that does not contain a divalent ring structure, the number of atoms in the linking chain can be determined by counting the number of atoms in the linking chain.

[0304] On the other hand, when the linking chain of the partial structure linking the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage is a structure containing a divalent ring structure, the number of atoms in the linking chain does not necessarily correspond to the above-mentioned length, and the length that can be defined by the number of atoms in the linking chain tends to be shorter than the above-mentioned length. Even in such a case, the number of atoms in the linking chain can be counted for convenience from the viewpoint of defining the length of the linking chain by the number of atoms. Specifically, the number of atoms in the linking chain in such a case may be determined by counting the number of atoms in the chain structure that does not contain a divalent ring structure in the linking chain, as described above, and the number of atoms in the shortest path connecting two bonds in the divalent ring structure.

[0305] Preferably, the linking chain of the partial structure linking the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage may be a chain structure that does not contain a divalent ring structure. In this case, L1-B can be appropriately designed so that the linking chain of the partial structure linking the terminal portion and R' generated by cleavage does not contain a divalent ring group.

[0306] In a specific embodiment, the partial structure represented by L1-B preferably does not contain a peptide moiety. In this case, the antibody having the functional substance of the present invention (e.g., antibody-drug conjugate) obtained by using the antibody having the bioorthogonal functional group of the present invention has the advantage that it does not contain a peptide moiety that may have immunogenicity as a linker.

[0307] 4-3-2. Specific structure of partial structure "L1-B" In the above formula (IV), L1 and B are structures that can be linked to each other. Therefore, in the above formula (IV), L1 and B can be defined as a partial structure represented by "L1-B".

[0308] In certain embodiments, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group.

[0309] Preferably, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group. In this case, the bioorthogonal functional group in (i') may be the same or different from the bioorthogonal functional group in (a). In terms of adopting a simple structure and / or improving reactivity to a single functional substance, ( The bioorthogonal functional group in i') may be the same as the bioorthogonal functional group in (a). On the other hand, in order to ensure differentiated reactivity to two or more functional substances and to avoid using some bioorthogonal functional groups in the reaction, the bioorthogonal functional group in (i') may be different from the bioorthogonal functional group in (a).

[0310] Alternatively, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B may be (b) a divalent group not containing a bioorthogonal functional group. In this case, the antibody or salt thereof having a bioorthogonal functional group represented by formula (IV) has a simpler structure and is therefore easier to synthesize.

[0311] In another specific embodiment, when L1 is (ii') a monovalent group that does not contain a bioorthogonal functional group, B is (a) a divalent group that contains a bioorthogonal functional group.

[0312] In certain embodiments, the structural unit represented by L1-B may be represented by the following formula (L1B'): [ka] [During the ceremony, The definitions, examples and preferred examples of C1, p, q, X, R1a, R1b, Y and Z are the same as those described above; ● (black circle) indicates a bond to R'.

[0313] Therefore, the above formula (IV) can be defined as the following formula (IV'). [ka] [During the ceremony, The definitions, examples and preferred examples of C1, p, q, X, R1a, R1b, Y, Z, R', and T are the same as those described above.

[0314] In the above formulas (L1B') and (IV'), the length from C to C1 in C=Z is the same as the length of the partial structure linking the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage. In these formulas, the length from C to C1 in C=Z can also be defined as the number of atoms constituting the linking chain (excluding hydrogen atoms and substituents) of the partial structure from C to C1 in C=Z. Such number of atoms is the same as the number of atoms constituting the partial structure (excluding hydrogen atoms and substituents) linking the L1 terminal portion (or C1 terminal) and R' generated by cleavage. The portion linking C to C1 in C=Z The linking chain of the structure (excluding hydrogen atoms and substituents) may or may not contain a ring structure, but preferably does not contain a ring structure. The linking chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide portion.

[0315] 4-4. Binding sites of substructures other than antibodies that an antibody has (regioselectivity) A partial structure other than an antibody (e.g., L1-B-R') can be regioselectively bound to the above-mentioned target region of an antibody (T). 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, the partial structure other than an antibody 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.

[0316] 4-5. Number of substructures other than antibodies contained in an antibody The number of partial structures (e.g., L1-B-R') other than the antibody possessed by the antibody (T) can vary. For example, when T is a multimeric protein containing a plurality of monomeric proteins, T can have partial structures other than T in a plurality of target regions corresponding to the plurality of monomeric proteins. As a result, T can have a plurality of partial structures other than T. Thus, the structure represented by formula (IVI), (IV'), or IV'') indicates that T may have one or a plurality of 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 antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number varies depending on the type of antibody, but may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[0317] In a specific embodiment, the number of partial structures other than an antibody possessed by an antibody may be more than one when the antibody is a multimeric protein composed of multiple monomeric proteins. According to the present invention, multiple partial structures other than an antibody can be introduced into the same target region of multiple monomeric proteins.

[0318] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples, and preferred examples of the antibody are as described above. In the present invention, the number of partial structures other than the antibody possessed by the antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[0319] 4-6. Manufacturing method The present invention provides a method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising: (C1) Cleaving the cleavable portion of an antibody or its salt comprising an affinity substance for the antibody and a cleavable portion to generate an antibody or its salt having a bioorthogonal functional group.

[0320] The affinity substance for the antibody and the antibody comprising the cleavable moiety are represented by formula (II), preferably formula (II'), more preferably formula (II"). The antibody having a bioorthogonal functional group is represented by formula (IV), preferably formula (IV'), more preferably formula (IV").

[0321] The antibody or its salt having an affinity for the antibody and the cleavable portion can be cleaved by the above-mentioned cleavage treatment because the antibody or its salt has a cleavable portion that can be cleaved by the above-mentioned cleavage treatment. Such a cleavage reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds), as described in "2-6. Production method" above. This can be done.

[0322] Examples of the cleavage treatment 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 physicochemical stimuli selected from the group consisting of light, or (c) leaving the cleavable linker containing a self-degrading cleavable moiety alone. The conditions for such cleavage treatment are common technical knowledge in the field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Jounal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99,423(2004).;DeSimone,JM,Journal of American Chemical Society.132,17928(2010);Thompson,DH,Journal of Controlled Release,91,187(2003);Schoenmarks,RG,Journal of Controlled Release,95,291(2004)).

[0323] The production of an antibody or a salt thereof having a bioorthogonal functional group 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. Confirmation of regioselectivity, confirmation of the number of partial structures other than the antibody, and purification can be appropriately performed by the methods described above in "2-6. Production method".

[0324] The present invention also provides a method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising: (C2) reacting a compound or a salt thereof containing an affinity substance for the antibody, a cleavable moiety and a reactive group with the antibody to produce an antibody or a salt thereof containing an affinity substance for the antibody and a cleavable moiety; and (C3) Cleaving the cleavable portion of the antibody or its salt comprising an affinity substance for the antibody and a cleavable portion to generate an antibody or its salt having a bioorthogonal functional group.

[0325] The above step (C2) can be carried out in the same manner as the above step (A1) described in "2-6. Manufacturing method". The above step (C3) can be carried out in the same manner as the above step (C1). The above steps (C2) and (C3) can be carried out separately. Alternatively, the above steps (C2) and (C3) can be carried out simultaneously depending on factors such as the combination (non-reactive) of the reactive group and the bioorthogonal functional group that can be generated by cleavage. That is, when the above steps (C2) and (C3) are carried out simultaneously, the product-containing reaction solution obtained in the above step (C2) may be subjected to the above step (C3) without isolating the product obtained in the above step (C2).

[0326] 5. Method for producing an antibody having a functional substance or a salt thereof 5-1. Overview The present invention provides a method for producing an antibody having a functional substance represented by formula (V) or a salt thereof. 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 that includes a bioorthogonal functional group, or (b) a divalent group that does not include a bioorthogonal functional group. is a divalent group not 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 or both of the bioorthogonal functional groups in (i') and (a), F is a functional material; R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody.

[0327] 5-2.Substructure "F-(L1-B)'" 5-2-1. Length of the partial structure "L1-B" connecting F and R' In formula (V), the length of the partial structure "L1-B" (the linear portion in "L1-B") linking F and R' (or, in an antibody or salt thereof having a functional substance site-selectively, not limited to a specific formula such as formula (V), the number of atoms in the main chain linking the functional substance and the side chain of a specific amino acid residue, or, in the case where the functional substance is bound to the antibody via a bioorthogonal functional group, the number of atoms in the main chain linking the bioorthogonal functional group and the side chain of a specific amino acid residue; the same applies below) can be appropriately designed depending on various factors such as those described above in "1-6-1. Length of the main chain in the partial structure "LB" linking A and R".

[0328] The length of the partial structure linking F and R' may be, for example, about 2.5 Å or more, preferably about 4 Å or more, more preferably about 5 Å or more. The length of the partial structure may also be, for example, about 15 Å or less, preferably about 12 Å or less, more preferably about 8 Å or less. More specifically, the length of the partial structure may be, for example, about 2.5 to 15 Å, preferably about 4 to 12 Å, more preferably about 5 to 8 Å.

[0329] More specifically, the length of the partial structure linking F and R' can also be defined as the number of atoms constituting the linking chain of the partial structure (excluding hydrogen atoms and substituents). The number of atoms constituting the linking chain may be, for example, 2 (about 2.5 Å) or more, preferably 3 (about 4 Å) or more, more preferably 4 (about 5 Å) or more. The number of atoms constituting the linking chain may also be, for example, 10 (about 15 Å) or less, preferably 8 (about 12 Å) or less, more preferably 6 (about 8 Å) or less. More specifically, the number of atoms constituting the linking chain may be, for example, 2 to 10, preferably 3 to 8, more preferably 4 to 6.

[0330] When the connecting chain of the partial structure connecting F and R' is a chain structure not containing a divalent ring structure, the number of atoms in the connecting chain can be determined by counting the number of atoms in the connecting chain.

[0331] On the other hand, when the linking chain of the partial structure connecting F and R' is a structure containing a divalent ring structure, the number of atoms of the linking chain does not necessarily correspond to the above-mentioned length, and the length that can be defined by the number of atoms of the linking chain tends to be shorter than the above-mentioned length.Even in such a case, the number of atoms of the linking chain can be counted conveniently from the viewpoint of defining the length of the linking chain by the number of atoms.Specifically, the number of atoms of the linking chain in such a case may be determined by counting the number of atoms in the chain structure that does not contain a divalent ring structure in the linking chain, as described above, and the number of atoms of the shortest path connecting two bonds in the divalent ring structure.

[0332] Preferably, the linking chain of the partial structure linking F and R' may be a chain structure that does not include a divalent ring structure. L1-B can be appropriately designed so that the linking chain of the partial structure linking F and R' does not include a divalent ring group.

[0333] In a specific embodiment, the partial structure represented by L1-B preferably does not contain a peptide moiety. In this case, the antibody having the functional substance of the present invention (e.g., an antibody-drug conjugate) has the advantage that it does not contain a peptide moiety that may have immunogenicity as a linker.

[0334] 5-2-2. Specific structure of partial structure "F-(L1-B)'" In formula (V), L1 and B in F-(L1-B)' are structures that can be linked to each other. Therefore, in the above formula (V), L1 and B can be defined as a partial structure represented by "L1-B".

[0335] In addition, the structural unit represented by F-(L1-B)' is a moiety generated by the reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a). The bioorthogonal functional group subjected to the reaction does not exist in formula (V). Therefore, in formula (V), either one or both of the bioorthogonal functional groups in (i') and (a) do not exist. The moiety generated by the reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a) is the same as the moiety generated by the reaction between a functional substance and a bioorthogonal functional group. Therefore, in this specification, examples (names of residues) and specific examples (chemical structural formulas) of the moiety generated by the reaction between a functional substance and either one or both of the bioorthogonal functional groups in (i') and (a) are the same as those described above for the moiety generated by the reaction between a functional substance and a bioorthogonal functional group.

[0336] In one embodiment, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group.

[0337] In a preferred embodiment, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B is (a) a divalent group containing a bioorthogonal functional group. In this case, the bioorthogonal functional group 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, etc. From this point of view, the bioorthogonal functional group in (i') may be the same as the bioorthogonal functional group in (a). On the other hand, from the viewpoint of ensuring differentiated reactivity to two or more types of functional substances and not using some bioorthogonal functional groups in the reaction, the bioorthogonal functional group in (i') may be different from the bioorthogonal functional group in (a).

[0338] In another preferred embodiment, when L1 is (i') a monovalent group containing a bioorthogonal functional group, B may be (b) a divalent group not containing a bioorthogonal functional group. In this case, the antibody or salt thereof having a functional substance represented by formula (V) has a simpler structure and is therefore easier to synthesize.

[0339] In another embodiment, when L1 is (ii') a monovalent group that does not contain a bioorthogonal functional group, B is (a) a divalent group that contains a bioorthogonal functional group.

[0340] In certain embodiments, when the bioorthogonal functional groups in (i') and (a) are heterologous, the antibody or salt thereof having a functional substance represented by formula (V) may be represented by formula (V1) or (V2).

[0341] 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; B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody.

[0342] F-L1'-B-R'-T (V2) [During the ceremony, L1' is a divalent group containing a moiety generated by reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; F is a functional material; R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody.

[0343] L1' is a divalent group containing a moiety generated by the reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group. The moiety generated by the reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group is the same as the moiety generated by the reaction between a functional substance and a bioorthogonal functional group. Therefore, in this specification, examples (name of residue) and specific examples (chemical structure) of the moiety generated by the reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group are the same as those described above for the moiety generated by the reaction between a functional substance and a bioorthogonal functional group. Therefore, since the functional substance reacts with the bioorthogonal functional group, the divalent group (L1') containing a moiety generated by the reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group can also be expressed as a divalent group containing a moiety generated by the reaction between a functional substance and a bioorthogonal functional group (hereinafter the same). More specifically, the divalent group containing a moiety generated by the reaction between the functional substance and the bioorthogonal functional group may be (1) a divalent group containing a thiosuccinimide residue, a triazole residue, or a hydrazone residue, which are the residues mentioned in the above-mentioned preferred examples, or (2) a divalent group containing a residue selected from the group consisting of, but not limited to, a disulfide residue (this residue is the residue mentioned in the above-mentioned preferred examples), an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue, a tertiary alkyloxycarbamate residue, a silane residue, a hydrazone-containing residue, a 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, but (1) a divalent group containing a thiosuccinimide residue, a triazole residue, or a hydrazone residue is preferred.

[0344] In another specific embodiment, when the bioorthogonal functional groups in (i') and (a) are the same or different, the antibody or salt thereof having a functional substance represented by formula (V) may be represented by formula (V3).

[0345] Fa-L1'-B'(-Fb)-R'-T (V3) [During the ceremony, L1' is a divalent group containing a moiety generated by reaction between a functional substance and (i') a monovalent group containing a bioorthogonal functional group; B' is a trivalent group that includes a moiety formed by reaction between a functional agent and a bioorthogonal functional group; Fa and Fb are the same or different functional substances, R' is the moiety generated by reaction between the antibody and the reactive group, T is an antibody.

[0346] In a particular embodiment, in the above formula (V1), the structural unit represented by L1-B'(-F) may be represented by the following formula (L1B'(F)). [ka] [During the ceremony, The definitions, examples and preferred examples of F, C1, p, q, X, R1a, R1b and Z are the same as those described above; Y' is a residue obtained by removing one hydrogen atom from Y in formula (B-1), ● (black circle) indicates a bond to R'.

[0347] The definition, examples and preferred examples of the residue obtained by removing one hydrogen atom from Y in the above formula (B-1) are the same as those described above.

[0348] Therefore, the above formula (V1) can be defined as the following formula (V1'). [ka] [During the ceremony, The definitions, examples and preferred examples of F, C1, p, q, X, R1a, R1b, Z, R', and T are the same as those described above; Y' is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1).

[0349] In a particular embodiment, in the above formula (V2), the structural unit represented by F-L1'-B may be represented by the following formula (FL1'B): [ka] [During the ceremony, The definitions, examples and preferred examples of F, p, q, X, R1a, R1b, Y and Z are the same as those described above; C1' is a moiety generated by reaction between a functional substance and a bioorthogonal functional group; ● (black circle) indicates a bond to R'.

[0350] C1' is the moiety generated by the reaction between the functional substance and the bioorthogonal functional group. The definition, examples and preferred examples of the moiety generated by reaction between the functional substance and the bioorthogonal functional group in 1' are the same as those described above in "Divalent group containing a moiety generated by reaction between a functional substance and a bioorthogonal functional group" (hereinafter the same).

[0351] Therefore, the above formula (V2) can be defined as the following formula (V2'). [ka] [During the ceremony, The definitions, examples and preferred examples of F, p, q, X, R1a, R1b, Y, Z, R', and T are the same as those described above; C1' is the moiety generated by reaction between the functional substance and the bioorthogonal functional group.

[0352] In a specific embodiment, in the above formula (V3), the structural unit represented by Fa-L1'-B'(-Fb) may be represented by the following formula (FaL1'B'(Fb)). [ka] [During the ceremony, The definitions, examples and preferred examples of Fa, Fb, p, q, X, R1a, R1b and Z are the same as those described above; C1' is a moiety generated by reaction between a functional substance and a bioorthogonal functional group; Y' is a residue obtained by removing one hydrogen atom from Y in formula (B-1), ● (black circle) indicates a bond to R'.

[0353] Therefore, the above formula (V3) can be defined as the following formula (V3'). [ka] [During the ceremony, The definitions, examples and preferred examples of Fa, Fb, p, q, X, R1a, R1b, Z, R', and T are the same as those described above; C1' is a moiety generated by reaction between a functional substance and a bioorthogonal functional group; Y' is a residue obtained by removing one hydrogen atom from Y in the above formula (B-1).

[0354] The above formulas (L1B'(F)), (FL1'B), and (FaL1'B'(F2)), etc. In addition, in (V1'), (V2') and (V3'), the length from C to C1 in C=Z is the same as the length of the partial structure linking the L1 terminal portion (or C1 terminal portion) and R' generated by cleavage. In these formulas, the length from C to C1 in C=Z can also be defined as the number of atoms constituting the linking chain (excluding hydrogen atoms and substituents) of the partial structure from C to C1 in C=Z. Such number of atoms is the same as the number of atoms constituting the partial structure (excluding hydrogen atoms and substituents) linking the L1 terminal portion (or C1 terminal) and R' generated by cleavage. The linking chain (excluding hydrogen atoms and substituents) of the partial structure linking C to C1 in C=Z may or may not contain a ring structure, but preferably does not contain a ring structure. The linking chain (excluding hydrogen atoms and substituents) may preferably not contain a peptide portion.

[0355] 5-3. Binding sites of substructures other than antibodies that an antibody has (regioselectivity) A partial structure other than an antibody (e.g., F-(L1-B)'-R') can be regioselectively bound to the above-mentioned target region of an antibody (T). 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, the partial structure other than an antibody 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.

[0356] 5-4. Number of substructures other than antibodies contained in an antibody The number of partial structures other than the antibody (e.g., F-(L1-B)'-R') possessed by the antibody (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 target regions corresponding to the multiple monomeric proteins. As a result, T can have multiple partial structures other than T. Therefore, the structure represented by formula (V), (V1), (V2), (V3), (V1'), (V2'), or (V3') 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 antibody and the reaction ratio between the antibody and the structural unit introduced thereto. Such a number varies depending on the type of antibody, but may be, for example, 1 to 8, preferably 1 to 4, and more preferably 1 or 2.

[0357] In a specific embodiment, the number of partial structures other than an antibody possessed by an antibody may be more than one when the antibody is a multimeric protein composed of multiple monomeric proteins. According to the present invention, multiple partial structures other than an antibody can be introduced into the same target region of multiple monomeric proteins.

[0358] In a preferred embodiment, the antibody may be an antibody comprising multiple heavy chains. The definition, examples, and preferred examples of the antibody are as described above. In the present invention, the number of partial structures other than the antibody possessed by the antibody may be 1 or 2 (preferably 2) for IgG, IgE, and IgD, 1 to 4 (preferably 4) for IgA, and 1 to 8 (preferably 8) for IgM.

[0359] 5-5. Manufacturing method The present invention provides a method for producing an antibody or a salt thereof having a functional substance. The production method can be classified into (D) a method using a complex or a salt thereof having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody as a raw material (see reaction (3) in FIG. 2), and (E) a method using an antibody or a salt thereof having a bioorthogonal functional group as a raw material (see reaction (5) in FIG. 2).

[0360] (D) Methods of using a complex having an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, or a salt thereof, as a raw material include the following: (D1) Cleaving the cleavable portion of a complex or its salt having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody to produce an antibody or its salt having a functional substance.

[0361] The complex having an affinity substance for an antibody, a cleavable moiety, a functional substance and an antibody is represented by formula (III), preferably formula (III'), more preferably formula (III''). The antibody having a functional substance is represented by formula (V), preferably formula (V1), (V2), or (V3), more preferably formula (V1'), (V2'), or (V3').

[0362] The complex or its salt having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody can be cleaved by the above-mentioned cleavage treatment because it has a cleavable portion that can be cleaved. Such a cleavage reaction can be carried out in the manner described above in "4-6. Production method".

[0363] The production of an antibody or a salt thereof having a functional substance can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry, depending on the specific raw material and the molecular weight of the product. Confirmation of regioselectivity, confirmation of the number of partial structures other than the antibody, and purification can be appropriately performed by the methods described above in "2-6. Production method."

[0364] The present invention also provides a method for producing an antibody or a salt thereof having a functional substance, comprising: (D2) reacting an affinity substance for the antibody and an antibody or a salt thereof comprising a cleavable moiety with a functional substance to produce a complex or a salt thereof having an affinity substance for the antibody, a cleavable moiety, a functional substance, and an antibody; and (D3) Cleaving the cleavable portion of a complex or its salt having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody to produce an antibody or its salt having a functional substance.

[0365] The above step (D2) can be carried out in the same manner as the above step (B1) described in "3-7. Manufacturing method". The above step (D3) can be carried out in the same manner as the above step (D1). The above steps (D2) and (D3) can be carried out separately. Alternatively, the above steps (D2) and (D3) can be carried out simultaneously depending on factors such as the combination of the cleavable moiety, the functional substance, and the bioorthogonal functional group. That is, when the above steps (D2) and (D3) are carried out simultaneously, the product-containing reaction solution obtained in the above step (D2) may be subjected to the above step (D3) without isolating the product obtained in the above step (D2).

[0366] The present invention further provides a method for producing an antibody or a salt thereof having a functional substance, the method comprising: (D4) reacting a compound or a salt thereof comprising an affinity substance for the antibody, a cleavable moiety and a reactive group with the antibody to produce an antibody or a salt thereof comprising an affinity substance for the antibody and a cleavable moiety; (D5) reacting an affinity substance for the antibody and an antibody or a salt thereof comprising a cleavable moiety with a functional substance to produce a complex or a salt thereof having an affinity substance for the antibody, a cleavable moiety, a functional substance, and an antibody; and (D6) A complex or complex having an affinity substance for an antibody, a cleavable portion, a functional substance and an antibody or a salt thereof, to produce an antibody or a salt thereof having a functional substance.

[0367] The above step (D4) can be carried out in the same manner as the above step (A1) described in "2-6. Manufacturing method". The above steps (D5) and (D6) can be carried out in the same manner as the above steps (D2) and (D3). The above step (D5) can be carried out in the same manner as the above step (B1) described in "3-7. Manufacturing method". The above step (D6) can be carried out in the same manner as the above step (D1). The above steps (D4) to (D6) can be carried out separately. Alternatively, at least a part of the above steps (D4) to (D6) can be carried out simultaneously depending on factors such as the combination of reactive groups, cleavable moieties, functional substances, and bioorthogonal functional groups. That is, when two or more steps of the above steps (D4) to (D6) are carried out simultaneously, the product-containing reaction solution obtained in the previous step may be subjected to the next step without isolating the product obtained in the previous step.

[0368] (E) Methods using an antibody or a salt thereof having a bioorthogonal functional group as a starting material include the following: (E1) Reacting an antibody or a salt thereof having a bioorthogonal functional group with a functional substance to produce an antibody or a salt thereof having the functional substance.

[0369] The antibody having a bioorthogonal functional group is represented by formula (IV), preferably formula (IV'). The antibody having a functional substance is represented by formula (V), preferably formula (V1), (V2), or (V3), more preferably formula (V1'), (V2'), or (V3').

[0370] Since the antibody or its salt having a bioorthogonal functional group has a bioorthogonal functional group, it can react with a functional substance. Such a reaction can be carried out in the manner described in "3-7. Production method" above.

[0371] The production of an antibody or a salt thereof having a functional substance can be confirmed by the method described above. The confirmation of regioselectivity, the confirmation of the number of partial structures other than the antibody, and purification can be appropriately performed by the method described above in "2-6. Production method".

[0372] The present invention also provides a method for producing an antibody or a salt thereof having a functional substance, comprising: (E2) cleaving the cleavable portion of the antibody or its salt comprising an affinity substance for the antibody and a cleavable portion to generate an antibody or its salt having a bioorthogonal functional group; and (E3) Reacting the antibody or its salt having a bioorthogonal functional group with a functional substance to produce an antibody or its salt having the functional substance.

[0373] The above step (E2) can be carried out in the same manner as the above step (C1) described in "4-6. Manufacturing method". The above step (E3) can be carried out in the same manner as the above step (E1). The above steps (E2) and (E3) can be carried out separately. Alternatively, the above steps (E2) and (E3) can be carried out simultaneously depending on factors such as the combination of the cleavable moiety, the functional substance, and the bioorthogonal functional group. That is, when the above steps (E2) and (E3) are carried out simultaneously, the product-containing reaction solution obtained in the above step (E2) may be subjected to the above step (E3) without isolating the product obtained in the above step (E2).

[0374] The present invention further provides a method for producing an antibody or a salt thereof having a functional substance, the method comprising: (E4) A compound or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety and a reactive group with an antibody to produce an antibody or salt thereof comprising an affinity agent for the antibody and a cleavable moiety; (E5) cleaving the cleavable moiety of the antibody or its salt comprising an affinity substance for the antibody and the cleavable moiety to generate an antibody or its salt having a bioorthogonal functional group; and (E6) Reacting the antibody or its salt having a bioorthogonal functional group with a functional substance to produce an antibody or its salt having the functional substance.

[0375] The above step (E4) can be carried out in the same manner as the above step (A1) described in "2-6. Production method". The above steps (E5) and (E6) can be carried out in the same manner as the above steps (E2) and (E3). The above steps (E4) to (E6) can be carried out separately. Alternatively, at least a part of the above steps (E4) to (E6) can be carried out simultaneously depending on factors such as the combination of reactive groups, cleavable moieties, functional substances, and bioorthogonal functional groups. That is, when two or more steps among the above steps (E4) to (E6) are carried out simultaneously, the product-containing reaction solution obtained in the previous step may be subjected to the next step without isolating the product obtained in the previous step.

[0376] 6. Salt In the present invention, examples of salts include salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0377] 7.Applications The compound of the present invention having an affinity substance for an antibody, a cleavable moiety and a reactive group or a salt thereof is useful, for example, for site-selective modification of an antibody. Thus, the present invention provides a site-selective modification reagent for an antibody, comprising a compound having an affinity substance for an antibody, a cleavable moiety and a reactive group or a salt thereof.

[0378] The regioselective modification reagent of the present invention may be provided in the form of a composition further containing other components. Such other components include, for example, a solution and a stabilizer (e.g., an antioxidant, a preservative). The solution is preferably an aqueous solution. Examples of the aqueous solution include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), and buffer solutions (e.g., aqueous phosphate solution, Tris-hydrochloric acid buffer solution, carbonate-bicarbonate buffer solution, aqueous boric acid solution, glycine-sodium hydroxide buffer solution, citrate buffer solution), with buffer solutions being preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The regioselective modification reagent of the present invention can be provided in a liquid or powder form (e.g., lyophilized powder).

[0379] The antibody or a salt thereof of the present invention having an affinity substance for an antibody and a cleavable moiety (position-selectively), an affinity substance for an antibody, a cleavable moiety, a functional substance and an antibody (position-selectively) The conjugate of the present invention having a bioorthogonal functional group (regioselectively) or a salt thereof, and the antibody of the present invention having a bioorthogonal functional group (regioselectively) or a salt thereof are useful, for example, as intermediates for the preparation of an antibody having a functional substance (regioselectively) or a salt thereof.

[0380] The antibody or a salt thereof of the present invention having a functional substance (regioselectively) is useful, for example, as a pharmaceutical or a reagent (eg, a diagnostic agent, a research reagent).

[0381] In particular, the antibody or salt of the present invention having a functional substance (position-selectively) is useful as a medicine. It has been reported as above that changing the number and binding position of the drug in an antibody-drug conjugate (ADC) changes the pharmacokinetics, drug release rate, and effect. For these reasons, it is required to control the number and position of the drug to be conjugated in next-generation ADCs. It is believed that if the number and position are constant, the expected efficiency can be achieved, and the problems of variation in the conjugated drug and lot differences, so-called regulation, can be solved. The antibody or salt of the present invention having a functional substance can solve such regulation problems. Therefore, the antibody or salt of the present invention having a functional substance may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain a pharmaceutical acceptable carrier in addition to the antibody or salt having a functional substance. Examples of medicamentously acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cacao butter, polyethylene glycol, and white kerosene. However, these are not limited to these. The antibody or a salt thereof of the present invention having a functional substance may also have any modification (eg, PEGylation) that ensures stability.

[0382] Formulations suitable for oral administration include liquid preparations in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or granules; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0383] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0384] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to which the composition is administered, the drug tolerance, body weight, age, etc. of the subject, but can be determined appropriately. EXAMPLES

[0385] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples. It is not something that can be done.

[0386] Example 1: Design of modified protein A and its secretory expression in C. glutamicum (1-1) Overview of engineered protein A design In the present invention, it is necessary to bind a linker to a protein. That is, in order to achieve specific binding of the linker to a specific amino acid residue in a protein, it is necessary to design the protein so that there is only one amino group that can react with the linker. With this in mind, the following mutations were made. A) Mutate all K (lysine) in the protein to R (arginine) B) By changing the N-terminal amino acid to Q (glutamine), pyroglutamylation is performed to convert the N-terminal amino group into an amide. According to the above rules A) and B), the actual modified protein A was designed as follows.

[0387] (1-2) Preparation of modified protein A The following modified Protein A was prepared: (a) CspB6Tev-QZ34C QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 7) (b) QZ34C-60 QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 8) (c)QET-Z34C1 QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 9) (d) QET-Z34C2 QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10) (e) QET-Z34C3 QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO:22) (f) QET-Z34C4 QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23)

[0388] (1-3) Expression of modified protein A Corynex (registered trademark) was used to study the expression of these modified proteins A. In the expression using Corynex (registered trademark), the CspB fusion method (WO2013 / 062029), that is, a technique that improves the secretory production of a target protein by inserting a base sequence encoding an amino acid sequence containing the N-terminal three residues Gln-Glu-Thr (QET) of a CspB mature protein between a base sequence encoding a signal peptide and a base sequence encoding a target protein, was utilized. In addition, since the N-terminus of the CspB tag is Q, there is an advantage that after the signal sequence is cleaved, the first residue Q is pyroglutamylated to protect the N-terminal amino group. In particular, for QET-Z34C1 and QET-Z34C2, these findings were utilized to successfully achieve high secretory expression of modified protein A while minimizing the additional sequence. That is, in addition to the above rules A) and B), it is effective to consider the following rule C) in the expression of affinity proteins using Corynex (registered trademark). C) Addition of three QET residues to the N-terminus improves secretion efficiency with Corynex® Below, an example of expression study using Corynex® is described.

[0389] (1-4) Construction of secretory expression plasmids for CspB6Tev-QZ34C, QZ34C-60, QET-Z34C1, and QET-Z34C2 As modified protein A (modified Z34C), four types of amino acid sequences, CspB6Tev-QZ34C, QZ34C-60, QET-Z34C1, and QET-Z34C2, were designed, and the base sequences encoding these proteins were designed taking into account the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed to enable secretory expression by C. glutamicum.

[0390] CspB6Tev-QZ34C is a fusion protein (hereinafter referred to as "CspBss-CspB6Tev-QZ34C") of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 6 amino acid residues QETNPT (SEQ ID NO: 11) of the CspB mature protein derived from the same strain, the recognition sequence ENLYFQ (SEQ ID NO: 12) of the proTev protease, and QZ34C. The base sequence and amino acid sequence encoding the designed CspBss-CspB6Tev-QZ34C are shown in SEQ ID NOs: 13 and 14, respectively. CspB6Tev-QZ34C is cleaved with proTev protease after secretion expression to remove the CspB6Tev sequence QETNPTENLYFQ (SEQ ID NO: 15), so that the QZ34C protein can be obtained.

[0391] Nucleotide sequence encoding CspBss-CspB6Tev-QZ34C atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccaacccaaccgagaacctgtact tccagcagaagaacatgcagtgtcagcgtcgcttctacgaggccctccacgaccctaacctcaacgaggaacagcgtaacgcgcgtattcgctccatccgcgatgactgctaa (SEQ ID NO: 13)

[0392] Amino acid sequence of CspBss-CspB6Tev-QZ34C MFNNRIRTAALAGAIAISTAASGVAIPAFAQETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 14)

[0393] QZ34C-60 is a fusion protein of the 25 amino acid residues of the signal peptide of CspA derived from C. ammoniagenes ATCC6872 strain and QZ34C-60 (hereinafter referred to as "CspAss-QZ34C-60"). The nucleotide sequence and amino acid sequence encoding the designed CspAss-QZ34C-60 are shown in SEQ ID NOs: 16 and 17, respectively.

[0394] Nucleotide sequence encoding CspAss-QZ34C-60 atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcacagaccgcagacaaccagaagaacatgcagtgtcagcgtcgcttctacgaggccctcca cgaccctaacctcaacgaggaacagcgtaacgcgcgtattcgctccatccgcgatgactgctcccagtccgcaaacctcctcgcagaggcacagcagctcaacgacgcacaggcaccacaggcataa (SEQ ID NO: 16)

[0395] Amino acid sequence of CspAss-QZ34C-60 MKRMKSLAAALTVAGAMLAAPVATAQTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 17)

[0396] QET-Z34C1 is a fusion protein of the 30 amino acid residue signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the mature CspB protein derived from the same strain, and Z34C1 (hereinafter referred to as "CspBss-QET-Z34C1"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-QET-Z34C1 are shown in SEQ ID NOs: 18 and 19, respectively.

[0397] Nucleotide sequence encoding CspBss-QET-Z34C1 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccaagaaca tgcagtgtcagcgtcgcttctacgaggccctccacgaccctaacctcaacgaggaacagcgtaacgcgcgtattcgctccatccgcgatgactgctaa (SEQ ID NO: 18)

[0398] Amino acid sequence of CspBss-QET-Z34C1 MFNNRIRTAALAGAIAISTAASGVAIPAFAQETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 19)

[0399] QET-Z34C2 was designed as a fusion protein of the 30 amino acid residue signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the mature CspB protein derived from the same strain, and Z34C2 (hereinafter referred to as "CspBss-QET-Z34C2"), and a base sequence encoding the fusion protein. The base sequence and amino acid sequence encoding the designed CspBss-QET-Z34C2 are shown in SEQ ID NOs: 20 and 21, respectively.

[0400] Nucleotide sequence encoding CspBss-QET-Z34C2 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccttcaaca agcagtgtcagcgtcgcttctacgaggccctccacgaccctaacctcaacgaggaacagcgtaacgcgcgtattcgctccatccgcgatgactgctaa (SEQ ID NO: 20)

[0401] Amino acid sequence of CspBss-QET-Z34C2 MFNNRIRTAALAGAIAISTAASGVAIPAFAQETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 21)

[0402] We designed and synthesized four expression cassettes for modified protein A, each of which had the promoter of the cspB gene derived from C. glutamicum ATCC13869 linked upstream of the nucleotide sequences described in CspBss-CspB6Tev-QZ34C, CspAss-QZ34C-60, CspBss-QET-Z34C1, and CspBss-QET-Z34C2, and further had a KpnI site added to the 5'-end and a BamHI site added to the 3'-end. The totally synthesized DNA fragment (expression cassette for modified protein A) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to obtain the modified protein A secretion expression plasmids pPK4_CspBss-CspB6Tev-QZ34C, pPK4_CspAss-QZ34C-60, pPK4_CspBss-QET-Z34C1, and pPK4_CspBss-QET-Z34C2, respectively. The results of sequencing the inserts confirmed that the expression cassettes for modified protein A were constructed as designed. The sequences were determined using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3130 Genetic Analyzer (Applied Biosystems).

[0403] (1-5) Secretory expression of each modified protein A in C. glutamicum Using the pPK4_CspBss-CspB6Tev-QZ34C, pPK4_CspAss-QZ34C-60, pPK4_CspBss-QET-Z34C1, and pPK4_CspBss-QET-Z34C2 constructed above, C. glutamicum described in WO2016 / 171224 was The YDK010::phoS(W302C) strain was transformed to obtain the YDK010::phoS(W302C) / pPK4_CspBss-CspB6Tev-QZ34C strain, the YDK010::phoS(W302C) / pPK4_CspAss-QZ34C-60 strain, the YDK010::phoS(W302C) / pPK4_CspBss-QET-Z34C1 strain, and the YDK010::phoS(W302C) / pPK4_CspBss-QET-Z34C2 strain. The C. glutamicum YDK010::phoS(W302C) strain was obtained by replacing the wild-type phoS gene of C. glutamicum YDK010 described in WO2002 / 081694 with the mutant phoS(W302C) gene. The mutant phoS(W302C) gene encodes a PhoS protein (W302C) in which the tryptophan residue at position 302 is replaced with a cysteine ​​residue. The YDK010 strain is a deletion strain of the cell surface protein PS2 (CspB) of the C. glutamicum AJ12036 strain (FERM BP-734). The AJ12036 strain was originally deposited as an international deposit at the Microbial Research and Development Institute, Agency of Industrial Science and Technology (currently the Patent Organism Depositary Center, National Institute of Technology and Evaluation, Japan; postal code: 292-0818; address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on March 26, 1984, and has been assigned the accession number FERM BP-734.

[0404] The resulting transformants were grown in MMTG liquid medium (120 g glucose, 3 g magnesium sulfate heptahydrate, 30 g ammonium sulfate, 1.5 g potassium dihydrogen phosphate, 0.03 g iron sulfate heptahydrate, 0.03 g manganese sulfate pentahydrate, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 1.0 mg DL-methionine) containing 25 mg / L kanamycin. The culture was performed at 30°C for 72 hours using 0.15 g of the yeast, soybean hydrochloric acid hydrolyzate (total nitrogen content 0.2 g), 50 g of calcium carbonate, and water (total volume 1 L, pH adjusted to 7.0).

[0405] After the culture was completed, each culture solution was centrifuged, and 6.5 μL of the resulting culture supernatant was subjected to reducing SDS-PAGE and stained with Quick-CBB (Wako). As a result, a protein band estimated to be CspB6Tev-QZ34C was observed in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-CspB6Tev-QZ34C strain (Fig. 5, lane 2), a protein band estimated to be QZ34C-60 was observed in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspAss-QZ34C-60 strain (Fig. 5, lane 3), and a protein band estimated to be QET-Z34C1 was observed in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-QET-Z34C1 strain (Fig. 5, lane 4). 4), and a protein band estimated to be QET-Z34C2 was detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-QET-Z34C2 strain (Fig. 5, lane 5).

[0406] (1-6) Construction of secretory expression plasmids for QET-Z34C3 and QET-Z34C4 As modified protein A (modified Z34C), two types of amino acid sequences, QET-Z34C3 and QET-Z34C4, were desi...

Claims

1. A compound or a salt thereof having an affinity substance for an antibody, a cleavable moiety, and a reactive group, The compound has the following formula (I): A-L-B-R (I) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group to the antibody; A compound or a salt thereof, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

2. 2. The compound or salt thereof according to claim 1, wherein the polypeptide comprises a tripeptide (QET) consisting of glutamine residue (Q)-glutamic acid (E)-threonine residue (T) at the N-terminus.

3. 3. The compound or salt thereof according to claim 1 or 2, wherein the glutamine residue is pyroglutamylated.

4. The compound or salt thereof according to any one of claims 1 to 3, wherein the polypeptide has 16 to 123 amino acid residues.

5. The compound or salt thereof according to any one of claims 1 to 3, wherein the polypeptide has 20 to 70 amino acid residues.

6. The compound or salt thereof according to any one of claims 1 to 5, wherein the polypeptide is selected from the group consisting of the following (1) to (4): 【Chemistry 1】 [Q is an N-terminal glutamine residue, E is a glutamic acid residue, T is a threonine residue, the solid line is a bond, the spacer portion is a portion consisting of 1 to 50 amino acid residues, and the affinity substance for the antibody is a portion consisting of 15 to 70 amino acid residues.]

7. The compound or salt thereof according to claim 6, wherein the polypeptide moiety having affinity for an antibody is selected from the group consisting of Protein A, Protein G, Protein L, Protein Z, and fragments thereof having affinity for an antibody, and mutants thereof having affinity for an antibody.

8. The compound or salt thereof according to claim 6 or 7, wherein the affinity polypeptide portion for the antibody comprises an amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNAXIXSIRDDC (SEQ ID NO: 5) (wherein each of the two Xs is independently an amino acid residue selected from the group consisting of an arginine residue, a glutamic acid residue, and a glutamine residue), in which any one of the amino acid residues is substituted with a lysine residue, and which has an identity of 90% or more to the amino acid sequence of SEQ ID NO:

5.

9. The compound according to any one of claims 6 to 8, or a salt thereof, wherein the affinity polypeptide portion for the antibody comprises an amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 6) in which any one of the amino acid residues is substituted with a lysine residue and which has 90% or more identity to the amino acid sequence of SEQ ID NO:

6.

10. The compound or salt thereof according to any one of claims 1 to 9, wherein the affinity substance for an antibody comprises an amino acid sequence selected from the group consisting of: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 7); (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 8); (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 9); (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 10); (5) QETFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDC (SEQ ID NO: 22); (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 23); (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:51); (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:52); (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO:53); (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 24); and (11) QETRGNCAYHKGQIIWCTYH (sequence number 25).

11. A regioselective modification reagent for an antibody, comprising: The following formula (I): A-L-B-R (I) [During the ceremony, A is an affinity substance for the antibody, L is a cleavable linker that is a divalent group that contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R is a reactive group to the antibody.], and A site-selective modification reagent for an antibody, wherein the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

12. An affinity substance for an antibody, and an antibody or a salt thereof having a cleavable moiety, The following formula (II): A-L-B-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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is a moiety resulting from the reaction between the antibody and the reactive group, T is an antibody; An antibody or a salt thereof, wherein the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

13. A complex or a salt thereof comprising an affinity substance for an antibody, a cleavable moiety, a functional substance, and an antibody, The following formula (III): A-L-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 contains a cleavable moiety; B' is a trivalent group that includes a moiety that is generated by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; R' is a moiety resulting from the reaction between the antibody and the reactive group, T is an antibody; A conjugate or a salt thereof, wherein the affinity substance for an antibody is a polypeptide containing a glutamine residue (Q) at the N-terminus.

14. A method for producing an antibody or a salt thereof having a bioorthogonal functional group, comprising the steps of: The following formula (II): A-L-B-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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is a moiety resulting from the reaction between the antibody and the reactive group, and T is an antibody.], and the cleavable portion of the antibody or its salt having the cleavable portion is cleaved to obtain 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 in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

15. A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: The following formula (III): A-L-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 contains a cleavable moiety; B' is a trivalent group that includes a moiety that is generated by reaction between a functional agent and a bioorthogonal functional group; F is a functional material; R' is a moiety resulting from the reaction between the antibody and the reactive group, and T is an antibody.], by cleaving the cleavable portion of a complex or a salt thereof having an affinity substance for an antibody, a cleavable portion, a functional substance, and an antibody, 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; B', F, R' and T are the same as those of the formula (III), A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

16. A method for producing an antibody or a salt thereof having a functional substance, comprising the steps of: (A) a compound represented by the following formula (II): A-L-B-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 contains a cleavable moiety; B is (a) a divalent group that contains a bioorthogonal functional group, or (b) a divalent group that does not contain a bioorthogonal functional group; R' is a moiety resulting from the reaction between the antibody and the reactive group, and T is an antibody.], and the cleavable portion of the antibody or its salt having the cleavable portion is cleaved to obtain 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 antibody 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 material; 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), B' is a trivalent group that includes a moiety that is generated by reaction between a functional agent and a bioorthogonal functional group; F a and F b are the same or different functional substances, A method in which the affinity substance for the antibody is a polypeptide containing a glutamine residue (Q) at its N-terminus.

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