Affinity substances, compounds, antibodies, and salts thereof

Abstract

The objective is to develop a technology that allows for the easy chemical modification of only one of the heavy chains in an antibody's constituent unit (in other words, an immunoglobulin unit containing two heavy chains and, if necessary, two light chains). [Solution] The present invention provides a technique for easily chemically modifying only one of the heavy chains in an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the present invention provides (A) an affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and (B) a compound or salt thereof comprising a reactive group for the antibody.

Description

[Technical Field]

[0001] The present invention relates to affinity substances and their salts, as well as compounds containing affinity substances, antibodies, and salts thereof. [Background technology]

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

[0003] ADCs, including T-DM1, have been plagued by heterogeneity since their initial development. For example, because small-molecule drugs are randomly reacted with approximately 70-80 lysine residues in the antibody, the drug-antibody ratio (DAR) and conjugation site are not constant. Typically, such random conjugation methods result in a DAR ranging from 0 to 8, producing multiple antibody drugs with different drug binding numbers. Recently, it has been reported that changing the number and binding site of drugs in an ADC alters pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs require control over the number and location of conjugated drugs. It is believed that maintaining a constant number and location will resolve issues such as expected efficacy, variations in conjugated drugs, and lot-to-lot differences, or regulation problems.

[0004] While regioselective modification methods for antibodies are being studied worldwide, most of these methods involve genetic engineering or enzymatic modification. Regarding genetic engineering, although regioselectivity and number selectivity can be controlled, problems have been pointed out, such as a decrease in the expression efficiency of the antibody itself (a decrease in the total yield when preparing ADCs). Furthermore, the long time required to construct antibody expression systems is also a problem.

[0005] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed, enabling regioselective modification of antibodies using chemical synthesis techniques (Patent Document 1). This method successfully modifies antibodies regioselectively by reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide. However, in ADCs produced by this method, the antibody and drug are linked via a linker containing the peptide portion. The peptide portion has potential immunogenicity and is easily hydrolyzed in the blood. Therefore, there is room for improvement in ADCs produced by this method because they contain a peptide portion in the linker.

[0006] As an improvement to the above C-CAP method, a technique has been reported that allows for the preparation of antibodies that regioselectively contain a functional substance (e.g., a drug) without including the peptide portion as a linker, by a chemical synthesis method using a predetermined compound containing an affinity peptide (Patent Documents 2-5). Furthermore, Patent Document 6 discloses a technique for the large-scale and simple production of affinity peptides by utilizing an affinity peptide containing glutamine-glutamic acid-threonine (QET) at its N-terminus in the preparation of compounds containing affinity peptides. Avoiding the use of linkers containing the peptide portion is desirable in clinical applications. These techniques propose multiple positions in antibodies that can be regioselectively modified with drugs, corresponding to various amino acid residues in the CH2 and CH3 domains (e.g., lysine residues, tyrosine residues, serine residues, and threonine residues). However, the position of the antibody with a functional substance... Selective modification and controlling the binding ratio between the antibody and the functional substance to a desired range is not always easy. In particular, it is not easy to specifically modify only one of the heavy chains in the constituent unit of the antibody (an immunoglobulin unit containing two heavy chains).

[0007] Incidentally, Patent Document 6 discloses that (i) a peptide molecule containing first and second physiologically active peptide sites and a peptide linker can regulate the activity of a protein by non-covalently binding to the protein, and (ii) a peptide linker of a predetermined length containing an amino acid sequence consisting of amino acid residues of proline (P), alanine (A), and serine (S). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2016 / 186206 [Patent Document 2] International Publication No. 2018 / 199337 [Patent Document 3] International Publication No. 2019 / 240287 [Patent Document 4] International Publication No. 2019 / 240288 [Patent Document 5] International Publication No. 2020 / 090979 [Patent Document 6] International Publication No. 2021 / 112249 [Overview of the project] [Problems that the invention aims to solve]

[0009] The objective of the present invention is to develop a technique that allows for the easy chemical modification of only one of the heavy chains in an antibody's constituent unit (in other words, an immunoglobulin unit containing two heavy chains and, optionally, two light chains).

[0010] A further object of the present invention is to develop regioselectively modified antibodies that can be easily chemically modified in only one of the heavy chains in the constituent units of an antibody. [Means for solving the problem]

[0011] As a result of diligent research, the inventors have found that by using (A) affinity substances containing first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and (B) a compound containing a reactive group for the antibody or a salt thereof, it is possible to easily chemically modify only one of the heavy chains in the constituent units of an antibody.

[0012] The compound or salt of the present invention can associate with two heavy chains in an antibody unit via an affinity substance (A) containing first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and then react specifically with the side chain of a specific amino acid residue in one of the heavy chains in the antibody unit via a reactive group (R) for the antibody to produce an affinity substance-modified antibody or salt thereof in which only one of the two heavy chains in the antibody unit is modified (Figure 1). Although we do not wish to be constrained by theory, the mechanism by which the compound or salt of the present invention modifies only one of the heavy chains in an antibody unit is as follows: Because the affinity substance contained in the compound or salt of the present invention (including first and second affinity moieties having affinity for the constant region of the antibody heavy chain) can stably associate with the constant region of the two heavy chains in the antibody unit, the reactive group contained in the compound or salt of the present invention can modify only one of the heavy chains (Figure 1). In this case, the constant region of the other heavy chain (the constant region of the unmodified heavy chain) has affinity moieties associated with it, resulting in steric hindrance. Therefore, other molecules (the compound of the present invention or its salt) cannot associate with the constant region of the other heavy chain via the affinity substance they contain. The compound of the present invention or its salt can associate with the constant regions of the two heavy chains via the two affinity moieties contained in the affinity substance, and therefore can stably associate with the constituent units of the antibody, thereby highly suppressing the association of other molecules with the constituent units of the antibody. Consequently, the compound of the present invention or its salt can highly suppress modification of the constant region of the other heavy chain, and can modify only the constant region of one heavy chain (Figure 1).

[0013] Patent documents 1 to 5 disclose that antibodies can be regioselectively modified with functional substances by using affinity substances and compounds containing reactive groups for antibodies. However, they do not describe or suggest (1) the challenge of developing a technique to easily chemically modify only one heavy chain in the constituent units of an antibody, nor (2) the use of a substance as an affinity substance that contains first and second affinity moieties having affinity for the constant region of the heavy chain of the antibody (in particular, the technical concept of chemically modifying only one heavy chain in the constituent units of an antibody by using such affinity substance and a compound containing a reactive group for antibodies or a salt thereof).

[0014] Patent Document 6 discloses that the activity of a protein can be regulated by a peptide molecule containing first and second physiologically active peptide sites on a protein, as well as a predetermined peptide linker, binding to the protein non-covalently. However, it does not describe or suggest (1) the challenge of developing a technique to easily chemically modify only one of the heavy chains in the constituent unit of an antibody, nor (2) the use of a substance containing first and second affinity sites that have affinity for the constant region of the heavy chain of the antibody as an affinity substance (in particular, the technical concept of chemically modifying only one of the heavy chains in the constituent unit of an antibody by using such an affinity substance and a compound or salt thereof containing a reactive group for the antibody).

[0015] The inventors have also succeeded in generating antibodies in which only one heavy chain in an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains) is chemically modified, by using the compounds of the present invention or salts thereof. Such antibodies are characterized by comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a modification unit (e.g., the affinity substance, bioorthogonal functional group, or functional substance), and (c) the modification unit being introduced only in the constant region of one heavy chain in the immunoglobulin unit.

[0016] In other words, the present invention is as follows: [1] A compound or salt thereof comprising (A) an affinity substance having first and second affinity moieties having affinity for the constant region of the heavy chain of an antibody, and (B) a reactive group for the antibody. [2] A compound of [1] or a salt thereof, wherein the constant region is the Fc region. [3] A compound or salt of [1] or [2], wherein the constant region is a CH2 domain. [4] Any compound or salt of [1] to [3] whose constant region is the human constant region. [5] Any compound or salt thereof from [1] to [4], wherein the antibody is IgG. [6] Any compound or salt thereof from [1] to [5], wherein the first and second affinity moieties are different affinity moieties. [7] The affinity substance is given by the following formula (A): AP1-L A -AP2 (A) [During the ceremony, AP1 exhibits a first affinity peptide that has affinity for the constant region in the heavy chain of the antibody. AP2 exhibits a second affinity peptide that has affinity for the constant region in the heavy chain of the antibody. L A indicates a linker. A compound or salt thereof from any of [1] to [6] represented by ]. [8] A compound or salt thereof from any of [1] to [7], wherein the affinity substance (i)(i-1) contains only one specific reactive group and (i-2) is linked to a reactive group for the antibody via the specific reactive group. [9] Any compound from [1] to [8] or a salt thereof, wherein the affinity substance is an affinity polypeptide comprising first and second affinity peptides having affinity for the constant region in the heavy chain of the antibody.

[10] Affinity polypeptide is given by the following formula (A'): AP1-PL A -AP2 (A') [During the ceremony, AP1 exhibits affinity for the constant region of the antibody heavy chain and represents a first affinity peptide located at the N-terminus of the affinity polypeptide. AP2 exhibits affinity for the constant region of the antibody heavy chain and represents a second affinity peptide located at the C-terminus of the affinity polypeptide. PL A indicates a peptide linker. A compound or salt thereof from any of [1] to [6] represented by ].

[11] A compound or salt of any of [1] to

[10] wherein the affinity polypeptide (i) contains only one amino acid residue having an amino group in its side chain, and (i-2) is linked to a reactive group for the antibody via the amino group, or (ii) is linked to a reactive group for the antibody via the N-terminal amino group in the first affinity peptide.

[12] A compound or salt of

[11] wherein the amino acid residue having an amino group in its side chain is a lysine residue.

[13] A compound or salt thereof from any of [1] to

[12] , wherein the affinity polypeptide further comprises a tripeptide consisting of Gln-Glu-Thr(QET) at its N-terminus.

[14] A compound of

[10] or a salt thereof, wherein the peptide linker has a length of 20 or more amino acid residues.

[15] One of the first and second affinity peptides is an affinity peptide that has affinity for the constant region of the antibody heavy chain and has one lysine residue, and A compound or salt thereof from any of [1] to

[14] , wherein the other of the first and second affinity peptides is an affinity peptide that has affinity for the constant region of the antibody heavy chain and does not contain a lysine residue.

[16] Affinity peptides having affinity for the constant region of the antibody heavy chain and having one lysine residue are as follows (1)~(4): (1) Affinity peptide containing the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (2) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​residues are substituted with other amino acid residues other than lysine and cysteine ​​residues in the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (3) Affinity peptides containing the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and (4) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​residues are substituted with other amino acid residues other than lysine and cysteine ​​residues in the amino acid sequence of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39), It is one of the following (where the two cysteine ​​residues in the amino acid sequence may be crosslinked by a disulfide bond), and / or Affinity that has affinity for the constant region of the antibody heavy chain and does not contain lysine residues. Peptides are as follows (5)~(10): (5) Affinity peptide containing the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40); (6) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than cysteine ​​residues are substituted with lysine residues and other amino acid residues other than cysteine ​​residues in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40), and (7) Affinity peptide containing the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41); (8) An affinity peptide comprising an amino acid sequence in which one or two amino acid residues other than cysteine residues in the amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41) are substituted with another amino acid residue other than lysine residues and cysteine residues, and having an affinity for the constant region in the heavy chain of an antibody; (9) An affinity peptide comprising the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and (10) An affinity peptide comprising an amino acid sequence in which one or two amino acid residues other than cysteine residues in the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78) are substituted with another amino acid residue other than lysine residues and cysteine residues, and having an affinity for the constant region in the heavy chain of an antibody; Any one of the above (wherein the two cysteine residues contained in the amino acid sequence may be crosslinked by a disulfide bond), the compound of

[15] or a salt thereof.

[17] The compound is represented by the following formula (I): [Chemical formula] [In the formula, R represents the reactive group, L represents a linker, A represents the affinity substance.], a compound of any one of [1] to

[16] or a salt thereof.

[18] The compound or a salt thereof further comprises a cleavable moiety between (i) the affinity substance and (ii) the reactive group, a compound of any one of [1] to

[17] or a salt thereof.

[19] The cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, the compound of

[18] or a salt thereof.

[20] The compound is represented by the following formula (Ia): [Chemical formula] [In the formula, R represents the reactive group, L1 represents the first linker, L2 represents the second linker, CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side upon cleavage, A represents the affinity substance. ] The compound of

[19] or a salt thereof represented by.

[21] The compound is represented by the following formula (Ia-1):

Chemical formula

[19] or a salt thereof represented by.

[22] The compound of

[21] or a salt thereof in which the leaving group is selected from the following: (a) R A -S (where R A represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and S represents a sulfur atom.); (b) R A -O (where R A represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and O represents an oxygen atom.); (c) R A -(R B -)N (where R A and R B each independently represent a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent, and N represents a nitrogen atom.); or (d) A halogen atom. The compound or its salt according to any one of [1] to

[17] , wherein the compound or its salt further comprises a bioorthogonal functional group between (ii) the reactive group and (iii) the cleavable moiety. 〔24〕The compound is represented by the following formula (Ib):

Chemical formula

[23] . 〔25〕The compound is represented by the following formula (Ib-1): <0OO0955>

Chemical formula

[23] . 〔26〕The compound or its salt according to any one of

[23] to

[25] , wherein the bioorthogonal functional group is an azide residue, an alkyne residue, a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a furan residue, or a halocarbonyl residue.

[0017] 〔27〕A reagent for antibody derivatization, comprising the compound or its salt according to any one of [1] to

[26] . [[ID=5,4]]

[0018]

[28] An affinity substance-modified antibody or a salt thereof, comprising an affinity substance containing first and second affinity moieties having affinity for the constant region of the antibody heavy chain, within the constant region of the antibody heavy chain.

[29] A salt of the affinity substance-modified antibody or salt of the antibody or salt of the antibody or salt of the antibody or salt of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody of the antibody, wherein (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) the affinity substance is introduced only in a constant region of one of the heavy chains of the immunoglobulin unit.

[30] The affinity substance modified antibody or salt thereof according to

[29] , wherein the affinity substance is introduced only into the constant region of the one heavy chain by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the one heavy chain.

[31] Affinity-modified antibody or salt of

[30] , wherein one or more of the above positions are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[32] Affinity-modified antibody is given by the following formula (II): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. L indicates the linker. A represents the affinity substance, The average modification percentage r of the immunoglobulin units by the affinity substance is 65 to 135. An affinity-modified antibody or salt thereof, containing a structural unit represented by

[28] to

[31] .

[33] An affinity substance-modified antibody or salt thereof according to any of

[28] to

[32] , wherein the antibody or salt thereof further comprises (i') the affinity substance and (ii') the antibody and (iii') the antibody, wherein the antibody or salt thereof further comprises (iii') a cleavable moiety.

[34] An affinity-modified antibody or salt of

[33] , wherein the cleavable portion is a cleavable portion capable of generating a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[35] Affinity-modified antibody is given by the following formula (IIa): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L1 indicates the first linker. L2 indicates the second linker. CLE(B) represents a cleavable portion that can generate bioorthogonal functional groups on the immunoglobulin unit side by cleavage. A represents the affinity substance, The average percentage r of modification of the immunoglobulin unit by the affinity substance is 65-135%.

[34] Affinity substance-modified antibody or salt thereof containing a structural unit represented by

[36] formula (IIa-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. L3 indicates the third linker. L4 indicates the fourth linker. S represents a sulfur atom. A represents the affinity substance, The average percentage r of modification of the immunoglobulin unit by the affinity substance is 65-135%.

[34] An affinity substance-modified antibody or salt thereof of

[34] containing a structural unit represented by ].

[37] An affinity substance-modified antibody or salt thereof of

[33] further comprising (ii') a bioorthogonal functional group between the antibody and (iii') a cleavable moiety.

[38] Affinity-modified antibody is given by the following formula (IIb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L5 indicates the fifth linker. L6 indicates the sixth linker. B represents a group containing a bioorthogonal functional group. CLE indicates the cleavage portion. A represents the affinity substance, The average percentage r of modification of the immunoglobulin unit by the affinity substance is 65-135%.

[37] Affinity substance-modified antibody or salt thereof containing a structural unit represented by

[39] formula (IIb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. L7 indicates the seventh linker. L8 indicates the 8th linker. B represents a group containing a bioorthogonal functional group. V represents an oxygen atom or a sulfur atom. A represents the affinity substance, The average percentage r of modification of the immunoglobulin unit by the affinity substance is 65-135%.

[37] Affinity substance-modified antibody or salt thereof, comprising a structural unit represented by

[37] .

[40] Any affinity substance-modified antibody or salt thereof from

[28] to

[39] , wherein the affinity substance-modified antibody further comprises an additional modification moiety.

[41] The affinity-modified antibody or salt thereof of

[40] , wherein the additional modification portion is an additional affinity substance comprising a third affinity portion having affinity for a constant region in the heavy chain of the antibody, and the additional affinity substance is contained in the constant region in the heavy chain of the antibody.

[42] The affinity-modified antibody or salt thereof of

[41] , wherein the additional affinity substance is introduced into the constant region of the two heavy chains by modifying the amino group in the side chain of a lysine residue located at one or more positions in the constant region of the two heavy chains.

[43] One or more positions in the constant region of the two heavy chains are positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering.

[42] Affinity-modified antibody or a salt thereof.

[0019]

[44] A method for producing an affinity-modified antibody or a salt thereof, comprising reacting (A) an affinity substance having first and second affinity moieties having affinity for the constant region of the heavy chain of the antibody, and (B) a compound having a reactive group for the antibody or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and optionally two light chains, to produce an affinity-modified antibody or a salt thereof containing the affinity substance in the constant region of the heavy chain of the immunoglobulin unit.

[45] The method of

[44] , wherein the compound or a salt thereof further comprises (i) the affinity substance and (ii) the reactive group, and (iii) a cleavable moiety.

[46] The method of

[45] , wherein the cleavable portion is a cleavable portion that can generate a bioorthogonal functional group on the reactive group side by cleavage.

[47] The method of

[44] , wherein the compound or a salt thereof further comprises (ii) a bioorthogonal functional group between (ii) the reactive group and (iii) the cleavable moiety.

[48] ​​The method of

[44] , wherein the affinity-modified antibody or a salt thereof is one of the affinity-modified antibodies or a salt thereof from

[29] to

[43] .

[0020]

[49] An antibody derivative or salt thereof comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a bioorthogonal functional group, wherein (c) the bioorthogonal functional group is introduced only in the constant region of one of the heavy chains of the immunoglobulin unit.

[50] An antibody derivative or salt of

[49] , wherein a bioorthogonal functional group is introduced only into the constant region of the one heavy chain by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the one heavy chain.

[51] An antibody derivative or salt thereof of

[49] or

[50] , wherein one or more positions in the constant region of one of the heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[52] An antibody derivative or salt containing a bioorthogonal functional group is given by the following formula (IIIa): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L1 indicates the first linker. B represents a group containing a bioorthogonal functional group. The average modification percentage r of the immunoglobulin unit by bioorthogonal functional groups is 65-135%. An antibody derivative or salt of any of

[49] -

[51] containing a structural unit represented by ].

[53] Antibody derivatives or salts containing bioorthogonal functional groups are given by the following formula (IIIa-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W1 represents an oxygen atom or a sulfur atom. L3 indicates the third linker. SH indicates a thiol group. The average modification percentage r of the immunoglobulin unit by bioorthogonal functional groups is 65-135%. An antibody derivative or salt of any of

[49] -

[51] containing a structural unit represented by ].

[54] Antibody derivatives or salts containing bioorthogonal functional groups, as shown in formula (IIIb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L5 indicates the fifth linker. B represents a group containing a bioorthogonal functional group. T1 indicates a monovalent group. The average modification percentage r of the immunoglobulin unit by bioorthogonal functional groups is 65-135%. An antibody derivative or salt of any of

[49] -

[51] containing a structural unit represented by ].

[55] Antibody derivatives or salts containing bioorthogonal functional groups, as shown in formula (IIIb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W1 and W2 each independently represent either an oxygen atom or a sulfur atom. L7 indicates the seventh linker. B represents a group containing a bioorthogonal functional group. T2 indicates a monovalent group. The average modification percentage r of the immunoglobulin unit by bioorthogonal functional groups is 65-135%. An antibody derivative or salt of any of

[49] -

[51] containing a structural unit represented by ].

[56] Any antibody derivative or salt thereof from

[49] to

[55] , wherein the antibody derivative further comprises an additional modification moiety.

[57] The additional modification portion is an additional modification portion containing a bioorthogonal functional group, and the bioorthogonal An antibody derivative or salt thereof of

[56] , wherein an additional modification moiety containing an interconnecting functional group is included in the constant region of the antibody heavy chain.

[58] An antibody derivative or salt of

[57] wherein the additional modification moiety containing the bioorthogonal functional group is introduced into the constant region of the two heavy chains by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the two heavy chains.

[59] An antibody derivative or salt of

[58] wherein one or more positions in the constant region of the two heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[0021]

[60] A conjugate of an antibody and a functional substance or a salt thereof, comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a functional substance, wherein (c) the functional substance is introduced only into a constant region of one of the heavy chains in the immunoglobulin unit.

[61] A conjugate or salt of

[60] wherein the functional substance is introduced only into the steady region of the one heavy chain by modification of an amino group in the side chain of a lysine residue located at one or more positions in the steady region of the one heavy chain.

[62] A conjugate or salt of

[60] or

[61] , wherein one or more positions in the constant region of one of the heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[63] The conjugate or its salt is given by the following formula (IVa): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L1 indicates the first linker. Z indicates a functional substance. The average percentage modification r of the immunoglobulin unit by the functional substance is 65-135%. A conjugate or salt of any of

[60] -

[62] containing the structural unit represented by [ ].

[64] The conjugate or its salt is given by the following formula (IVa-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W1 represents an oxygen atom or a sulfur atom. L3 indicates the third linker. Z indicates a functional substance. The average percentage modification r of the immunoglobulin unit by the functional substance is 65-135%. A conjugate or salt of any of

[60] -

[62] containing the structural unit represented by [ ].

[65] The conjugate or its salt is given by the following formula (IVb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L5 indicates the fifth linker. Z indicates a functional substance. T1 indicates a monovalent group. The average percentage modification r of the immunoglobulin unit by the functional substance is 65-135%. A conjugate or salt of any of

[60] -

[62] containing the structural unit represented by [ ].

[66] The conjugate or its salt is given by the following formula (IVb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W1 and W2 each independently represent either an oxygen atom or a sulfur atom. L7 indicates the seventh linker. Z indicates a functional substance. T2 indicates a monovalent group. The average percentage modification r of the immunoglobulin unit by the functional substance is 65-135%. A conjugate or salt of any of

[60] -

[62] containing the structural unit represented by [ ].

[67] A conjugate or salt thereof of any of

[60] to

[66] , wherein the functional substance is a drug, labeling substance, affinity substance, transport substance, or stabilizer.

[68] A conjugate or salt of

[67] wherein the affinity substance is a full-length antibody or a fragment thereof.

[69] Any conjugate or salt thereof from

[60] to

[68] , wherein the conjugate further includes additional modifying parts.

[70] A conjugate or salt thereof of

[69] , wherein the additional modification portion is an additional modification portion containing a functional substance, and the additional modification portion containing the functional substance is included in a constant region of the heavy chain of the antibody.

[71] A conjugate of

[70] or a salt thereof, wherein the additional modified portion containing the functional substance is introduced into the constant region of the two heavy chains by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the two heavy chains.

[72] A conjugate or salt of

[71] , wherein one or more positions in the constant region of the two heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[0022]

[73] A method for producing an antibody or salt that does not contain an affinity substance, comprising (A) an affinity substance having first and second affinity moieties having affinity for a constant region in the heavy chain of an antibody, and (B) an antibody, and (C) an affinity substance-modified antibody or salt thereof having a cleavable moiety between (A) the affinity substance and (B) the antibody, by cleaving the cleavable moiety to produce an antibody or salt that does not contain an affinity substance.

[74] The cleavable portion is a cleavable portion that can generate bioorthogonal functional groups on the antibody side by cleavage, The method of

[73] , wherein the antibody or salt thereof that does not contain affinity substances is an antibody derivative or salt thereof that contains a bioorthogonal functional group.

[75] The method of

[74] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group is any of the above antibody derivatives or salt thereof.

[76] An antibody or salt thereof that does not contain affinity substances further contains a bioorthogonal functional group between the antibody and the cleavage portion, The method of

[73] , wherein the antibody or salt thereof that does not contain affinity substances is an antibody derivative or salt thereof that contains a bioorthogonal functional group.

[77] The method of

[76] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group is any of the above antibody derivatives or salt thereof.

[78] A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) To produce an antibody derivative or salt thereof containing a bioorthogonal functional group by the method of

[74] : and (2) Reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance.

[79] The method of

[78] , wherein the conjugate or a salt thereof is any of the above conjugates or a salt thereof.

[80] A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) To produce an antibody derivative or salt thereof containing a bioorthogonal functional group by the method of

[76] : and (2) Reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance.

[81] The method of

[80] , wherein the conjugate or a salt thereof is any of the above conjugates or a salt thereof.

[0023]

[82] A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, The process involves reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance. An antibody derivative or salt containing a bioorthogonal functional group is an antibody derivative or salt containing a bioorthogonal functional group, wherein the antibody derivative comprises (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a bioorthogonal functional group, and (c) the bioorthogonal functional group is introduced only in the constant region of one of the heavy chains in the immunoglobulin unit. A method comprising an antibody and a functional substance conjugate or a salt thereof, wherein the conjugate or salt thereof comprises (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a functional substance, and (c) the functional substance is introduced only into the constant region of one of the heavy chains in the immunoglobulin unit.

[83] The method of

[82] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group is any of the above antibody derivatives or salt thereof.

[84] The method of

[82] , wherein the conjugate or a salt thereof is any of the above conjugates or a salt thereof.

[0024]

[85] An affinity substance or a salt thereof comprising first and second affinity moieties having affinity for the constant region of the heavy chain of an antibody.

[86] The affinity substance is given by the following formula (A): AP1-L A -AP2 (A) [During the ceremony, AP1 exhibits a first affinity peptide that has affinity for the constant region in the heavy chain of the antibody. AP2 exhibits a second affinity peptide that has affinity for the constant region in the heavy chain of the antibody. L A indicates a linker. An affinity substance of

[85] or a salt thereof, represented by ].

[87] A salt of the affinity substance of

[85] or

[86] , wherein the affinity substance contains only one specific reactive group.

[88] Any affinity substance or salt thereof from

[85] to

[87] , wherein the affinity substance is an affinity polypeptide comprising first and second affinity peptides having affinity for the constant region in the heavy chain of the antibody.

[89] Affinity polypeptide is given by the following formula (A'): AP1-PL A -AP2 (A') [During the ceremony, AP1 exhibits affinity for the constant region of the antibody heavy chain and represents a first affinity peptide located at the N-terminus of the affinity polypeptide. AP2 exhibits affinity for the constant region of the antibody heavy chain and represents a second affinity peptide located at the C-terminus of the affinity polypeptide. PL A This indicates a peptide linker. It is an affinity substance or salt thereof from any of

[85] to

[88] , represented by ].

[90] Affinity polypeptide comprising (i) only one amino acid residue having an amino group in its side chain, or (ii) an unprotected N-terminal amino group, any affinity substance or salt thereof of

[85] to

[89] .

[91] An affinity substance of

[90] or a salt thereof, wherein the amino acid residue having an amino group in its side chain is a lysine residue.

[92] Affinity polypeptide further comprising a tripeptide consisting of Gln-Glu-Thr(QET) at its N-terminus, any affinity substance or salt thereof from

[85] to

[89] .

[93] A peptide linker having a length of 20 or more amino acid residues, which is an affinity substance or salt thereof of any of

[89] to

[92] .

[94] One of the first and second affinity peptides is an affinity peptide that has affinity for the constant region in the heavy chain of the antibody and has one lysine residue, and An affinity substance or salt thereof from any of

[85] to

[93] , wherein the other of the first and second affinity peptides is an affinity peptide that has affinity for the constant region of the antibody heavy chain and does not contain a lysine residue.

[0025]

[95] A polynucleotide encoding an affinity polypeptide comprising first and second affinity peptides having affinity for the constant region of the heavy chain of an antibody.

[0026] An expression vector comprising the polynucleotides

[96] and

[95] and a promoter operably ligated thereto.

[0027] A host cell comprising an expression unit containing the polynucleotides

[97] and

[95] and a promoter operably linked thereto.

[0028]

[98] An affinity-modified antibody or a salt thereof comprising a first modification portion comprising a first affinity substance comprising a first affinity substance comprising first and second affinity portions having affinity for the constant region of the antibody heavy chain, and a second modification portion comprising a second affinity substance comprising third and fourth affinity portions having affinity for the constant region of the antibody heavy chain, the first and second modification portions comprising the first and second modification portions comprising a first affinity substance having affinity for the constant region of the antibody heavy chain, the first modification portion comprising the first and second modification portions comprising a first affinity substance having affinity for the constant region of the antibody heavy chain, the first modification portion comprising a first affinity substance comprising a first affinity substance having first and second affinity portions having affinity for the constant region of the antibody heavy chain.

[99] (a) an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains, and (b) comprising the first and second modified portions, (c) The first modification portion is introduced into the steady region of the first heavy chain in the immunoglobulin unit, (d) an affinity-modified antibody or salt thereof of

[98] , wherein the second modification is introduced into the constant region of the second heavy chain in the immunoglobulin unit.

[100] Affinity-modified antibody or salt thereof of

[98] or

[99] , wherein the first modified portion is introduced into the constant region of the first heavy chain via modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the first heavy chain, and the second modified portion is introduced into the constant region of the second heavy chain via modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the second heavy chain.

[101] An affinity-modified antibody or salt thereof of

[100] , wherein one or more positions in the constant region of the first heavy chain and one or more positions in the constant region of the second heavy chain are positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering, and combinations thereof.

[102] An affinity-modified antibody containing the first and second modification moieties is given by the following formula (V): [ka] [During the ceremony, Ig represents an immunoglobulin unit consisting of two heavy chains, a first and a second heavy chain, and optionally two light chains. L L and L R Each of these independently indicates a linker. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L、 and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An affinity-modified antibody or salt thereof from any of

[98] to

[101] containing a structural unit represented by ].

[103] An affinity-modified antibody or salt thereof according to any of

[98] to

[102] , wherein the antibody or salt thereof further comprises (i') a first affinity substance and (ii') the immunoglobulin unit, and / or (i'') a second affinity substance and (ii'') the immunoglobulin unit, and (iii'') a second cleavage moiety.

[104] The affinity-modified antibody or salt thereof of

[103] , wherein the first and second cleavable portions are cleavable portions capable of generating bioorthogonal functional groups on the immunoglobulin unit side by cleavage.

[105] An affinity-modified antibody containing the first and second modification moieties is given by the following formula (Va): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L1 and L R1 Each of these independently indicates the first linker. L L2 and L R2 Each of these independently indicates the second linker. CLE(B) L and CLE(B)R Each of these independently represents a cleavable portion capable of generating bioorthogonal functional groups on the immunoglobulin unit side by cleavage. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An affinity-modified antibody or salt thereof from any of

[98] to

[104] containing a structural unit represented by ].

[106] An affinity-modified antibody containing the first and second modification moieties is given by the following formula (Va-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 Each of these independently represents either an oxygen atom or a sulfur atom. L L3 and L R3 Each of these independently indicates the third linker. L L4 and L R4 Each of these independently indicates the fourth linker. S represents a sulfur atom. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion REach of these is 65-135%. An affinity-modified antibody or salt thereof from any of

[98] to

[104] containing a structural unit represented by ].

[107] An affinity-modified antibody or salt of any of

[98] to

[106] wherein the antibody or salt further comprises (ii') a first bioorthogonal functional group between (ii') the immunoglobulin unit and (iii') the first cleavable moiety, and / or (ii'') a second bioorthogonal functional group between (iii'') the immunoglobulin unit and (iii'') the second cleavable moiety.

[108] An affinity-modified antibody containing the first and second modification moieties is given by the following formula (Vb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L5 and L R5 Each of these independently indicates the fifth linker. L L6 and L R6 Each of these independently indicates the sixth linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, CLE L and CLE R Each of these independently indicates a cleavage portion. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An affinity-modified antibody or salt thereof from any of

[98] to

[107] containing a structural unit represented by ].

[109] An affinity-modified antibody containing the first and second modification moieties is given by the following formula (Vb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 Each of these independently represents either an oxygen atom or a sulfur atom. L L7 and L R7 Each of these independently indicates the seventh linker. L L8 and L R8 Each of these independently indicates the eighth linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, V L and V R Each of these independently represents either an oxygen atom or a sulfur atom. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An affinity-modified antibody or salt thereof from any of

[98] to

[108] containing a structural unit represented by ].

[110] The antibody or a salt thereof further comprises (i') a first affinity substance and (ii') a first cleavageable moiety between the immunoglobulin unit and (ii'') a first bioorthogonal functional group between the immunoglobulin unit and (iii'') a second cleavageable moiety, An affinity-modified antibody or salt thereof from any of

[98] to

[109] , wherein the first cleavable portion is a cleavable portion capable of generating a second bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[111] Affinity-modified antibody containing the first and second modification moieties is given by the following formula (Vc): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L R1 This indicates the first linker, L R2 This indicates the second linker, L L5 This indicates the fifth linker, L L6 This indicates the sixth linker, B L This indicates a group containing a first bioorthogonal functional group, CLE L This indicates the first cleavable portion, CLE(B) R This represents a second cleavable portion that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage, A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An affinity-modified antibody or salt thereof from any of

[98] to

[110] containing a structural unit represented by ].

[112] An affinity substance-modified antibody containing the first and second modification parts has the following formula (Vc-1): [Chemical formula] [In the formula, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represents an oxygen atom or a sulfur atom, L R3 represents a third linker, L R4 represents a fourth linker, L L7 represents a seventh linker, L L8 represents an eighth linker, B L represents a group containing a first bioorthogonal functional group, V L represents an oxygen atom or a sulfur atom, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r L of the immunoglobulin unit by the first modification part, and the average modification percentage r R of the immunoglobulin unit by the second modification part are each 65 - 135%.], an affinity substance-modified antibody according to any one of

[98] to

[111] or a salt thereof containing a structural unit represented by the formula.

[113] An affinity substance-modified antibody according to any one of

[98] to

[112] or a salt thereof, wherein the affinity substance-modified antibody further contains an additional modification part.

[114] The affinity-modified antibody or salt thereof of

[113] , wherein the additional modification portion is an additional affinity substance comprising a fifth affinity portion having affinity for a constant region in the heavy chain of the antibody, and the additional affinity substance is contained in the constant region in the heavy chain of the antibody.

[115] The affinity-modified antibody or salt thereof of

[114] , wherein the additional affinity substance is introduced into the constant region of the two heavy chains by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the two heavy chains.

[116] Affinity-modified antibody or salt thereof of

[115] , wherein one or more positions in the constant region of the two heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[0029]

[117] Method for producing an affinity-modified antibody or a salt thereof containing a first and second modified moiety, including the following: (1) Reacting an antibody comprising an immunoglobulin unit comprising two heavy chains and optionally two light chains with a first modified portion comprising the first affinity substance in the constant region of the heavy chain of the antibody, (A) a first affinity substance comprising first and second affinity moieties having affinity for the constant region of the heavy chain of the antibody, and (B) a compound or salt thereof comprising a first reactive group for the antibody, to an antibody to produce an affinity substance-modified antibody or salt thereof comprising a first modified moiety containing the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit; and (2) Reacting an affinity-modified antibody or a salt thereof containing the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with a second affinity substance containing third and fourth affinity moieties having affinity for the constant region of the heavy chain of the antibody, and (B) a compound or a salt thereof containing a second reactive group for the antibody, to produce an affinity-modified antibody or a salt thereof containing the first and second modification moieties in the constant region of the heavy chain of the immunoglobulin unit.

[118] The method of

[117] , wherein the affinity-modified antibody or a salt thereof containing the first and second modification portions is any of the affinity-modified antibodies or salt thereof described above.

[0030]

[119] (a) an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains, and (b) a first modified portion comprising a first bioorthogonal functional group and a second modified portion comprising a second bioorthogonal functional group, (c) The first modification portion is introduced into the steady region of the first heavy chain, (d) The second modification portion is introduced into the steady region of the second heavy chain, (e) An antibody derivative or salt thereof comprising first and second modified moieties, wherein the first and second modified moieties are different from each other.

[120] An antibody derivative or salt of

[119] , wherein the first modified portion is introduced into the steady region of the first heavy chain by modification of an amino group in the side chain of a lysine residue located at one or more positions in the steady region of the first heavy chain, and the second modified portion is introduced into the steady region of the second heavy chain by modification of an amino group in the side chain of a lysine residue located at one or more positions in the steady region of the second heavy chain.

[121] An antibody derivative or salt thereof of

[119] or

[120] , wherein one or more positions in the constant region of the first heavy chain and one or more positions in the constant region of the second heavy chain are positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering, and combinations thereof.

[122] An antibody derivative or salt thereof containing the first and second modified moieties is given by the following formula (VIa): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L1 and L R1 Each of these independently indicates the first linker. B L This represents a first group containing a first bioorthogonal functional group, B RThis indicates a second group containing a second bioorthogonal functional group, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An antibody derivative or salt of any of

[119] -

[121] containing the structural unit represented by ].

[123] An antibody derivative or salt thereof containing the first and second modified moieties is given by the following formula (VIa-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W R1 Each of these independently represents either an oxygen atom or a sulfur atom. L L3 and L R3 Each of these independently indicates the third linker. SH represents a thiol group, which is a bioorthogonal functional group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An antibody derivative or salt of any of

[119] -

[122] containing the structural unit represented by ].

[124] An antibody derivative or salt thereof containing the first and second modified moieties is given by the following formula (VIb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L5 and L R5 Each of these independently indicates the fifth linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, T L1 and T R1 Each of these independently represents a monovalent group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An antibody derivative or salt of any of

[119] -

[123] containing the structural unit represented by ].

[125] An antibody derivative or salt thereof containing the first and second modified moieties is given by the following formula (VIb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W L2 , and W R1 and W R2 Each of these independently represents either an oxygen atom or a sulfur atom. L L7 and L R7 Each of these independently indicates the seventh linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, T L2 and T R2 Each of these independently represents a monovalent group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An antibody derivative or salt of any of

[119] -

[124] containing the structural unit represented by ].

[126] An antibody derivative or salt thereof containing the first and second modified moieties is given by the following formula (VIc): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L R1 This indicates the first linker, L L5 This indicates the fifth linker, B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, T L1 This indicates a monovalent group, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An antibody derivative or salt of any of

[119] -

[125] containing the structural unit represented by ].

[127] An antibody derivative or salt thereof containing the first and second modified moieties is given by the following formula (VIc-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W L2 , and W R1 Each of these independently represents either an oxygen atom or a sulfur atom. L R3 This indicates the third linker. L L7 This indicates the seventh linker, B L This represents a first group containing a first bioorthogonal functional group, SH represents a thiol group, which is a second bioorthogonal functional group. T L2This indicates a monovalent group, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. An antibody derivative or salt of any of

[119] -

[126] containing the structural unit represented by ].

[128] Any antibody derivative or salt thereof from

[119] to

[127] , wherein the antibody derivative further comprises an additional modification moiety.

[129] An antibody derivative or salt thereof of

[128] , wherein the additional modification portion is an additional modification portion containing a bioorthogonal functional group, and the additional modification portion containing the bioorthogonal functional group is included in the constant region of the antibody heavy chain.

[130] An antibody derivative or salt of

[129] , wherein the additional modification moiety containing the bioorthogonal functional group is introduced into the constant region of the two heavy chains by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the two heavy chains.

[131] An antibody derivative or salt of

[130] , wherein one or more positions in the constant region of the two heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering.

[0031]

[132] (a) an immunoglobulin unit comprising two heavy chains consisting of a first and a second heavy chain and optionally two light chains, and (b) a first modified portion comprising a first functional substance and a second modified portion comprising a second functional substance, (c) The first modification portion is introduced into the steady region of the first heavy chain, (d) The second modification portion is introduced into the steady region of the second heavy chain, (e) An antibody in which the first and second modified parts are different from each other, and a conjugate of the first and second modified parts or a salt thereof.

[133] A conjugate or salt of

[132] , wherein the first modified portion is introduced into the steady region of the first heavy chain via modification of an amino group in the side chain of a lysine residue located at one or more positions in the steady region of the first heavy chain, and the second modified portion is introduced into the steady region of the second heavy chain via modification of an amino group in the side chain of a lysine residue located at one or more positions in the steady region of the second heavy chain.

[134] A conjugate or salt thereof of

[132] or

[133] , wherein one or more positions in the steady region of the first heavy chain and one or more positions in the steady region of the second heavy chain are positions selected from the group consisting of positions 246 / 248, 288 / 290, and 317 of a human IgG heavy chain according to EU numbering, and combinations thereof.

[135] The conjugate or its salt is of the following formula (VIIa): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L1 and L R1 Each of these independently indicates the first linker. Z L This indicates the first functional substance, Z R This indicates a second functional substance, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. A conjugate or salt of any of

[132] -

[134] containing the structural unit represented by ].

[136] The conjugate or its salt is of the following formula (VIIa-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W R1 Each of these independently represents either an oxygen atom or a sulfur atom. L L3 and L R3 Each of these independently indicates the third linker. Z L This indicates the first functional substance, Z R This indicates a second functional substance, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. Conjugates or salts of

[132] -

[135] containing the structural unit represented by ].

[137] The conjugate or its salt is of the following formula (VIIb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L5 and L R5 Each of these independently indicates the fifth linker. Z L This indicates the first functional substance, Z R This indicates a second functional substance, T L1 and T R1 Each of these independently represents a monovalent group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. Conjugates or salts of

[132] -

[136] containing the structural unit represented by ].

[138] The conjugate or its salt is of the following formula (VIIb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W L2 , and W R1 and W R2 Each of these independently represents either an oxygen atom or a sulfur atom. L L7 and L R7 Each of these independently indicates the seventh linker. Z L This indicates the first functional substance, Z R This indicates a second functional substance, T L2 and T R2 Each of these independently represents a monovalent group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. Conjugates or salts of

[132] -

[137] containing the structural unit represented by ].

[139] The conjugate or its salt is of the following formula (VIIc): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L R1 This indicates the first linker, L L5 This indicates the fifth linker, Z L This indicates the first functional substance, Z R This indicates a second functional substance, T L1 This indicates a monovalent group, The average modification percentage r of the immunoglobulin units by the first modification portion L, and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. Conjugates or salts of

[132] -

[138] containing the structural unit represented by ].

[140] The conjugate or its salt is given by the following formula (VIIc-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W L2 , and W R1 Each of these independently represents either an oxygen atom or a sulfur atom. L R3 This indicates the third linker. L L7 This indicates the seventh linker, Z L This indicates the first functional substance, Z R This indicates a second functional substance, T L2 This indicates a monovalent group, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R Each of these is 65-135%. Conjugates or salts of

[132] -

[139] containing the structural unit represented by ].

[141] Conjugates or salts thereof of

[132] -

[140] , wherein the first and second functional substances are each independently a drug, a labeling substance, an affinity substance, a transport substance, or a stabilizer.

[142] Either one of the first and second functional substances is a full-length antibody or a fragment thereof, or both of the first and second functional substances are, independently, full-length antibodies as well k is a fragment of the conjugate or salt of

[132] -

[141] .

[143] The conjugate or salt of

[132] -

[142] , in which the conjugate further includes additional modifying parts.

[144] A conjugate or salt thereof of

[143] , wherein the additional modification portion is an additional modification portion containing a functional substance, and the additional modification portion containing the functional substance is included in the constant region of the heavy chain of the antibody.

[145] A conjugate or salt of

[144] wherein the additional modified portion containing the functional substance is introduced into the constant region of the two heavy chains by modification of an amino group in the side chain of a lysine residue located at one or more positions in the constant region of the two heavy chains.

[146] A conjugate or salt of

[145] , wherein one or more positions in the constant region of the two heavy chains are positions 246 / 248, 288 / 290, or 317 of a human IgG heavy chain according to EU numbering. [Effects of the Invention]

[0032] According to the present invention, only one of the heavy chains in an antibody constituent unit (an immunoglobulin unit containing two heavy chains) can be easily modified. Furthermore, according to the present invention, it is possible to provide an antibody that is regioselectively modified while easily modifying only one of the heavy chains in the constituent units of the antibody. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 is a schematic diagram showing the modification of an antibody constituent unit by the compound of the present invention represented by formula (I) or a salt thereof. [Figure 2] Figure 2 is a diagram illustrating an overview of one embodiment of the present invention. [Figure 3] Figure 3 is a diagram illustrating an overview of a different embodiment of the present invention. [Figure 4] Figure 4 shows an overview of yet another embodiment of the present invention. [Figure 5] Figure 5 shows an overview of yet another embodiment of the present invention. [Figure 6] Figure 6 shows an overview of yet another embodiment of the present invention. [Figure 7] Figure 7 shows an overview of one embodiment of the present invention. [Figure 8] Figure 8 is a diagram illustrating an overview of another embodiment of the present invention. [Figure 9] Figure 9 shows an overview of yet another embodiment of the present invention. [Figure 10] Figure 10 shows an overview of yet another embodiment of the present invention. [Figure 11] Figure 11 shows an overview of yet another embodiment of the present invention. [Figure 12] Figure 12 shows an overview of yet another embodiment of the present invention. [Figure 13] Figure 13 shows an overview of yet another embodiment of the present invention. [Figure 14] Figure 14 shows an overview of yet another embodiment of the present invention. [Figure 15] Figure 15 shows an overview of yet another embodiment of the present invention. [Figure 16] Figure 16 shows the expression of each polypeptide in each transformant. [Figure 17-1] Figure 17-1 is a sensorgram showing the affinity between polypeptide QET-Z34CM-PA32-Fc3K (SEQ ID NO: 1) and the Fc region of the IgG1 antibody. [Figure 17-2] Figure 17-2 is a sensorgram showing the affinity between polypeptide QET-Z34CM-PA48-Fc3K (SEQ ID NO: 2) and the Fc region of the IgG1 antibody. [Figure 17-3] Figure 17-3 is a sensorgram showing the affinity between polypeptide QET-Fc3-PA32-Z34CM (SEQ ID NO: 3) and the Fc region of the IgG1 antibody. [Figure 17-4] Figure 17-4 is a sensorgram showing the affinity between polypeptide QET-Fc3K-PA48-Z34CM (SEQ ID NO: 4) and the Fc region of the IgG1 antibody. [Figure 17-5] Figure 17-5 is a sensorgram showing the affinity between polypeptide QET-Fc3K-PA32-ProAR (SEQ ID NO: 5) and the Fc region of the IgG1 antibody. [Figure 17-6] Figure 17-6 is a sensorgram showing the affinity between polypeptide QET-Fc3K-PA48-ProAR (SEQ ID NO: 6) and the Fc region of the IgG1 antibody. [Figure 17-7] Figure 17-7 is a sensorgram showing the affinity between polypeptide QET-ProAR-PA32-Z34CK (SEQ ID NO: 7) and the Fc region of the IgG1 antibody. [Figure 17-8] Figure 17-8 is a sensorgram showing the affinity between polypeptide QET-ProAR-PA48-Z34CK (SEQ ID NO: 8) and the Fc region of the IgG1 antibody. [Figure 18] Figure 18 shows the results of the peptide / antibody binding ratio verification. [Figure 19-1] Figure 19-1 shows the modification sites of lysine residues within the constant region of the antibody heavy chain, as determined by LC-MS / MS. [Figure 19-2] Figure 19-2 shows the modification of the lysine residue at position 246 or 248 of the heavy chain according to EU numbering, as determined by CID spectroscopy. [Figure 19-3] Figure 19-3 shows the selectivity of modification to the lysine residue at position 248, as determined by BioPharma Finder. [Figure 20-1] Figure 20-1 is a sensorogram showing the affinity between EGFR and cetuximab. [Figure 20-2] Figure 20-2 is a sensorgram showing the affinity between fetal Fc receptor (FcRn) and cetuximab. [Figure 20-3] Figure 20-3 is a sensorogram showing the pH-dependent affinity between FcRn and cetuximab. [Figure 21-1] Figure 21-1 is a sensorogram showing the affinity between EGFR and the bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3). [Figure 21-2] Figure 21-2 is a sensorogram showing the affinity between HER2 and the bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3). [Figure 21-3] Figure 21-3 is a sensorogram showing the affinity between FcRn and the bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3). [Figure 21-4] Figure 21-4 is a sensorgram showing the pH-dependent affinity between FcRn and the bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3). [Figure 22-1] Figure 22-1 is a sensorogram showing the affinity between EGFR and the bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4). [Figure 22-2] Figure 22-2 is a sensorogram showing the affinity between HER2 and the bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4). [Figure 22-3] Figure 22-3 is a sensorgram showing the affinity between FcRn and the bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4). [Figure 22-4] Figure 22-4 is a sensorogram showing the pH-dependent affinity between FcRn and the bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4). [Figure 23-1] Figure 23-1 is a sensorogram showing the affinity between EGFR and the tri-specific antibody (M-2). [Figure 23-2] Figure 23-2 is a sensorgram showing the affinity between HER2 and the tri-specific antibody (M-2). [Figure 23-3] Figure 23-3 is a sensorgram showing the affinity between PD-1 and the tri-specific antibody (M-2). [Figure 23-4]Figure 23-4 is a sensorgram showing the affinity between FcRn and the Tri-specific antibody (M-2). [Figure 23-5] Figure 23-5 is a sensorogram showing the pH-dependent affinity between FcRn and the Tri-specific antibody (M-2). [Figure 24-1] Figure 24-1 shows the positive rates of SKBR-3 cells (HER2-positive) and A-431 cells (EGFR-positive) conjugated with cetuximab (Cmab), trastuzumab (Tmab), bi-specific antibodies (Cetuximab-Trastuzumab Fab) (M-3) (Trastuzumab-Cetuximab Fab) (M-4), and tri-specific antibody (Pembrolizumab Fab-Cetuximab-Trastuzumab Fab) (M-2), calculated by flow cytometry. [Figure 24-2] Figure 24-2 shows the evaluation of cetuximab (Cmab) binding to SKBR-3 cells (HER2-positive), A-431 cells (EGFR-positive), and T cells (PD-1-positive) by flow cytometry. [Figure 24-3] Figure 24-3 shows the evaluation of trastuzumab (Tmab) binding to SKBR-3 cells (HER2-positive) and A-431 cells (EGFR-positive) by flow cytometry. [Figure 24-4] Figure 24-4 shows the evaluation of the binding of the bi-specific antibody (Cetuximab-Trastuzumab Fab) (M-3) to SKBR-3 cells (HER2-positive) and A-431 cells (EGFR-positive) by flow cytometry. [Figure 24-5] Figure 24-5 shows the evaluation of the binding of a bi-specific antibody (Trastuzumab-Cetuximab Fab) (M-4) to SKBR-3 cells (HER2-positive) and A-431 cells (EGFR-positive) by flow cytometry. [Figure 24-6]Figure 24-6 shows the evaluation of pembrolizumab (Pbl) binding to T cells (PD-1 positive) by flow cytometry. [Figure 24-7] Figure 24-7 shows the evaluation of the binding of the tri-specific antibody (Pembrolizumab Fab-Cetuximab-Trastuzumab Fab) (M-2) to SKBR-3 cells (HER2-positive), A-431 cells (EGFR-positive), and T cells (PD-1-positive) by flow cytometry. [Figure 24-8] Figure 24-8 shows the positivity rates of T cells (PD-1 positive) bound to cetuximab (Cmab), pembrolizumab (Pbl), and tri-specific antibody (Pembrolizumab Fab-Cetuximab-Trastuzumab Fab) (M-2), calculated by flow cytometry. [Figure 25] Figure 25 shows the SDS-PAGE of CD3-VHH-PA24H6-AzF(V-1) expression. [Figure 26] Figure 26 shows the results of the affinity evaluation of CD3-VHH(V-1). [Figure 27] Figure 27 shows the results of the affinity evaluation of CD3-VHH(V-1). [Figure 28] Figure 28 shows the SDS-PAGE of six affinity peptides. [Modes for carrying out the invention]

[0034] 1. Definitions of general terms In this specification, terms and expressions used to describe a particular invention or subject may also be used to describe another invention or subject. The definitions, examples, and preferred examples of terms and expressions used in the description may also be the same for other inventions or matters described by such terms and expressions.

[0035] In this invention, the term "antibody" is defined as follows. The term "immunoglobulin unit" corresponds to the bivalent monomer unit that constitutes such an antibody, and is an immunoglobulin unit containing two heavy chains and optionally two light chains. Therefore, the definitions, examples, and preferred examples of terms and expressions for immunoglobulin units, such as their origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, amino acid residue (e.g., lysine residue) position, and regioselectivity, are the same as those for antibodies described below and are interchangeable with the expression "antibody."

[0036] The origin of the antibodies is not particularly limited and may be derived from animals such as mammals or birds (e.g., chickens). Preferably, the immunoglobulin units are derived from mammals. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g., dogs, cats), livestock (e.g., cattle, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, and more preferably humans.

[0037] The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a predetermined glycan has been added (e.g., antibodies modified to have a glycan-binding consensus sequence such as an N-linked glycan-binding consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide-bonded reduct antibodies. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, as the monoclonal antibody, a full-length antibody or an antibody fragment containing a variable region and CH1 and CH2 domains can be used, but a full-length antibody is preferred. The antibody is preferably a human IgG monoclonal antibody, and more preferably a human full-length IgG monoclonal antibody.

[0038] Any antigen can be used as the antigen for the antibody. For example, such antigens include proteins (including oligopeptides and polypeptides; proteins modified with biomolecules such as sugars (e.g., glycoproteins)), glycans, nucleic acids, and small molecule compounds. Preferably, the antibody may be an antibody that uses a protein as its antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0039] More specifically, the antigen protein of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0040] (1) Oncology PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1(CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137(4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue Factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, Transfeline Receiver, TGFβ, IL-17, 5T4, RTK, Immune Suppressor Protein, NaPi2b, Lysil B Antigen, A34, Lysil-Oxidase, DLK-1, TROP-2, α9 Integrin, TAG-72 (CA72-4), CD70,

[0041] (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

[0042] (3) Neurological diseases CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0043] (4) Infectious disease Clostridium Difficile toxin B, cytomegalovirus, RSV, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0044] (5) Hereditary and rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0045] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0046] (7) Bone and orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0047] (8) Blood disorders vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0048] (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

[0049] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab) and certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, allentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pembrolizumab (IgG4), mepolizumab, elotuzumab, daratumumab) Examples include ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab, and specific human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, laxibakumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, nesitumumab, brodalumab (IgG2), olaratumab) (whereas the IgG subtype is not mentioned, it is assumed to be IgG1).

[0050] In this invention, specific amino acid residues in the constant region of the heavy chain in an antibody can be regioselectively modified. Examples of such specific amino acid residues include lysine residues, tyrosine residues, serine residues, and threonine residues. For example, in human IgG such as human IgG1, the following amino acid residues present in the constant region of the heavy chain can be exposed on the antibody surface, and these amino acid residues can be used to introduce specific cleavage sites (the positions of amino acid residues are based on EU numbering; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). (1) Exposed lysine residues CH2 domains (e.g., 246th, 248th, 274th, 288th, 290th, 317th, 320th, 322nd) CH3 domains (e.g., ranked 360th, 414th, 439th) (2) Exposed tyrosine residues CH2 domains (e.g., ranked 278th, 296th, 300th) CH3 domain (e.g., ranked 436th) (3) Exposed serine residues CH2 domains (e.g., ranks 254, 267, and 298) CH3 domains (e.g., ranks 400, 415, and 440) (4) Exposed threonine residues CH2 domains (e.g., ranked 256th, 289th) CH3 domains (e.g., ranked 335th, 359th)

[0051] The positions of amino acid residues in antibodies and the positions of constant regions in the heavy chain (e.g., the CH2 domain) follow EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, when targeting human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the human IgG CH2 domain, the lysine residue at position 248 corresponds to the 18th amino acid residue in the human IgG CH2 domain, the lysine residue at position 288 corresponds to the 58th amino acid residue in the human IgG CH2 domain, the lysine residue at position 290 corresponds to the 60th amino acid residue in the human IgG CH2 domain, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the human IgG CH2 domain. The notation 246 / 248 indicates that the target is the lysine residue at position 246 or 248. The notation 288 / 290 indicates that the target is the lysine residue at position 288 or 290.

[0052] Preferably, specific amino acid residues in the constant region of the heavy chain that are regioselectively modified can regioselectively modify lysine residues (e.g., lysine residues at positions 246 / 248 or 288 / 290). In this specification, "regioselectivity" means that, even though specific amino acid residues are not eclectically distributed in specific regions of the antibody, a predetermined structural unit capable of binding to a specific amino acid residue in the antibody is eclectically distributed in specific regions of the antibody. Therefore, expressions related to regioselectivity such as "regioselectively present," "regioselective binding," and "regioselective binding" mean that the possession or binding rate of a predetermined structural unit in a target region containing one or more specific amino acid residues is significantly higher than the possession or binding rate of the same structural unit in a non-target region containing multiple amino acid residues of the same type as the specific amino acid residue in the target region. Such regioselectivity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%. According to the present invention, specific lysine residues in the heavy chain of an antibody can be regioselectively modified without using a linker containing a peptide. The peptide portion has potential immunogenicity and is readily hydrolyzed in the blood. Therefore, avoiding the use of a linker containing a peptide portion is desirable in clinical applications.

[0053] In this invention, as long as specific amino acid residues in the constant region of the heavy chain (e.g., lysine residues at specific positions) are regioselectively modified, specific amino acid residues at other positions may be further regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in International Publications 2018 / 199337, 2019 / 240288, 2019 / 240287, and 2020 / 090979. Such specific amino acid residues can be amino acid residues having easily modifiable side chains (e.g., amino group, carboxyl group, amide group, hydroxyl group, thiol group) (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine ​​residues), but preferably lysine residues having a side chain containing an amino group, tyrosine residues having a side chain containing a hydroxyl group, serine residues, and threonine residues, or cysteine ​​residues having a side chain containing a thiol group, and more preferably lysine residues (i.e., two lysine residues among the lysine residues at positions 246 / 248, 288 / 290, and 317 may be regioselectively double-modified, or three lysine residues may be regioselectively triple-modified).

[0054] (Halogen atom) Examples of halogen atoms include fluorine, chlorine, bromine, and iodine.

[0055] (Monovalent base) Examples of monovalent groups include monovalent hydrocarbon groups and monovalent heterocyclic groups.

[0056] The monovalent group may be substituted with one or more substituents (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) as described below.

[0057] (Monovalent hydrocarbon group, and related terminology) Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0058] A monovalent linear hydrocarbon group refers to a hydrocarbon group composed solely of a linear structure, and whose main chain does not contain a cyclic structure. However, the linear structure may be linear or branched. Examples of monovalent linear hydrocarbon groups include alkyl, alkenyl, and alkynyl groups. Alkyl, alkenyl, and alkynyl groups may be linear or branched.

[0059] As alkyls, alkyls having 1 to 12 carbon atoms are preferred, alkyls having 1 to 6 carbon atoms are more preferred, and alkyls having 1 to 4 carbon atoms are even more preferred. If the alkyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon number. Examples of alkyls 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.

[0060] As the alkenyl, alkenyls having 2 to 12 carbon atoms are preferred, alkenyls having 2 to 6 carbon atoms are more preferred, and alkenyls having 2 to 4 carbon atoms are even more preferred. If the alkenyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of alkenyls having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0061] As for the alkynyl, alkynyls having 2 to 12 carbon atoms are preferred, alkynyls having 2 to 6 carbon atoms are more preferred, and alkynyls having 2 to 4 carbon atoms are even more preferred. If the alkynyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of alkynyls having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0062] Alkyl groups are preferred as monovalent chain hydrocarbon groups.

[0063] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain aromatic rings, and the alicyclic hydrocarbon may be monocyclic or polycyclic. Furthermore, it is not necessary to consist solely of alicyclic hydrocarbons; it may also contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl groups, which may be monocyclic or polycyclic.

[0064] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0065] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkenyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0066] As the cycloalkynyl, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkynyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0067] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0068] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. If the monovalent aromatic hydrocarbon group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0069] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0070] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups.

[0071] (Monovalent heterocyclic groups and related terminology) A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. A monovalent heterocyclic group is either a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heterocyclic group preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0072] As monovalent aromatic heterocyclic groups, those having 1 to 15 carbon atoms are preferred, those having 1 to 9 carbon atoms are more preferred, and those having 1 to 6 carbon atoms are even more preferred. If the monovalent aromatic heterocyclic group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoox Examples include sazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazoninyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0073] As monovalent non-aromatic heterocyclic groups, non-aromatic heterocyclic groups having 2 to 15 carbon atoms are preferred, non-aromatic heterocyclic groups having 2 to 9 carbon atoms are more preferred, and non-aromatic heterocyclic groups having 2 to 6 carbon atoms are even more preferred. If the monovalent non-aromatic heterocyclic group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinil, azetidinil, oxetanil, thietanil, pyrrolidinil, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinil, oxazolidinil, piperidinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinil, dihydrooxazinil, tetrahydrooxazinil, dihydropyrimidinil, and tetrahydropyrimidinil.

[0074] Among these, a 5-membered or 6-membered heterocyclic group is preferred as the monovalent heterocyclic group.

[0075] (Divalent base) Divalent groups include divalent linear hydrocarbon groups, divalent cyclic hydrocarbon groups, divalent heterocyclic groups, -C(=O)-, -C(=S)-, -NR1-, -C(=O)-NR1-, -NR1-C(=O)-, -C(=S)-NR1-, -NR1-C(=S)-, -O-, -S-, and -(O-R2). n -, and -(S-R2) m -A group having a main chain structure comprising one group selected from the group consisting of -, or two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2 or 3). R1 ​​represents a hydrogen atom or a substituent described later. R2 represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. n and m are integers from 1 to 10, preferably from 1 to 8, more preferably from 1 to 6, even more preferably from 1 to 5, and particularly preferably from 1 to 3.

[0076] Divalent linear hydrocarbon groups are linear alkylenes, linear alkenylenes, or linear alkylenes. The linear alkylenes are linear alkylenes having 1 to 6 carbon atoms, with linear alkylenes having 1 to 4 carbon atoms being preferred. Examples of linear alkylenes include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. Linear alkenylenes are linear alkenylenes having 2 to 6 carbon atoms, with linear alkenylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkenylenes include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. Linear alkylenes are linear alkylenes having 2 to 6 carbon atoms, with linear alkylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkylenes include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. As the divalent linear hydrocarbon group, linear alkylenes are preferred.

[0077] Divalent cyclic hydrocarbon groups are arylenes or divalent non-aromatic cyclic hydrocarbon groups. As for the arylene, arylene having 6 to 14 carbon atoms is preferred, arylene having 6 to 10 carbon atoms is more preferred, and arylene having 6 carbon atoms is particularly preferred. Examples of arylene include phenylene, naphthylene, and anthracenylene. As for the divalent non-aromatic cyclic hydrocarbon group, a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms is preferred, a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms is more preferred, and a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms is particularly preferred. Examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. Arylene is preferred as the divalent cyclic hydrocarbon group.

[0078] A divalent heterocyclic group is either a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably include one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms. As for the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As for the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxylandiyl, aziridindiyl, azetidinediyl, oxetanediyl, thietandiyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

[0079] Preferably, the divalent group is alkylene, arylene, -C(=O)-, -NR1-, -C(=O)-NR1-, -NR1-C(=O)-, -O-, and -(O-R2) n -A divalent group having a main chain structure containing one group selected from the group consisting of -, Alkylene, arylene, -C(=O)-, -NR1-, -C(=O)-NR1-, -NR1-C(=O)-, -O-, and -(O-R2) n -A divalent group having a main chain structure containing two or more groups selected from the group consisting of, R1 is a hydrogen atom or an alkyl group. R2 is either alkylene or arylene. n may be an integer between 1 and 5 (i.e., 1, 2, 3, 4, or 5). Alkylenes, arylenes, and alkyls are the same as those described above.

[0080] The main chain structure at the divalent group may be substituted with one or more substituents (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) described later.

[0081] (substituent) Examples of substituents include: (i) halogen atom; (ii) Monovalent hydrocarbon group; (iii) monovalent heterocyclic groups; (iv) Aralkir; (v)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or a monovalent hydrocarbon group. ); or (vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents a hydrogen atom or a monovalent hydrocarbon group, either identical or distinct. (vii) Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

[0082] The definitions, examples, and preferred examples of halogen atoms, monovalent hydrocarbon groups, and monovalent heterocyclic groups in the above substituents are the same as those described above.

[0083] An aralkyl refers to an arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyls are as described above. Preferred aralkyls have 3 to 15 carbon atoms. Examples of such aralkyls include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0084] Preferably, the substituents may be: (i) halogen atom; (ii) alkyl, phenyl, or naphthyl atoms having 1 to 12 carbon atoms; (iii) Aralkyl groups with 3 to 15 carbon atoms; (iv) A complex ring with 5 or 6 members; (v)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 12 carbon atoms. );(vi)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 12 carbon atoms. (vii) The same base as those listed in (vii) above.

[0085] More preferably, the substituents may be: (i) halogen atom; (ii) Alkyl atoms having 1 to 12 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 12 carbon atoms. (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 12 carbon atoms. (v) The same base as those listed in (vii) above.

[0086] More preferably, the substituents may be: (i) halogen atom; (ii) Alkyl atoms having 1 to 6 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 6 carbon atoms. (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R cThis represents, either identical or different, a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. (v) The same base as those listed in (vii) above.

[0087] Particularly preferred, the substituents may be: (i) halogen atom; (ii) Alkyl atoms having 1 to 4 carbon atoms; (iii)R a -O-, R a -C(=O)-, R a -OC(=O)-, or R a -C(=O)-O-(R a This represents a hydrogen atom or an alkyl group with 1 to 4 carbon atoms. (iv)NR b R c -, NR b R c -C(=O)-, NR b R c -C(=O)-O- or R b -C(=O)-NR c -(R b and R c This represents, either identical or distinct, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. (v) The same base as those listed in (vii) above.

[0088] (Biocorthogonal functional groups) Bioorthogonal functional groups are groups that do not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bioorthogonal functional groups are well known in the art (see, for example, Sharpless KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

[0089] In this invention, a bioorthogonal functional group for proteins is used as the bioorthogonal functional group. This is because the antibody to be derivatized by the reagent of this invention is a protein. A bioorthogonal functional group for proteins is a group that does not react with the side chains of the 20 natural amino acid residues that make up proteins, or reacts slowly with those side chains, but reacts with the desired functional group. 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 (K). Of these 20 naturally occurring amino acids, glycine, which lacks a side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., their side chains do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inert to normal reactions. Therefore, bioorthogonal functional groups for proteins are those that do not react with the side chains of these amino acids, which have side chains that are inert to normal reactions, as well as those that react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, or react slowly, but do react with the functional group of interest.

[0090] Examples of such bioorthogonal functional groups 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 with an intercarbon double bond; the same applies below), alkyne residues (in other words, any residue that has an ethynylene moiety, which is the smallest unit with an intercarbon triple bond; 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., carbonyl residues with a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position; the same applies below), isonitrile residues, cydonone residues, and selenium residues. It is possible.

[0091] Bioorthogonal functional groups may be protected or unprotected. A bioorthogonal functional group refers to either an unprotected or protected bioorthogonal functional group. An unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above. A protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleaving a protecting group. Cleavage of the protecting group can be performed by specific treatments under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (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) standing up with a cleavage linker containing a self-degrading cleavageable moiety. Such protecting groups and their cleavage conditions 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., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society. 132, 17928 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)). The reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are described below.

[0092] Examples of protected bioorthogonal functional groups include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinaldiol residues.

[0093] Preferably, the bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0094] More preferably, the bioorthogonal functional group may be a specific bioorthogonal functional group that exhibits excellent reactivity (e.g., reaction level and / or reaction specificity) with other bioorthogonal functional groups. Examples of such bioorthogonal functional groups include azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with substituents as described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, furan residues, and halocarbonyl residues. Examples of combinations of two bioorthogonal functional groups that can react with each other include combinations of azide residues and alkyne residues, combinations of tetrazine residues and alkene residues, combinations of tetrazine residues and alkyne residues, combinations of thiol residues and maleimide residues, combinations of furan residues and maleimide residues, combinations of thiol residues and halocarbonyl residues (where the halogen is replaced by a thiol through a substitution reaction), and combinations of a thiol residue and another thiol residue (where a disulfide bond is formed).

[0095] (Functional substance) The functional substance is not particularly limited as long as it is a substance that confers any function to the antibody, and examples include drugs, labeling substances, affinity substances, transport substances, and stabilizers, but preferably it may be a drug, labeling substance, affinity substance, or transport substance. The functional substance may also be a single functional substance or a substance in which two or more functional substances are linked together.

[0096] The drug may be any drug for any disease. Such diseases include, for example, 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, inflammatory diseases ( Examples include allergic diseases, rheumatoid arthritis, systemic lupus erythematosus, neurological diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood disorders (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, and digestive system diseases). The drugs may be for the prevention or treatment of diseases, or for the mitigation of side effects.

[0097] More specifically, the drug may be an anticancer agent. Examples of anticancer agents include chemotherapeutic agents, toxins, radioisotopes or substances containing them. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., lysine). Examples of radioisotopes include radioisotopes of hydrogen atoms (e.g., 3 H), radioactive isotopes of carbon atoms (e.g., 14 C) Radioactive isotopes of the phosphorus atom (e.g., 32 P), radioactive isotopes of the sulfur atom (e.g., 35 S ), radioactive isotopes of yttrium (e.g., 90 Y), radioactive isotopes of technetium (e.g., 99m Tc), radioactive isotopes of indium (e.g., 111 In), radioactive isotopes of the iodine atom (e.g., 123 I, 125 I, 129 I, 131 I) Radioactive isotopes of samarium (e.g., 153 Sm), Lenniu Radioactive isotopes of (e.g., 186 Re), radioactive isotopes of astatine (e.g., 211 At), radioactive isotopes of bismuth (e.g., 212Bi) is one example. More specifically, drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calichemycin, duocalmycin, eribulin, anthracycline, dmDNA31, and tubulisin.

[0098] Labeling substances are substances that enable the detection of targets (e.g., tissues, cells, materials). 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 mentioned above), or substances containing them.

[0099] An affinity substance is a substance that has affinity for a target. Examples of affinity substances include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary chains to target nucleic acids. Preferably, the affinity substance is an affinity protein or affinity peptide, and more preferably, an antibody. The type of animal from which the antibody used as a functional substance originates is the same as described above.

[0100] The type of antibody used as a functional substance may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a predetermined glycan has been attached (e.g., antibodies modified to have a glycan-binding consensus sequence such as an N-linked glycan-binding consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond-reducing antibodies. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgE). Examples include IgG3, IgG4, IgM, IgA, IgD, IgE, and IgY. Antibodies used as functional substances include, for example, full-length antibodies and their fragments (fragment antibodies). Fragment antibodies only need to maintain their ability to bind to the desired antigen, and examples include Fab, Fab', F(ab')2, scFv, and VHH antibodies.

[0101] The antigenicity of the antibody used as a functional substance may be the same as or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, and it is preferable that it be different. Furthermore, the origin of the antibody used as a functional substance may be the same as or different from the origin of the immunoglobulin unit, and it is preferable that it be different. Therefore, the antibody used as a functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody, or an antibody derived therefrom, as mentioned in the specific examples of monoclonal antibodies described above. The antibody used as a functional substance may also be IgG1, IgG2, IgG3, or IgG4, or an antibody derived therefrom, as mentioned in the specific examples of monoclonal antibodies described above.

[0102] The transport material is a substance that has the ability to transport compounds. Preferred transport materials are substances that can encapsulate compounds within a protein shell (e.g., multimers) (e.g., ferritin, virus particles, virus-like particles).

[0103] Stabilizers are substances that enable the stabilization of antibodies. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0104] Functional substances may also be peptides, proteins, nucleic acids, organic compounds, inorganic compounds, glycans, lipids, high molecular weight polymers, metals (e.g., gold), or chelators. Examples of peptides include cell membrane permeable peptides, blood-brain barrier permeable peptides, and peptide pharmaceuticals. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, antibodies, and ferritin. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Other examples of nucleic acids include RNA interference-inducible nucleic acids (e.g., siRNA), aptamers, and antisense compounds. Examples of organic compounds include low molecular weight organic compounds such as proteolytic chimeric molecules, dyes, and photodegradable compounds. Examples of inorganic compounds include silica, talc, and alumina.

[0105] (salt) In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and salts with acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or an organic acid (e.g., t). It is a salt of (lifluoroacetic acid).

[0106] 2. Affinity substances or their salts, and related inventions 2-1. Affinity substances or their salts The present invention provides an affinity substance or a salt thereof comprising first and second affinity moieties having affinity for a constant region in the heavy chain of an antibody.

[0107] The affinity substance used in the present invention comprises first and second affinity moieties having affinity for the constant region of the heavy chain in the antibody constituent unit. The antibody constituent unit is an immunoglobulin unit comprising two heavy chains and optionally two light chains. Therefore, the antibody constituent unit is an immunoglobulin unit comprising two heavy chains and two light chains, or an immunoglobulin unit comprising two heavy chains but not two light chains. Examples of antibodies comprising an immunoglobulin unit comprising two heavy chains and two light chains include bivalent antibodies (e.g., IgG, IgD, IgE), or quadrivalent or higher antibodies (e.g., IgA antibody, IgM antibody), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and disulfide-bonded reductantibodies. Examples of antibodies comprising an immunoglobulin unit comprising two heavy chains but not two light chains include Fc region proteins, Fc fusion proteins, and disulfide-bonded reductantibodies. The Fc region contains the CH2 and CH3 domains as heavy chains, but does not contain a light chain.

[0108] The first and second affinity moieties may be the same or different. If the first and second affinity moieties are different, they may be affinity substances having affinity for different regions in the constant region of the antibody heavy chain (e.g., one of the first and second affinity moieties having affinity for the CH2 domain and the other having affinity for the CH3 domain), or they may be different affinity moieties having affinity for the same region in the constant region of the antibody heavy chain (e.g., both the first and second affinity moieties having affinity for the CH2 domain or the CH3 domain), and it is preferable that they be different affinity moieties having affinity for the same region in the constant region of the antibody heavy chain. Furthermore, it is preferable that the first and second affinity moieties have a relationship in which the first association site of the first affinity moiety to the constant region of the heavy chain and the second association site of the second affinity moiety to the constant region of the heavy chain sterically interfere with each other, so that the association of one can suppress the association of the other, in order to suppress the association of the first and second affinity moieties to the constant region of a single heavy chain. In such a relationship, it is possible to suppress the competitive association of two molecules (first and second molecules) of an affinity substance and a compound containing a reactive group for the antibody within a single antibody unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), thereby promoting the specific modification of only the constant region of one of the heavy chains. Examples of the first and second affinity moieties include polymeric substances of a predetermined structural unit [e.g., peptides (including oligopeptides, polypeptides, and proteins), nucleic acids (including oligonucleotides and polysaccharides), sugars (including oligosaccharides and polysaccharides)], and non-polymeric substances (e.g., small molecule compounds).Numerous substances have been reported that can be used as first and second affinity moieties that have affinity for the constant region of the antibody heavy chain, including peptides, nucleic acids, sugars, and small molecule compounds (e.g., published publications such as International Publication Nos. 2007 / 004748, 2008 / 054030, 2013 / 027796, 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979; see Nomura Y et al., Nucleic Acids Res., 2010 Nov;38(21):7822-9; Miyakawa S et al., RNA., 2008 Jun;14(6):1154-63, and the scientific papers mentioned below). The first and second affinity portions are preferably identical.

[0109] Furthermore, substances that can be used as first and second affinity moieties having affinity for the constant region of the antibody heavy chain can be obtained by any method. For example, such substances can be obtained by screening for substances having affinity for the constant region of the antibody heavy chain from a library of any substance (e.g., small molecule compound library, peptide library, aptamer library, sugar library, phage library, mRNA library, cDNA library) (e.g., high-throughput screening method, phage display method, SELEX method, mRNA display method, ribosome display method, cDNA display method, yeast display method). In addition, when screening for substances having affinity for the Fc region or specific regions within the constant region of an antibody (e.g., CH2 domain, CH3 domain), substances that can selectively bind to such regions can be efficiently obtained by using partial peptides present in specific regions (e.g., CH1, CH2, CH3) of the Fc region of various antibodies (e.g., IgG, IgA, IgM, IgD, IgE).

[0110] The first and second affinity moieties may have affinity for the constant region of the antibody heavy chain. For example, the first and second affinity moieties may have affinity for the Fc region of the antibody heavy chain. Alternatively, the first and second affinity moieties may have affinity for the CH1 domain, CH2 domain, or CH3 domain, or a region spanning them (e.g., adjacent regions of the CH1 and CH2 domains, adjacent regions of the CH2 and CH3 domains), as the constant region of the heavy chain. The first and second affinity moieties may have affinity for the same or different CHX domains (X is 1, 2, or 3), but it is preferable that they have affinity for the same domain. Here, "having affinity for a CHX domain" is not particularly limited as long as it has affinity for at least a portion of the region within the CHX domain, and may have affinity for a partial region within the CHX domain, or affinity for a region spanning a CHX domain and other CHX domains (e.g., adjacent regions). Therefore, the first and second affinity portions having affinity for the CH2 domain may have affinity only for a subdomain within the CH2 domain, or affinity for a region spanning the CH2 domain and the CH1 or CH3 domain (e.g., adjacent regions of the CH1 and CH2 domains, adjacent regions of the CH2 and CH3 domains). Preferably, the first and second affinity portions having affinity for the CH2 domain may have affinity only for a subdomain within the CH2 domain, or affinity for a region spanning the CH2 and CH3 domains (e.g., adjacent regions of the CH2 and CH3 domains), and more preferably, affinity only for a subdomain within the CH2 domain.

[0111] The affinity substance or salt thereof of the present invention may have the first and second affinity moieties directly linked, or a linker may be inserted between the first affinity substance and the second affinity moiety. If the binding sites of the first and second affinity moieties to the constant region of the antibody heavy chain are close together, and the sizes of the first affinity substance and the second affinity moiety are sufficiently large, and there is no need to adjust the distance between the first and second affinity moieties, the first and second affinity moieties can be directly linked. On the other hand, if the binding sites of the first and second affinity moieties to the constant region of the antibody heavy chain are not close together, and the sizes of the first affinity substance and the second affinity moiety are not sufficiently large, and there is a need to adjust the distance between the first and second affinity moieties, a linker can be inserted between the first affinity substance and the second affinity moiety. A divalent group can be used as the linker. The divalent group may be substituted or not. Examples of divalent groups include those described above. Examples of substituents when a divalent group is substituted include those mentioned above. Examples of linkers include peptides and nucleic acids. Substances such as sugars, other polymeric substances (e.g., polyethylene glycol), and divalent hydrocarbon groups (e.g., alkyl chains) may be used. Those skilled in the art can appropriately determine the presence or absence of a linker and the type of linker depending on the types of first and second affinity moieties used. Preferably, a linker may be inserted from the viewpoint of optimizing the binding of the first and second affinity moieties to the constant region of the antibody heavy chain.

[0112] The constant regions in the heavy chain of the antibody to which the first and second affinity portions have affinity may be derived from animals as described above (e.g., mammals, birds). The constant regions in the heavy chain of the antibody may preferably be mammalian constant regions, more preferably primate constant regions or rodent constant regions, and even more preferably human constant regions.

[0113] The constant regions in the heavy chain of the antibody to which the first and second affinity portions have affinity may be the constant regions of a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Such constant regions are preferably the constant regions of a bivalent antibody (e.g., IgG, IgD, IgE), and more preferably the constant regions of IgG.

[0114] Preferably, the first and second affinity moieties may be affinity peptides having affinity for the constant region of the antibody heavy chain. Numerous peptides have been reported as such affinity peptides. For example, examples of such affinity peptides are as follows: (1) Various IgG-binding peptides that have affinity for specific regions (CH2 domains) of human IgG in general (i.e., human IgG1, IgG2, IgG3, and IgG4; the same applies hereafter) (see, e.g., International Publication No. 2008 / 054030, International Publication No. 2013 / 027796, International Publication No. 2016 / 186206); (2) Protein A that has affinity for a specific region (CH2 domain) of human IgG in general Mimetic (PAM) peptide (see e.g. Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, VOL. 6, 564-569); (3) EPIHRSTLTALL (SEQ ID NO: 27) which has affinity for a specific region (CH2 domain) of human IgG in general (see, for example, Ehrlich GK et al., J. Biochem. Biophys. Methods, 2001, VOL. 49, 443-454); (4) (NH2-Cys1-X1-X2-X3-X4)2-Lys-Gly-OH that has affinity for a specific region (Fc region) of human IgG in general (see, for example, Ruvo M et al., ChemBioChem, 2005, VOL.6, 1242-1253); (5) FARLVSSIRY (SEQ ID NO: 28), FGRLVSSIRY (SEQ ID NO: 29), and TWKTSRISIF (SEQ ID NO: 30) which have affinity for specific regions (Fc regions) of human IgG in general (see, for example, Krook M et al., Journal of Immunological Methods, 1998, VOL.221, 151-157); (6) QSYP (Sequence ID 31) that has affinity for specific regions of human IgG in general (see, e.g., Jacobs JM et al., Bio.Techniques, 2003, VOL.34, 132-141); (7) HWRGWV (SEQ ID NO: 32), HYFKFD (SEQ ID NO: 33), and HFRRHL (SEQ ID NO: 34) which have affinity for specific regions (Fc regions) of human IgG in general (see, e.g., Carbonell RG et al., Journal of Chromatography A, 2009, VOL.1216, 910-918); (8) DAAGs (SEQ ID NO: 35) that have affinity for specific regions (Fc regions) of human IgG in general (see, for example, Lund LN et al., Journal of Chromatography A, 2012, VOL.1225, 158-167); (9) Fc-I, Fc-II, and Fc-III which have affinity for specific regions (Fc regions) of human IgG in general (e.g., Warren L. Delano et al., Science, 2000, VOL.287, 1279-1283; see International Publication 2001 / 045746); and (10) NARKFYKG (SEQ ID NO: 36) and NKFRGKYK (SEQ ID NO: 37) (e.g., Biochemical See Engineering Journal, 2013, VOL.79, 33-40. (11) Protein A, Protein G, Protein L, or Protein Z, or fragments thereof, that have affinity for a specific region (Fc region) of human IgG in general (e.g., Moks T et al.,Eur J Biochem.1986 May 2;156(3):637-43;Sjobring UJ et al.,Biol Chem. 1991 Jan 5;266(1):399-405;Graille M el al.,Structure. 2001 Aug;9(8):679-87;Nilsson B et al.,Protein Eng. 1987 Feb-Mar;1(2):107-13); (12) Various IgG-binding peptides that have affinity for specific regions (Fc region or CH2 domain) of human IgG in general (see, e.g., International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, International Publication No. 2020 / 090979).

[0115] Furthermore, affinity peptides can be obtained by the screening methods described above (e.g., the method using the library described above, or the display method described above).

[0116] For affinity peptides, amino acid residues from the 20 common natural amino acids that make up proteins, or from non-natural amino acids, can be used. Examples of the 20 common natural amino acids that make up proteins include, for example, L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (the notation L will be omitted below).

[0117] In certain embodiments, one of the first and second affinity peptides may be an affinity peptide having one lysine residue, while the other may be an affinity peptide not having a lysine residue.

[0118] Numerous peptides have been reported as affinity peptides that have affinity for the constant region of the antibody heavy chain and contain one lysine residue (see, e.g., International Publication Nos. 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979). Therefore, in the present invention, such peptides can be used as one of the first and second affinity peptides.

[0119] More specifically, the following may be used as affinity peptides that have affinity for the constant region of the antibody heavy chain and have one lysine residue: (1) Affinity peptides containing the amino acid sequences of SEQ ID NOs. 39-72 of International Publication No. 2018 / 199337; (2) Affinity peptides containing the amino acid sequences of Sequence IDs 5, 6, and 37-100 of International Publication No. 2019 / 240288; (3) Affinity peptides containing the amino acid sequences of SEQ ID NOs. 5, 8-57, and 68-92 of International Publication No. 2019 / 240287; (4) Affinity peptides having QET at the N terminus (SEQ ID NOs. 7-10, 22-25, 52, 53 of International Publication No. 2020 / 090979); and (5) Among the affinity peptides listed above as examples of affinity peptides (1) to (12), an affinity peptide having one lysine residue.

[0120] In certain embodiments, affinity peptides having affinity for the constant region of the antibody heavy chain and containing one lysine residue include, for example, the following (1) to (4): (1) Affinity peptide containing the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (2) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​residues are substituted with other amino acid residues other than lysine and cysteine ​​residues in the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (3) Affinity peptides containing the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and (4) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​residues are substituted with other amino acid residues other than lysine and cysteine ​​residues in the amino acid sequence of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39), Here, the two cysteine ​​residues in the above amino acid sequence may be cross-linked by a disulfide bond.

[0121] Numerous affinity peptides have been reported that possess affinity for the constant region of the antibody heavy chain and do not contain a lysine residue. Furthermore, in the above affinity peptides that possess affinity for the constant region of the antibody heavy chain and contain one lysine residue, the lysine residue is often introduced not to maintain affinity for the constant region of the antibody heavy chain, but to derivatize the affinity substance by covalently bonding with other parts (e.g., partial compounds containing reactive groups) (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979). Therefore, even if such a lysine residue is replaced with another amino acid residue, the affinity for the constant region of the antibody heavy chain can be maintained. Therefore, as an affinity peptide that has affinity for the constant region of the antibody heavy chain and does not contain a lysine residue, an affinity peptide that has affinity for the constant region of the antibody heavy chain and contains one lysine residue, in which the lysine residue is substituted with another amino acid residue (preferably a normal natural amino acid residue that constitutes a protein, other than a lysine residue or a cysteine ​​residue), can be used that has affinity for the constant region of the antibody heavy chain.

[0122] More specifically, the following affinity peptides may be used as affinity peptides that have affinity for the constant region of the antibody heavy chain and do not contain lysine residues: (1) Sequence IDs 20-38, 73-75 (when Xaa1 is not a lysine residue), and 92 of International Publication No. 2018 / 199337; (2) Sequence IDs 7, 11-14, 108 of International Publication No. 2019 / 240288; (3) Having affinity for the constant region of the antibody heavy chain and having one lysine residue Among the examples of affinity peptides listed above (1) to (4), affinity peptides in which the lysine residue is substituted with another amino acid residue (preferably another amino acid residue other than a cysteine ​​residue); and (4) Among the affinity peptides listed above as examples of affinity peptides (1) to (12), an affinity peptide that does not contain a lysine residue.

[0123] In certain embodiments, examples of affinity peptides that do not contain lysine residues include the following (5) to (10): (5) Affinity peptide containing the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40); (6) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than cysteine ​​residues are substituted with lysine residues and other amino acid residues other than cysteine ​​residues in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40), and (7) Affinity peptide containing the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41); (8) Affinity peptide containing an amino acid sequence in which one or two amino acid residues other than cysteine ​​residues in the amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41) are substituted with lysine residues and other amino acid residues other than cysteine ​​residues, and which has affinity for the constant region of the antibody heavy chain; (9) Affinity peptides containing the amino acid sequence RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and (10) An affinity peptide having affinity for the constant region of the antibody heavy chain, comprising an amino acid sequence in which one or two amino acid residues other than cysteine ​​residues are substituted with lysine residues and other amino acid residues other than cysteine ​​residues in the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78), Here, the two cysteine ​​residues in the above amino acid sequence may be cross-linked by a disulfide bond.

[0124] Amino acid substitutions may be conservative substitutions. The term "conservative substitution" refers to the substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in this field. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Amino acids having uncharged polar side chains and amino acids having nonpolar side chains are sometimes collectively referred to as neutral amino acids. Preferably, the conservative substitution of amino acids may be a substitution between aspartic acid and glutamic acid, a substitution between arginine, lysine and histidine, a substitution between tryptophan and phenylalanine, a substitution between phenylalanine and valine, a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.

[0125] When the first and second affinity portions are affinity peptides, the affinity substance or salt of the present invention may have the first and second affinity peptides directly linked together, or a linker may be inserted between the first and second affinity peptides. However, from the viewpoint of optimizing the binding of the first and second affinity peptides to the constant region of the antibody heavy chain, it is preferable that a linker is inserted. When the first and second affinity portions are affinity peptides, a peptide linker is preferred as the linker.

[0126] The number of amino acid residues constituting the peptide linker can be appropriately set according to conditions such as the type of amino acid residue (e.g., α-amino acids, β-amino acids, γ-amino acids; preferably α-amino acids). For example, the peptide linker may consist of 20 or more amino acid residues. The peptide linker may consist of 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, 32 or more, 34 or more, 36 or more, 38 or more, or 40 or more amino acid residues. The peptide linker may also consist of fewer than 50, fewer than 49, fewer than 48, fewer than 47, fewer than 46, or fewer than 45 amino acid residues.

[0127] The amino acid residues constituting the peptide linker can be the aforementioned natural amino acids or non-natural amino acid residues. Preferably, the amino acid residues constituting the peptide linker may consist only of the aforementioned natural amino acid residues. Suitable amino acid residues for the peptide linker include, but are not limited to, alanine, proline, serine, and glycine. Other peptide linkers that can be used are those disclosed in International Publication No. 2021 / 112249 and International Publication No. 2011 / 144756.

[0128] Preferably, the affinity substance or salt of the present invention may be an affinity substance or salt thereof in which the first and second affinity portions are affinity peptides and a linker is contained between the first affinity peptide and the second affinity peptide. Such an affinity substance is given by the following formula (A): AP1-L A -AP2 (A) [During the ceremony, AP1 exhibits a first affinity peptide that has affinity for the constant region in the heavy chain of the antibody. AP2 exhibits a second affinity peptide that has affinity for the constant region in the heavy chain of the antibody. L A This indicates a linker. It can be represented as ].

[0129] In formula (A) and other formulas presented in connection with the present invention, the hyphen (-) indicates that the two units on either side are covalently bonded. Thus, in formula (A), AP1 is covalently bonded to L, L is covalently bonded to both AP1 and AP2, and AP2 is covalently bonded to L.

[0130] The definitions, examples, and preferred examples of the first and second affinity peptides, as shown in AP1 and AP2, respectively, are as described above.

[0131] L A The linker shown is a divalent group. The divalent group may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above. Preferably, as the linker, substances such as peptides, nucleic acids, sugars, other polymeric substances (e.g., polyethylene glycol), or divalent hydrocarbon groups (e.g., alkyl chains) may be used.

[0132] More preferably, the affinity substance or salt of the present invention may be an affinity polypeptide or salt thereof, wherein the first and second affinity portions are affinity peptides, and a peptide linker is included between the first affinity peptide and the second affinity peptide. Such an affinity polypeptide is given by the following formula (A'): AP1-PL A -AP2 (A') [During the ceremony, AP1 exhibits affinity for the constant region of the antibody heavy chain and represents a first affinity peptide located at the N-terminus of the affinity polypeptide. AP2 exhibits affinity for the constant region of the antibody heavy chain and represents a second affinity peptide located at the C-terminus of the affinity polypeptide. PL A This indicates a peptide linker. It can be represented as [ ].

[0133] Affinity substances such as affinity polypeptides or salts thereof may contain the aforementioned natural or non-natural amino acid residues as constituent amino acid residues. If the affinity substance contains only the aforementioned natural amino acid residues, it can be produced, for example, by a polypeptide expression system using host cells, a cell-free synthesis system, or an organic synthesis system (e.g., solid-phase synthesis). If the affinity substance contains non-natural amino acid residues, it can be produced, for example, by an organic synthesis system (e.g., solid-phase synthesis). Preferably, the affinity substance may contain only natural amino acid residues to enable the production of large quantities of affinity substances by a polypeptide expression system using host cells or a cell-free synthesis system.

[0134] When the affinity substance of the present invention is an affinity substance such as an affinity polypeptide or a salt thereof, the amino group and carboxyl group at the terminus of the affinity substance can be appropriately protected. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., acetyl groups, propoxy groups, tert-butoxycarbonyl groups, etc.), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl group), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl group). Preferably, the N-terminal amino group may be alkylated, formylated, or acetylated. Examples of protecting groups for the C-terminal carboxyl group include groups capable of forming esters or amides. Groups capable of forming esters or amides include, for example, alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups.

[0135] Furthermore, if the N-terminal amino acid of the affinity substance is glutamic acid (E) or glutamine (Q), the N-terminus can be protected by utilizing their side chains. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid can have a pyroglutamic acid cyclic structure. When the N-terminal amino acid is glutamine, the protected N-terminal glutamine can have a pyroglutamic acid-type cyclic structure by the reaction (pyroglutamylation) of the N-terminal amino group (NH2) with the amide group present in its side chain. Therefore, the N-terminal amino acid may preferably be glutamic acid or glutamine.

[0136] If the affinity substance is an affinity polypeptide, the affinity polypeptide may further contain a tripeptide consisting of Gln-Glu-Thr (QET) at its N-terminus. In this case, a polypeptide expression system using host cells enables large-scale and simple secretion production of the affinity polypeptide, along with protection of the N-terminal amino group by pyroglutamylation of Q (see examples, International Publication No. 2013 / 062029, International Publication No. 2020 / 090979). In this case, MFNNRIRTAALAGAIAISTAASGVAIPAFA (distributed A signal peptide, such as the signal peptide (CspBss) consisting of the amino acid sequence in column 42), can be added to the N-terminus of QET (see Examples, International Publication No. 2013 / 062029, and International Publication No. 2020 / 090979).

[0137] The affinity substance or a salt thereof of the present invention can be used, for example, as a synthetic intermediate for the compound or a salt thereof of the present invention, which contains the affinity substance and a reactive group for antibodies. Therefore, the affinity substance or a salt thereof of the present invention may be derivatized to contain only one specific reactive group that enables a specific reaction with a partial compound containing a reactive group for antibodies, in order to easily realize the uniform synthesis of the compound or a salt thereof of the present invention, which contains the reactive group for antibodies, in addition to the affinity substance. When the affinity substance contains only one specific reactive group, both the affinity substance and the reactive group for antibodies can be specifically reacted via the specific reactive group in the affinity substance, so that the compound or a salt thereof of the present invention, which contains the affinity substance and the reactive group for antibodies, can be easily produced as a uniform compound.

[0138] Examples of such specific reactive groups include the following: (1) Amino group (NH2, NHR3, NR3R4. R3 and R4 are each independently a monovalent group as described above, preferably a monovalent hydrocarbon group, more preferably an alkyl group, and even more preferably an alkyl group having 1 to 6 carbon atoms); (2) Residues that can react with an amino group. For example, 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-methoxyethyl residues, diazonium terephthalic acid residues; (3) Carboxyl group (COOH); (4) Residues that can react with a carboxyl group. For example, amino groups as described above; (5) Hydroxyl groups (OH) (including alcoholic and phenolic hydroxyl groups); and (6) Residues that can react with a hydroxyl group. For example, diazonium residues, diazodical boxlate residues, and 2,3-dihydro-1H-pyrazine-6-one residues. The specific reactive group may preferably be (1) to (4). More preferably, the specific reactive group may be (1) or (2), or (3) or (4). Alternatively, the specific reactive group may be (1) or (3). The specific reactive group is even more preferably (1), and particularly preferably an amino group (NH2).

[0139] The affinity substance or its salt may contain only one amino acid residue containing a specific reactive group. In such cases, the affinity polypeptide may contain (a) only one amino acid residue having a specific reactive group (e.g., an amino group, a carboxyl group, a hydroxyl group) in its side chain (e.g., a lysine residue, an aspartic acid residue, a glutamic acid residue, a tyrosine residue, a threonine residue, or a serine residue). If the affinity polypeptide contains only one lysine residue having an amino group in its side chain, the N-terminus of the affinity polypeptide is preferably protected (the C-terminus may also be protected). If the affinity polypeptide contains only one amino acid residue having a carboxyl group in its side chain (e.g., an aspartic acid residue or a glutamic acid residue), the C-terminus of the affinity polypeptide is preferably protected (the N-terminus may also be protected).

[0140] Alternatively, affinity polypeptides may contain only one amino group as a specific reactive group, provided they do not contain amino acid residues with an amino group in their side chain (e.g., lysine residues) and have an amino group at their N-terminus. Affinity polypeptides may also contain amino acid residues with a carboxyl group in their side chain (e.g., aspartic acid residues, glutamic acid residues). Firstly, by having a carboxyl group at the C-terminus, the polypeptide may contain only one carboxyl group as a specific reactive group.

[0141] Preferably, an affinity polypeptide containing only one specific reactive group may be a polypeptide containing an amino acid residue having an amino group in its side chain. When the affinity polypeptide is produced by an organic synthesis system (e.g., solid-phase synthesis), not only lysine residues, which are natural amino acids that make up proteins, but also other amino acid residues having an amino group in their side chain (e.g., ornithine) can be used. More preferably, an affinity polypeptide containing only one specific reactive group may be a polypeptide containing only one lysine residue having an amino group (NH2) in its side chain, so that it can be easily produced not only by organic synthesis systems but also by polypeptide expression systems using host cells and cell-free synthesis systems. In this case, the lysine residue may be contained in the first affinity peptide, the second affinity peptide, or in the peptide linker. When the lysine residue is contained in the peptide linker, it may be located in close proximity to the first affinity peptide or the second affinity peptide (e.g., at a position of 10 or fewer, 5 or fewer, or 1 to 3 amino acid residues from the first or second affinity peptide). Preferably, the lysine residue is contained in either the first affinity peptide or the second affinity peptide.

[0142] Preferably, the affinity substance represented by formula (A) or a salt thereof, and the first and second affinity peptides represented by AP1 and AP2, respectively, and L A The linker shown may further have the following characteristics (1) or (2), preferably the characteristic of (1).

[0143] (1) Affinity substances or salts thereof that contain only one amino group as a specific reactive group: (1-1)(i) an affinity peptide having a first affinity peptide containing only one amino acid residue (preferably a lysine residue) having an amino group in its side chain and having a protected N-terminus, and (ii) an affinity substance or a salt thereof having a second affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker that does not contain an amino group; (1-2)(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain amino acid residues having an amino group in its side chain and has an unprotected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain amino acid residues having an amino group in its side chain, and the linker is a linker that does not contain an amino group; (1-3)(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue (preferably a lysine residue) having an amino group in its side chain, and the linker is a linker that does not contain an amino group; (1-4)(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker containing only one amino group. A affinity substance or a salt thereof containing only one amino group as a specific reactive group may also be an affinity substance or a salt thereof that does not contain any groups that can react with an amino group (e.g., a carboxyl group) in order to suppress undesirable reactions (e.g., intramolecular or intermolecular reactions). Therefore, in (1-1) to (1-4) above, the first affinity peptide may not contain an amino acid residue having a carboxyl group in its side chain, the second affinity peptide may not contain an amino acid residue having a carboxyl group in its side chain and / or may have a protected C-terminus, and the linker may be a linker that does not contain a carboxyl group.

[0144] (2) Affinity substances or salts thereof that contain only one carboxyl group as a specific reactive group: (2-1)(i) an affinity peptide in which the first affinity peptide contains only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid), and (ii) an affinity peptide in which the second affinity peptide does not contain an amino acid residue having a carboxyl group in its side chain, and the linker is a linker that does not contain a carboxyl group; an affinity substance or a salt thereof; (2-2)(i) an affinity substance or a salt thereof wherein the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) and has a protected C-terminus, and the linker is a linker that does not contain a carboxyl group; (2-3)(i) an affinity peptide in which the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and (ii) an affinity peptide in which the second affinity peptide does not contain an amino acid residue having a carboxyl group in its side chain and has an unprotected C-terminus, and the linker is a linker that does not contain a carboxyl group, wherein the affinity substance or a salt thereof; (2-4) (i) an affinity peptide that does not contain amino acid residues having a carboxyl group in its side chain, and (ii) an affinity peptide in which the second affinity peptide does not contain amino acid residues having a carboxyl group in its side chain and has a protected C-terminus, and the linker is a linker containing only one carboxyl group, wherein the affinity substance or a salt thereof. A affinity substance or a salt thereof containing only one carboxyl group as a specific reactive group may also be an affinity substance or a salt thereof that does not contain any groups that can react with a carboxyl group (e.g., an amino group) in order to suppress undesirable reactions (e.g., intramolecular or intermolecular reactions). Therefore, in (2-1) to (2-4) above, the first affinity peptide may not contain an amino acid residue having an amino group in its side chain and / or may have a protected N-terminus, the second affinity peptide may not contain an amino acid residue having an amino group in its side chain, and the linker may be a linker that does not contain an amino group.

[0145] Preferably, the affinity substance represented by formula (A') or a salt thereof, and the first and second affinity peptides represented by AP1 and AP2, respectively, and PL A The peptide linker shown may further have the following properties (1') or (2'), preferably property (1').

[0146] (1') Affinity substances or salts thereof that contain only one amino group as a specific reactive group: (1-1')(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide containing only one amino acid residue (preferably a lysine residue) having an amino group in its side chain and having a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker that does not contain an amino acid residue having an amino group in its side chain; (1-2')(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain amino acid residues having an amino group in its side chain and has an unprotected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain amino acid residues having an amino group in its side chain, and the peptide linker is a peptide linker that does not contain amino acid residues having an amino group in its side chain; (1-3')(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue (preferably a lysine residue) having an amino group in its side chain, and the peptide linker is a peptide linker that does not contain an amino acid residue having an amino group in its side chain; (1-4')(i) The first affinity peptide is an affinity peptide that does not contain an amino acid residue with an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide An affinity substance or a salt thereof, wherein the peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker that contains only one amino acid residue having an amino group in its side chain. A affinity substance or a salt thereof containing only one amino group as a specific reactive group may also be an affinity substance or a salt thereof that does not contain any groups that can react with an amino group (e.g., a carboxyl group) in order to suppress undesirable reactions (e.g., intramolecular or intermolecular reactions). Therefore, in (1-1') to (1-4') above, the first affinity peptide may not contain an amino acid residue having a carboxyl group in its side chain, the second affinity peptide may not contain an amino acid residue having a carboxyl group in its side chain and / or may have a protected C-terminus, and the peptide linker may be a peptide linker that does not contain an amino acid residue having a carboxyl group in its side chain.

[0147] (2') Affinity substances or salts thereof that contain only one carboxyl group as a specific reactive group: (2-1')(i)An affinity substance or salt thereof in which the first affinity peptide is an affinity peptide containing only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid), and (ii)the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and the peptide linker is a peptide linker that does not contain an amino acid residue having a carboxyl group in its side chain; (2-2')(i)An affinity substance or salt thereof in which the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and (ii)the second affinity peptide is an affinity peptide containing only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) and has a protected C-terminus, and the peptide linker is a peptide linker that does not contain an amino acid residue having a carboxyl group in its side chain; (2-3')(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain amino acid residues having a carboxyl group in its side chain, and (ii) the second affinity peptide is an affinity peptide that does not contain amino acid residues having a carboxyl group in its side chain and has an unprotected C-terminus, and the peptide linker is a peptide linker that does not contain amino acid residues having a carboxyl group in its side chain; (2-4')(i) an affinity substance or a salt thereof, wherein the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain and has a protected C-terminus, and the peptide linker is a linker containing only one amino acid residue having a carboxyl group in its side chain. A affinity substance or a salt thereof containing only one carboxyl group as a specific reactive group may not contain any groups that can react with a carboxyl group (e.g., an amino group) in order to suppress undesirable reactions (e.g., intramolecular or intermolecular reactions). Therefore, in (2-1') to (2-4') above, the first affinity peptide may not contain an amino acid residue having an amino group in its side chain and / or may have a protected N-terminus, the second affinity peptide may not contain an amino acid residue having an amino group in its side chain, and the peptide linker may be a linker that does not contain an amino acid residue having an amino group in its side chain.

[0148] 2-2. Related Inventions of Affinity Substances or Salts thereof If the affinity substance or salt thereof of the present invention is an affinity polypeptide comprising first and second affinity peptides having affinity for the constant region of the heavy chain of an antibody, such an affinity polypeptide can be prepared using a host cell comprising an expression unit including a polynucleotide encoding the affinity polypeptide and a promoter operably linked thereto, or using a cell-free system, etc. The present invention also provides such a polynucleotide and host cell, as well as an expression vector that can be used to prepare the host cell.

[0149] The polynucleotide of the present invention is a polynucleotide that encodes the affinity polypeptide of the present invention. The polynucleotide of the present invention may be DNA or RNA, but DNA is preferred.

[0150] The host cells of the present invention can be prepared, for example, by a method using an expression vector containing the polynucleotide of the present invention (e.g., competent cell method, electroporation method), or by genome modification technology. If the expression vector is an integrative vector that undergoes homologous recombination with the host cell's genomic DNA, the expression units can be incorporated into the host cell's genomic DNA by transformation. On the other hand, if the expression vector is a non-integrative vector that does not undergo homologous recombination with the host cell's genomic DNA, the expression units are not incorporated into the host cell's genomic DNA by transformation and can exist independently of the genomic DNA within the host cell in the state of the expression vector. Alternatively, genome editing technology (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)) makes it possible to incorporate expression units into the host cell's genomic DNA and to modify the expression units inherently present in the host cell.

[0151] The present invention also provides an expression vector comprising the polynucleotide of the present invention and a promoter operably linked thereto. The expression vector of the present invention may further include elements such as a terminator that functions in a host cell, a ribosome binding site, and a drug resistance gene. Examples of drug resistance genes include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0152] The expression vector may also further include a region that enables homologous recombination with the host cell's genome for homologous recombination with the host cell's genomic DNA. For example, the expression vector may be designed so that the expression units contained therein are located between a pair of homologous regions (e.g., homology arms, or loxP, or FRT, homologous to a specific sequence in the host cell's genome). The genomic region of the host cell to which the expression units should be introduced (the target of the homologous region) is not particularly limited, but may be a locus of a gene that is highly expressed in the host cell.

[0153] The expression vector may be a plasmid, viral vector, phage, or artificial chromosome. The expression vector may also be an integrative or non-integrative vector. An integrative vector may be a vector that is entirely integrated into the host cell's genome. Alternatively, an integrative vector may be a vector in which only a portion (e.g., an expression unit) is integrated into the host cell's genome. The expression vector may also be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and their derivatives.

[0154] Host cells for expressing the affinity polypeptide of the present invention include, for example, Escherichia coli and other Escherichia species, and Corynebacterium species (e.g., Corynebacterium glutamicum). Various prokaryotic cells can be used, including bacteria of the genus Bacillus (e.g., Bacillus subtilis), bacteria of the genus Saccharomyces (e.g., Saccharomyces cerevisiae), bacteria of the genus Pichia (e.g., Pichia stipitis), and bacteria of the genus Aspergillus (e.g., Aspergillus oryzae). Alternatively, insect cells, plant cells, and animal cells (e.g., mammalian cells such as Chinese hamster ovary (CHO) cells) can be used as hosts. As a host, strains lacking a specific gene may be used. Examples of host cells include host cells that possess an expression vector in the cytoplasm, and host cells into which the target gene has been introduced into the genome.

[0155] When the affinity polypeptide of the present invention contains a tripeptide consisting of Gln-Glu-Thr (QET) at its N-terminus, it is preferable to use an affinity polypeptide that can be prepared by a polypeptide secretion production method using Corynebacterium as a host (International Publication No. 2013 / 062029). This method is suitable for preparing affinity polypeptides because it can add the three N-terminal residues Gln-Glu-Thr (QET) of a mature Csp protein to the N-terminus of the target polypeptide, and moreover, it can easily and abundantly prepare polypeptides containing a glutamine residue (Q) at the N-terminus. In this case, various signal peptides such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 42) can be added to the N-terminal side of QET (see Examples, International Publication No. 2013 / 062029, and International Publication No. 2020 / 090979). Examples of coryneform bacteria that can be used in this method include bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum, Corynebacterium statyonis) and bacteria of the genus Brevibacterium.

[0156] The host cells of the present invention can be cultured in a culture medium having, for example, the composition described below, using a predetermined culture apparatus (e.g., test tubes, flasks, jar fermenters). Culture conditions can be set as appropriate. Specifically, the culture temperature may be 10°C to 37°C, the pH may be 6.5 to 7.5, and the culture time may be 1h to 100h. Culture may also be performed while controlling the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture medium may be used as a control indicator. The aeration and stirring conditions can be controlled so that the relative dissolved oxygen concentration (DO value), assuming an atmospheric oxygen concentration of 21%, does not fall below, for example, 1 to 10%, preferably 3% to 8%. Furthermore, the culture may be performed as batch culture or fed-batch culture. In the case of fed-batch culture, the culture can be continued by continuously or discontinuously adding a solution containing a sugar source or a solution containing phosphate to the culture medium.

[0157] As promoters for expressing the polynucleotides of the present invention, promoters commonly used for heterologous protein production in E. coli can be used. Examples of strong promoters include PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and T5 promoter, with PhoA, PhoC, and lac being preferred. As vectors, for example, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and their derivatives may be used.

[0158] Furthermore, a terminator, which is a transcription termination sequence, is linked downstream of the polynucleotide of the present invention. This is also acceptable. Examples of such terminators include the T7 terminator, the fd phage terminator, the T4 terminator, the tetracycline resistance gene terminator, and the E. coli trpA gene terminator.

[0159] As the culture medium, you may use a medium commonly used for culturing E. coli, such as M9-casamino acid medium or LB medium. The medium may contain a specified carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, you may use peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc. Furthermore, the culture conditions and production induction conditions should be appropriately selected according to the type of vector marker, promoter, host bacteria, etc. used.

[0160] The affinity polypeptide of the present invention can be recovered by the following methods. The affinity polypeptide of the present invention can be obtained as a lysate and lysate by recovering the transformed cells of the present invention and then disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the bacterial cells. If the affinity polypeptide is secreted or leaked outside the cell, a disinfectant solution containing the affinity polypeptide can be obtained from the culture medium by centrifugation or membrane filtration. The affinity polypeptide of the present invention can be obtained by subjecting such lysates, lysates and disinfectant solutions to extraction, precipitation, filtration, column chromatography, or other methods.

[0161] 3. Compounds or their salts The compounds or salts thereof of the present invention comprise (A) affinity substances comprising first and second affinity moieties having affinity for the constant region of the heavy chain of an antibody, and (B) reactive groups for the antibody. The definitions, examples, and preferred examples of affinity substances and their constituent elements (e.g., affinity moieties such as affinity peptides, and linkers between affinity moieties such as peptide linkers) are as described above.

[0162] As a reactive group for antibodies, a reactive group for amino acid residues with readily reactive side chains among the amino acid residues constituting the antibody (protein) can be used. Of the 20 natural amino acids that make up proteins as described above, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, which have hydrocarbon side chains, are inactive to normal reactions. Therefore, a reactive group for antibodies is a group that can react to the side chains of one or more (e.g., two, three, or four) of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Depending on conditions such as the amino acid composition of the antibody, one or more (e.g., two, three, or four) reactive groups may be included in the compound of the present invention or its salt, but preferably, one reactive group may be included in the compound of the present invention or its salt.

[0163] Preferably, the antibody-reactive group is a group that can react with the side chain of any one of the 14 amino acids that make up the protein as described above. More preferably, the antibody-reactive group is a reactive group that is specific to the side chain of any one of the amino acids lysine, tyrosine, tryptophan, or cysteine, and even more preferably, a reactive group that is specific to the side chain of any one of the amino acids lysine, tyrosine, or tryptophan, and particularly preferably, a reactive group that is specific to the side chain of lysine or tyrosine, and especially to the side chain of lysine. For details of such reactive groups, see, for example, International Publication Nos. 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979.

[0164] Reactive groups specific to the side chain of a lysine residue are groups that can specifically react with the amino group (NH2) present in the side chain of the lysine residue. Examples 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-methoxyethyl residues, diazonium terephthalic acid residues, α-halogen-substituted acetamides, and α-halogen-substituted methyl ketones. The reaction between the above-mentioned reactive groups specific to the side chain of a lysine residue and the amino group (NH2) present in the side chain of the lysine residue can generate ligation sites such as amide residues, urea residues, pyridine residues, carbamate residues, and sulfonamide residues.

[0165] In certain embodiments, the compound or salt of the present invention is of the following formula (I): [ka] [During the ceremony, R indicates a reactive group for antibodies. L indicates the linker. A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of the antibody. It may also be represented as ]. The definitions, examples, and preferred examples of the reactive group for the antibody represented by R and the affinity substance represented by A are as described above.

[0166] The linker is a divalent group. The divalent group may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above.

[0167] In certain embodiments, the compound or salt of the present invention may further contain a cleavage moiety between the affinity substance and the antibody-reactive group. In this case, the compound or salt represented by formula (I) may contain a linker containing the cleavage moiety.

[0168] The cleavable portion is a site that can be cleaved by specific treatments under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). Therefore, the cleavable portion can be said to be a site (bonds other than amide bonds) that can be cleaved by specific cleavage treatments 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 such as light, or (c) incubation when using a cleavable linker containing a self-degradable cleavable portion. Such linkers that can cleave and their cleavage conditions 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., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society. 132, 17928 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release). (Rolled Release, 95, 291 (2004)). Reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are as described below. Examples of cleavable parts 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), phosphoamidate residues, aconityl residues, trityl residues, azo residues, vicinaldiol residues, selenium residues, aromatic ring-containing residues with electron-withdrawing groups, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0169] The aromatic ring group having an electron-withdrawing group is preferably an aromatic ring group selected from the group consisting of aryl, aralkyl, aromatic heterocyclic, and alkyl having an aromatic heterocyclic group, with aralkyl and alkyl having an aromatic heterocyclic group being more preferred. 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 electron-withdrawing groups include halogen atoms, alkyl groups substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl, and keto groups (e.g., acyl).

[0170] The definitions, examples, and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, and aralkyl, which are found as prefixes, suffixes, and other terms related to the names of residues as cleavable parts, are the same as those described above.

[0171] Examples of ester residues include ordinary ester residues composed of carbon atoms and oxygen atoms [e.g., alkyl esters (e.g., tertiary alkyloxycarbonyls such as tert-butyloxycarbonyl), aryl esters (e.g., phenacyl esters, 2-(diphenylphosphino)benzoate), glycosyl ester residues, orthoester residues], ester residues containing sulfur and oxygen atoms (e.g., thioester residues such as α-thiophenyl ester residues and alkylthioester residues), ester residues containing phosphorus and oxygen atoms (e.g., phosphodiester residues, phosphotriester residues), and activated ester residues (e.g., N-hydroxysuccinimide residues).

[0172] Examples of sulfone-containing residues include sulfone residues and quinolinylbenzenesulfonate residues.

[0173] 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) or diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane).

[0174] Alkoxyalkyl (i.e., alkyloxyalkyl) residues are groups formed by combining alkyloxy and alkyl as described above, and include, but are not limited to, methoxymethyl, ethoxymethyl, methoxyethyl, and ethoxyethyl residues.

[0175] Unsaturated bond-containing chain residues are residues containing an unsaturated bond portion consisting only of carbon atoms [e.g., vinyl (ethenyl), the smallest unit with a carbon-carbon double bond, or acetylenyl (ethynyl), the smallest unit with a carbon-carbon triple bond], or carbon atoms and heteroatoms These are residues containing an unsaturated bond portion (e.g., aldehyde, cyano) consisting of (e.g., nitrogen atom, sulfur atom, oxygen atom). Examples of unsaturated bond-containing chain residues include vinyl ether residues, cyanoethyl residues, ethylene residues, and malondialdehyde residues.

[0176] 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, dodecyl sulfate, N-dodecanoylsarcosinate, 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-(diphenylphosphinone)benzoate residues.

[0177] Examples of basic substances (also called nucleophiles) include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, and ammonium acetate, and organic basic substances such as hydroxylamine, triethylamine, and N,N'-diisopropylamine. 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.

[0178] Examples of reducing agents include cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Examples of sites that can be cleaved by reducing agents include disulfide residues, alkoxyalkyl residues, and azo residues.

[0179] Examples of oxidizing agents include sodium periodate and oxidized glutathione. Examples of sites that can be cleaved by oxidizing agents include vicinaldiol residues and selenium residues.

[0180] Examples of enzymes include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloproteinase, lipase, endoglycosidase, and PN gauze F. Examples of sites that can be cleaved by these enzymes include ester residues, phosphodiester residues, and glycosyl residues.

[0181] Examples of sites that can be cleaved by light include 2-nitrobenzyl residues, phenacyl ester residues, 8-quinoline benzenesulfonate residues, coumarin residues, phosphotriester residues, bisarylhydrazone residues, and bimandithiopropionic acid residues.

[0182] Examples of self-degrading cleavable moieties include activated ester residues (e.g., N-hydroxysuccinimide residues).

[0183] If the compound or salt of the present invention contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthogonal functional group on the reactive group side upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including ordinary ester residues and other ester residues such as thioester residues), acetal residues (including ordinary ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinaldiol residues.

[0184] If the compound or salt of the present invention contains a cleavable moiety that can generate a bioorthothic functional group on the reactive group side by cleavage, then the following formula (Ia): [ka] [During the ceremony, R indicates a reactive group for antibodies. L1 indicates the first linker. L2 indicates the second linker. CLE(B) represents a cleavable moiety that can generate a bioorthothic functional group on the reactive group side by cleavage. A represents an affinity substance containing first and second affinity moieties having affinity for the constant region in the heavy chain of the antibody. It may also be represented as ]. The definitions, examples, and preferred examples of the reactive group for the antibody represented by R, the affinity substance represented by A, and the cleavable moiety represented by CLE(B), which can generate a bioorthothic functional group on the reactive group side by cleavage, are as described above.

[0185] The first linker, indicated by L1, and the second linker, indicated by L2, may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent groups are substituted include those listed above.

[0186] In certain embodiments, the total number of atoms constituting the main chain in the first and second linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0187] The number of atoms constituting the main chain in the first linker and the second linker may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0188] The main chains in the first and second linkers consist of a chain-like structure, a cyclic structure, or a combination thereof. If the main chain is a chain-like structure that does not include a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain-like structure. On the other hand, if the main chain includes a cyclic structure, the number of atoms in the main chain can be determined by counting a predetermined number of atoms that constitute the cyclic structure. Specifically, the number of atoms in the main chain in a cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (see, for example, the bolded paths in (a) to (d) below). If the main chain includes a combination of chain-like and cyclic structures, the number of atoms in the main chain can be determined by adding the number of atoms in the chain-like structure that does not include a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure. The method for counting the number of atoms in the main chain is the same for other linkers. [ka] • is a coupling. In case (a), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 2. In case (b), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 3. In case (c), since both paths are the shortest paths (equidistances), the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4. In case (d), since the condensation site is the shortest path, the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4.

[0189] In certain embodiments, the compound or salt of the present invention is given by the following formula (Ia-1): [ka] [During the ceremony, X represents a leaving group, W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. L3 indicates the third linker. L4 indicates the fourth linker. S represents a sulfur atom. A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of the antibody. It may also be a compound represented by ] or a salt thereof. The definition, examples, and preferred examples of the affinity substance represented by A are as described above.

[0190] The leaving group represented by X is a group that can be eliminated by a reaction between a carbon atom in the adjacent C=W1 group and an amino group. Those skilled in the art can appropriately define such a leaving group. Examples of such leaving groups include: (a)R A -S(here, R A represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom. (b)R A -O(here, R A represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom. (c)R A -(R B -)N(Here, R A and R B Each independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom. (d) Halogen atom.

[0191] Preferably, the leaving group represented by X may be one of the following: (a)R A -S(here, R A represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom. (b)R A -O(here, R A represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom. (c)R A -(R B -)N(Here, R A and R B Each of these independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom.

[0192] More preferably, the leaving group represented by X may be one of the following: (a)R A -S(here, R A represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom. (b)R A -O(here, R A (wherein ∫ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom.)

[0193] More preferably, the leaving group represented by X may be one of the following: (a)R A -S(here, R A (wherein ∫ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom.)

[0194] Particularly preferred, the leaving group represented by X may be one of the following: (a')R A -S(here, R A represents a monovalent aromatic hydrocarbon group (e.g., phenyl) which may have substituents, and S represents a sulfur atom.

[0195] W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. Preferably, W1, W2, and W3 may be oxygen atoms.

[0196] The third linker, indicated by L3, and the fourth linker, indicated by L4, may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent groups are substituted include those listed above.

[0197] In certain embodiments, the total number of atoms constituting the main chain in the third and fourth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0198] The number of atoms constituting the main chain in the third and fourth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0199] The compound or salt of the present invention may also further contain a bioorthogonal functional group between the antibody-reactive group and the cleavage moiety if it contains a cleavage moiety. The bioorthogonal functional group is As described above, the bioorthogonal functional groups are preferably azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with substituents as described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, furan residues, and halocarbonyl residues.

[0200] If the compound or salt of the present invention further contains a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety, then the formula (Ib) is as follows: [ka] [During the ceremony, R indicates a reactive group for antibodies. L5 indicates the fifth linker. L6 indicates the sixth linker. B represents a group containing a bioorthogonal functional group. CLE indicates the cleavage portion. A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of the antibody. It may also be represented as ]. The definitions, examples, and preferred examples of the antibody-reactive group represented by R, the affinity substance represented by A, and the cleavable moiety represented by CLE are as described above.

[0201] The fifth linker, indicated by L5, and the sixth linker, indicated by L6, may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent groups are substituted include those listed above.

[0202] In certain embodiments, the total number of atoms constituting the main chain in the fifth and sixth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0203] The number of atoms constituting the main chain in the fifth and sixth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0204] The group containing the bioorthogonal functional group represented by B may be a group consisting solely of the bioorthogonal functional group, or a group containing the bioorthogonal functional group and other parts. Examples of other parts include the linkage between the bioorthogonal functional group and the linker. The linkage is, for example, a divalent group. The divalent group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the bioorthogonal functional group, the divalent group, and the substituents when the divalent group is substituted are as described above.

[0205] In certain embodiments, the compound or salt of the present invention is given by the following formula (Ib-1): [ka] [During the ceremony, X represents a leaving group, W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. L7 indicates the seventh linker. L8 indicates the 8th linker. B represents a group containing a bioorthogonal functional group. V represents an oxygen atom or a sulfur atom. A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of the antibody. It may also be a compound represented by ] or a salt thereof. The definitions, examples, and preferred examples of the leaving group represented by X, the group containing the bioorthogonal functional group represented by B, and the affinity substance represented by A are as described above.

[0206] W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. Preferably, W1, W2, and W3 may be oxygen atoms.

[0207] The seventh linker, indicated by L7, and the eighth linker, indicated by L8, may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent groups are substituted include those listed above.

[0208] In certain embodiments, the total number of atoms constituting the main chain in the seventh and eighth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0209] The number of atoms constituting the main chain in the seventh and eighth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0210] V represents an oxygen atom or a sulfur atom. Preferably, V may be a sulfur atom.

[0211] The compounds or salts of the present invention can readily modify only one of the heavy chains in the constituent units of an antibody. The compounds or salts of the present invention can also provide a regioselectively modified antibody while readily modifying only one of the heavy chains in the constituent units of an antibody.

[0212] The above-mentioned series of compounds or salts thereof can be produced by reacting the affinity substance of the present invention with a partial compound containing a reactive group for antibodies. For example, such a reaction can be carried out using a suitable organic solvent system (e.g., alkyl halides such as CH2Cl2 (e.g., halogens)). The reaction can be carried out at a suitable temperature (e.g., about -10 to 30°C) in an organic solvent containing methyl methyl phosphate and an amine such as triethylamine. 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.

[0213] The formation of the above-mentioned series of compounds or their salts can be confirmed by methods such as electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific molecular weights of the raw materials and products. Such compounds or their salts can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0214] 4. Affinity-modified antibodies or their salts 4-1. Affinity-modified antibody or salt thereof containing at least one affinity substance (including first and second affinity moieties) The present invention provides an affinity substance-modified antibody or a salt thereof, wherein an affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody's heavy chain is contained within the constant region of the antibody's heavy chain. Definitions, examples, and preferred examples of affinity substances, antibodies, and their constituent elements (e.g., affinity moieties such as affinity peptides, linkers between affinity moieties such as peptide linkers, and constant regions) are as described above.

[0215] Preferably, the affinity-modified antibody or a salt thereof comprises (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) an affinity substance, and (c) the affinity substance is introduced only in the constant region of one of the heavy chains in the immunoglobulin unit (i.e., the affinity-modified antibody is introduced in the constant region of one heavy chain in the immunoglobulin unit, and not in the constant region of the other heavy chain). The definitions, examples, and preferred examples of the antibody, immunoglobulin unit, affinity substance, and their constituent elements (e.g., constant regions) are as described above.

[0216] Affinity-modified antibodies or salts thereof may contain affinity substances through modification of the functional groups in the side chains of one or more (e.g., two, three, or four) amino acid residues from the 14 amino acid residues consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are present in the constant region (preferably the Fc region or CH2 domain). Preferably, affinity-modified antibodies or salts thereof may contain affinity substances through modification of the functional groups in the side chains of one amino acid from the lysine, tyrosine, tryptophan, or cysteine ​​present in the constant region (preferably the Fc region or CH2 domain), more preferably through modification of the functional groups in the side chains of one amino acid from the lysine, tyrosine, or tryptophan, even more preferably through modification of the functional groups in the side chains of lysine or tyrosine, and particularly preferably through modification of the amino group in the side chain of lysine. The positions of these amino acid residues in the constant region are as described above. The modification sites of antibodies or their salts by affinity substances can be confirmed by peptide mapping. As mentioned above, the modifications may be regioselective. Therefore, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modification unit via the functional group in the side chain of the above-mentioned amino acid residue.

[0217] Preferably, affinity-modified antibodies have a constant region (preferably the Fc region) of one of the heavy chains in the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). The affinity substance can be included via modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the region or CH2 domain (in other words, the affinity substance is included via the amino group in the side chain of a lysine residue in the steady region of one heavy chain of the immunoglobulin unit, but not via the amino group in the side chain of a lysine residue in the steady region of the other heavy chain). More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979). The modification may be regioselective, as described above. Therefore, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modification unit via the amino group in the side chain of the lysine residue.

[0218] The production of affinity-modified antibodies or salts thereof can be carried out by reacting the compound or salt thereof of the present invention with an antibody or salt thereof that contains an immunoglobulin unit comprising two heavy chains and optionally two light chains. The equivalent amount of the compound or salt thereof of the present invention relative to the antibody in the reaction (compound or salt thereof / antibody) is not particularly limited as it varies depending on factors such as the type of compound or salt thereof and the antibody, but is for example 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 40, and particularly preferably 6 to 20.

[0219] Such reactions can be carried out under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). For example, such mild reactions can be carried out at room temperature (e.g., about 15-30°C) in a suitable reaction system, such as a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, and more preferably 6.0-8.0. The buffer may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details on such reactions, see, for example, GJLBernardes et al., Chem. Rev., 115, 2174 (2015); GJLBernardes et al., Chem. Asian. J., 4, 630 (2009); BGDavies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0220] In certain embodiments, the affinity-modified antibody or its salt is defined by the following formula (II): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. L indicates the linker. A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135. It is %. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the immunoglobulin unit represented by Ig, the linker represented by L, and the affinity substance represented by A, as well as the antibody, are as described above (for example, for the linker represented by L, see the linker represented by L in the compound of formula (I)).

[0221] The average modification percentage r of the above immunoglobulin units by affinity substances is 65-135%. The average modification percentage r may be 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 82% or more, 84% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, or 96% or more. The average modification percentage r may also be 130% or less, 125% or less, 120% or less, 115% or less, 110% or less, 105% or less, 100% or less, 98% or less, 96% or less, 94% or less, 92% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, or 80% or less. The average modification percentage r is calculated using mass spectrometry (DAR). A calculator (Agilent software) can be used in conjunction with this. (See the example.)

[0222] The average modification percentage r may also preferably be 65-100%, more preferably 70-100%, even more preferably 75-100%, particularly preferably 80-100%, 85-100%, 90-100%, or 95-100%. In these average modification percentages, the upper limit may be a value less than or equal to the above percentages, such as 98% or 96% or less. Alternatively, the average modification percentage r may be 96-100%, 97-100%, 98-100%, 99-100%, or 100%.

[0223] The degree of the average modification percentage r described above can be similarly applied to other average modification percentages r. That is, the degree of the average modification percentage r described above can be similarly applied not only to the average modification percentage r by affinity substances described later, but also to the average modification percentage r by any modification (e.g., bioorthothic functional groups, functional substances) described later.

[0224] The production of an antibody or salt containing a structural unit represented by formula (II) can be carried out by reacting a compound or salt represented by formula (I) with an antibody or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0225] In certain embodiments, affinity-modified antibodies or salts thereof may further contain a cleavage moiety between the affinity substance and the antibody (immunoglobulin unit). In this case, the antibody or salt thereof containing the structural unit represented by formula (II) above may contain a linker containing the cleavage moiety. The definition, examples, and preferred examples of the cleavage moiety are as described above.

[0226] If an affinity-modified antibody or its salt contains a cleavable moiety, the cleavable moiety may be capable of generating bioorthogonal functional groups on the antibody (immunoglobulin unit) side upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including ordinary ester residues and other ester residues such as thioester residues), acetal residues (including ordinary ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinaldiol residues.

[0227] If an affinity-modified antibody or its salt contains a cleavable moiety that can generate bioorthogonal functional groups on the antibody (immunoglobulin unit) side upon cleavage, then the following formula (IIa): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. L1 indicates the first linker. L2 indicates the second linker. CLE(B) represents a cleavable moiety capable of generating bioorthogonal functional groups on the immunoglobulin unit side upon cleavage. A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. The average modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the antibody are as described above: the immunoglobulin unit represented by Ig, the first linker represented by L1, the second linker represented by L2, the cleavable moiety represented by CLE(B), the affinity substance represented by A, and the average modification percentage represented by r.

[0228] The production of an antibody or salt thereof containing a structural unit represented by formula (IIa) can be carried out by reacting a compound or salt thereof represented by formula (Ia) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0229] In certain embodiments, the affinity-modified antibody or its salt is given by the following formula (IIa-1): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. L3 indicates the third linker. L4 indicates the fourth linker. S represents a sulfur atom. A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. The average modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the antibody are as described above: the immunoglobulin unit represented by Ig, the atoms represented by W1, W2, and W3, the third linker represented by L3, the fourth linker represented by L4, the affinity substance represented by A, and the average modification percentage represented by r.

[0230] The production of an antibody or salt containing a structural unit represented by formula (IIa-1) can be carried out by reacting a compound or salt represented by formula (Ia-1) with an antibody or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0231] If the affinity-modified antibody or its salt contains a cleavable moiety, it may further contain a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety. The bioorthogonal functional group is as described above. Preferably, the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with substituents as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a furan residue, and a halocarbonyl residue.

[0232] If an affinity-modified antibody or its salt further contains a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavage moiety, then the formula is as follows (IIb): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. L5 indicates the fifth linker. L6 indicates the sixth linker. B represents a group containing a bioorthogonal functional group. CLE indicates the cleavage portion. A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody. The average modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the antibody are as described above: the immunoglobulin unit represented by Ig, the fifth linker represented by L5, the sixth linker represented by L6, the group containing a bioorthogonal functional group represented by B, the cleavage moiety represented by CLE, the affinity substance represented by A, and the average modification percentage represented by r.

[0233] The production of an antibody or salt thereof containing a structural unit represented by formula (IIb) can be carried out by reacting a compound or salt thereof represented by formula (Ib) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0234] In certain embodiments, the affinity-modified antibody or its salt is given by the following formula (IIb-1): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. W1, W2, and W3 each independently represent either an oxygen atom or a sulfur atom. L7 indicates the seventh linker. L8 indicates the 8th linker. B represents a group containing a bioorthogonal functional group. V represents an oxygen atom or a sulfur atom, and A represents an affinity substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of the antibody. The average modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the antibody are as described above: the immunoglobulin unit represented by Ig, the atoms represented by W1, W2, and W3, the seventh linker represented by L7, the eighth linker represented by L8, the group containing a bioorthothic functional group represented by B, the atom represented by V, the affinity substance represented by A, and the average modification percentage represented by r.

[0235] The production of an antibody or salt containing a structural unit represented by formula (IIb-1) can be carried out by reacting a compound or salt represented by formula (Ib-1) with an antibody or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0236] Affinity-modified antibodies or salts thereof may further contain additional modification moieties. Various methods are known for modifying antibodies. Therefore, in the present invention, affinity-modified antibodies or salts thereof can be modified to further contain additional modification moieties. The additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0237] In certain embodiments, the additional modification moiety may be an additional affinity substance comprising a third affinity moiety having affinity for a constant region in the heavy chain of the antibody. The "affinity moiety" in "third affinity moiety" and the "affinity substance" in "additional affinity substance" are the same as those described above. The third affinity moiety may be the same as or different from the first and / or second affinity moiety described above, but it is preferable that it be different.

[0238] In certain embodiments, an additional modification moiety, including a third affinity moiety having affinity for the constant region of the antibody heavy chain, may be introduced into the constant region of the two heavy chains via modification of amino groups in the side chains of lysine residues located at one or more positions in the constant region of the two heavy chains. The affinity-modified antibody or a salt thereof is introduced into one or more (preferably 1 or 2) constant regions (preferably the Fc region or CH2 domain) of the two heavy chains in the antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains) of the two heavy chains. The additional modification moiety may be included via modification of the amino group in the side chain of the lysine residue, more preferably 1). More specifically, the positions of one or more (preferably 1 or 2, more preferably 1) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979). The position where the additional modification moiety, which includes a third affinity moiety having affinity for the constant region of the antibody heavy chain, is introduced is preferably different from the position where the affinity substance, which includes the first and second affinity moieties having affinity for the constant region of the antibody heavy chain, is introduced. For example, if the affinity substance is introduced at the 246 / 248 position lysine residue, the additional modification portion is preferably introduced at the 288 / 290 position or the 317 position lysine residue, with the 288 / 290 position being more preferred. If the affinity substance is introduced at the 288 / 290 position lysine residue, the additional modification portion is preferably introduced at the 246 / 248 position or the 317 position lysine residue, with the 246 / 248 position being more preferred. If the affinity substance is introduced at the 317 position lysine residue, the additional modification portion is preferably introduced at the 246 / 248 position or the 288 / 290 position lysine residue.

[0239] 4-2. Affinity-modified antibody or salt thereof containing at least two affinity substances (including the first, second, third, and fourth affinity moieties) The present invention also provides an affinity substance-modified antibody or a salt thereof, comprising a first modification portion containing a first affinity substance having first and second affinity moieties having affinity to the constant region of the antibody heavy chain, and a second modification portion containing a second affinity substance having third and fourth affinity moieties having affinity to the constant region of the antibody heavy chain, both of which are contained within the constant region of the antibody heavy chain. Definitions, examples, and preferred examples of affinity substances, antibodies, and their constituent elements (e.g., affinity moieties such as affinity peptides, linkers between affinity moieties such as peptide linkers, and constant regions) are as described above. The first and second modification portions may be the same or different, and are preferably different.

[0240] Preferably, such affinity-modified antibodies or salts thereof comprise (a) an antibody unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) the first and second modification portions, wherein (c) the first modification portion is introduced into the constant region of the first heavy chain in the immunoglobulin unit, and (d) the second modification portion is introduced into the constant region of the second heavy chain in the immunoglobulin unit. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, affinity substances, and their constituent elements (e.g., constant regions) are as described above.

[0241] Affinity-modified antibodies or salts thereof may include a first modified moiety containing a first affinity substance and a second modified moiety containing a second affinity substance, through modification of functional groups in the side chain of one or more (e.g., two, three, or four) amino acid residues from 14 amino acid residues consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are present in the constant region (preferably the Fc region or CH2 domain). Affinity-modified antibodies or salts thereof may preferably contain a first modified moiety containing a first affinity substance and a second modified moiety containing a second affinity substance, via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, tryptophan, or cysteine ​​present in the constant region (preferably the Fc region or CH2 domain), more preferably via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, or tryptophan, even more preferably via modification of a functional group in the side chain of lysine or tyrosine, and particularly preferably via modification of an amino group in the side chain of lysine. The positions of these amino acid residues in the constant region are as described above. The modification sites of antibodies or salts thereof by affinity substances can be confirmed by peptide mapping. The modifications may be regioselective, as described above. Therefore, in the formulas described later, the immunoglobulin unit may regioselectively have the corresponding modified unit via the functional group in the side chain of the amino acid residue.

[0242] Preferably, affinity-modified antibodies may include a first modified moiety containing the first affinity substance via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of the first heavy chain in the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and may also include a second modified moiety containing the second affinity substance via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of the second heavy chain. More specifically, the positions of the one or more (preferably one or two, more preferably one) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Therefore, in the formula described later, the immunoglobulin unit may regioselectively possess the corresponding modification unit via the amino group in the side chain of the lysine residue.

[0243] The production of affinity-modified antibodies or their salts can be carried out as follows: (1) Reacting the compound of the present invention or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an affinity-modified antibody or a salt thereof comprising a first modification moiety containing the first affinity substance in the constant region of the heavy chain in the immunoglobulin unit; and (2) Reacting an affinity-modified antibody or a salt thereof containing the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with the compound of the present invention or a salt thereof to produce an affinity-modified antibody or a salt thereof containing the first and second modification portions in the constant region of the heavy chain of the immunoglobulin unit. The compound or salt of the present invention used in step (1) and the compound or salt of the present invention used in step (2) may be the same or different, but it is preferable that they be different. The equivalent amount of the compound or salt of the present invention relative to the antibody in the reaction (compound or salt of the present invention / antibody) is not particularly limited as it varies depending on factors such as the type of compound or salt of the present invention and the antibody, but for example it is 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 40, and particularly preferably 6 to 20.

[0244] Such reactions can be carried out under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). For example, reactions under such mild conditions are similar to those described above.

[0245] In certain embodiments, such affinity-modified antibodies or salts thereof are expressed by the following formula (V): [ka] [During the ceremony, Ig represents an immunoglobulin unit consisting of two heavy chains, a first and a second heavy chain, and optionally two light chains. L L and L R Each of these independently indicates a linker. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L、 and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0246] In certain embodiments, the affinity-modified antibody or a salt thereof may further include (iii') a first cleavage moiety between (i') the first affinity substance and (ii') the immunoglobulin unit, and / or further include (iii'') a second cleavage moiety between (i'') the second affinity substance and (ii'') the immunoglobulin unit. The first and second cleavage moieties are the same as the cleavage moieties described above. The first and second cleavage moieties may be the same or different.

[0247] If an affinity-modified antibody or a salt thereof contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including ordinary ester residues and other ester residues such as thioester residues), acetal residues (including ordinary ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinaldiol residues.

[0248] If an affinity-modified antibody or its salt contains a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, then the following formula (Va): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L1 and L R1 Each of these independently indicates the first linker. L L2 and L R2 Each of these independently indicates the second linker. CLE(B) L and CLE(B) R Each of these independently represents a cleavable portion capable of generating bioorthogonal functional groups on the immunoglobulin unit side by cleavage. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0249] The production of an antibody or salt containing a structural unit represented by formula (Va) can be carried out by reacting a compound or salt represented by formula (Ia) with an antibody or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0250] In certain embodiments, the affinity-modified antibody or its salt is given by the following formula (Va-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 Each of these independently represents either an oxygen atom or a sulfur atom. L L3 and L R3 Each of these independently indicates the third linker. L L4 and L R4 Each of these independently indicates the fourth linker. S represents a sulfur atom. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L, and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0251] The production of an antibody or salt containing a structural unit represented by formula (Va-1) can be carried out by reacting a compound or salt represented by formula (Ia-1) with an antibody or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0252] The affinity-modified antibody or a salt thereof may further include (iv') a first bioorthogonal functional group between (ii') an immunoglobulin unit and (iii') the first cleavable moiety, and / or further include (iv'') a second bioorthogonal functional group between (ii'') an immunoglobulin unit and (iii'') the second cleavable moiety. The bioorthogonal functional groups are as described above. Preferably, the bioorthogonal functional groups include azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with substituents as described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, furan residues, and halocarbonyl residues.

[0253] If an affinity-modified antibody or its salt further contains a bioorthogonal functional group between the immunoglobulin unit and the cleavable moiety, then the formula (Vb) is as follows: [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L5 and L R5 Each of these independently indicates the fifth linker. L L6 and L R6 Each of these independently indicates the sixth linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, CLE L and CLE R Each of these independently indicates a cleavage portion. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0254] The production of an antibody or salt thereof containing a structural unit represented by formula (Vb) can be carried out by reacting a compound or salt thereof represented by formula (Ib) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0255] In certain embodiments, the affinity-modified antibody or its salt is given by the following formula (Vb-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 Each of these independently represents either an oxygen atom or a sulfur atom. L L7 and L R7 Each of these independently indicates the seventh linker. L L8 and L R8 Each of these independently indicates the eighth linker. B LThis represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, V L and V R Each of these independently represents either an oxygen atom or a sulfur atom. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0256] The production of an antibody or salt containing a structural unit represented by formula (Vb-1) can be carried out by reacting a compound or salt represented by formula (Ib-1) with an antibody or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0257] The affinity substance-modified antibody or a salt thereof may further include (iii') a first cleavable moiety between (i') a first affinity substance and (ii') the immunoglobulin unit, and (iv'') a first bioorthogonal functional group between (ii'') the immunoglobulin unit and (iii'') a second cleavable moiety. In this case, the first cleavable moiety may be a cleavable moiety capable of generating a second bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[0258] In certain embodiments, the affinity-modified antibody or its salt is defined by the following formula (Vc): [ka] [During the ceremony, Ig represents the immunoglobulin unit, LR1 This indicates the first linker, L R2 This indicates the second linker, L L5 This indicates the fifth linker, L L6 This indicates the sixth linker, B L This indicates a group containing a first bioorthogonal functional group, CLE L This indicates the first cleavable portion, CLE(B) R This represents a second cleavable portion that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage, A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0259] The production of antibodies or salts thereof containing a structural unit represented by formula (Vc) can be carried out by reacting a compound or salt thereof represented by formula (Ia) and a compound or salt thereof represented by formula (Ib) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0260] In certain embodiments, the affinity-modified antibody or its salt is defined by the following formula (Vc-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1, W R2 and W R3 Each of these independently represents either an oxygen atom or a sulfur atom. L R3 Each of these independently indicates the third linker. L R4 Each of these independently indicates the fourth linker. L L7 Each of these independently indicates the seventh linker. L L8 Each of these independently indicates the eighth linker. B L This indicates a group containing a first bioorthogonal functional group, V L This indicates an oxygen atom or a sulfur atom. A L This represents the affinity substance of the first earlier term, A R This represents the second affinity substance, The average modification percentage r of the immunoglobulin unit by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0261] The production of antibodies or salts thereof containing the structural unit represented by formula (Vc-1) can be carried out by reacting a compound or salt thereof represented by formula (Ia-1) and a compound or salt thereof represented by formula (Ib-1) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0262] In the above formulas (V), (Va), (Va-1), (Vb), (Vb-1), (Vc), and (Vc-1), the definition, examples, and preferred examples of the immunoglobulin unit represented by Ig and the antibody are as described above.

[0263] In this specification, the subscript "L" attached to any symbol is used for convenience for symbols positioned to the left of the immunoglobulin unit (Ig). On the other hand, the subscript "R" attached to any symbol is used for convenience for symbols positioned to the right of the immunoglobulin unit (Ig). The meaning of symbols with the subscripts "L" and "R" is the same as the meaning of symbols without the subscripts "L" and "R".

[0264] Therefore, in the above formulas (V), (Va), (Va-1), (Vb), (Vb-1), (Vc), and (Vc-1), the symbols with the subscripts "L" and "R" are as follows: W L1 and W R1 This is similar to W1, and an oxygen atom is preferred. W L2 and W R2 This is similar to W2, and an oxygen atom is preferred. W L3 and W R3 This is similar to W3, and an oxygen atom is preferred. L L and L R The linker indicated by is the same as the linker indicated by L. L and L R The linkers indicated by may be the same or different, but it is preferable that they be different. L L1 and L R1 The first linker indicated by is the same as the first linker indicated by L1. L1 and L R1 The first linker indicated by may be the same or different, but it is preferable that they be different. L L2 and L R2 The second linker indicated by is the same as the second linker indicated by L2. L2 and L R2 The second linker indicated by may be the same or different, but it is preferable that it be different. L L3 and LR3 The third linker indicated by is the same as the third linker indicated by L3. L3 and L R3 The third linker indicated by may be the same or different, but it is preferable that it be different. L L4 and L R4 The fourth linker indicated by is the same as the fourth linker indicated by L4. L4 and L R4 The fourth linker indicated by may be the same or different, but it is preferable that it be different. L L5 and L R5 The fifth linker indicated by is the same as the fifth linker indicated by L5. L5 and L R5 The first linker indicated by may be the same or different, but it is preferable that they be different. L L6 and L R6 The sixth linker indicated by is the same as the sixth linker indicated by L6. L6 and L R6 The sixth linker indicated by may be the same or different, but it is preferable that it be different. L L7 and L R7 The seventh linker indicated by is the same as the seventh linker indicated by L7. L7 and L R7 The seventh linker, indicated by [the symbol], may be the same or different, but it is preferable that it be different. L L8 and L R8 The eighth linker indicated by is the same as the eighth linker indicated by L8. L8 and L R8 The eighth linker indicated by may be the same or different, but it is preferable that it be different. CLE(B) L and CLE(B) R The cleavage portion indicated by is the same as the cleavage portion indicated by CLE(B). CLE(B) L and CLE(B) RThe cleavage portion indicated by the symbol may be the same or different, but it is preferable that they be different. B L A first group containing a first bioorthogonal functional group represented by, and B R The second group containing the second bioorthogonal functional group shown by is the same as the group containing the bioorthogonal functional group shown by B. L A first group containing a first bioorthogonal functional group represented by, and B R The second group, which includes the second bioorthogonal functional group indicated by , may be the same or different, and is preferably different. CLE L and CLE R The cleavage portion indicated by is the same as the cleavage portion indicated by CLE. L and CLE R The cleavage portion indicated by the symbol may be the same or different, but it is preferable that they be different. A L and A R The affinity substance indicated by is the same as the affinity substance indicated by A. L and A R The affinity substances indicated by the symbol may be the same or different. r L and r R The degree of the average modification percentage shown by and the method of determining it are the same as those for the average modification percentage shown by r. L and r R The average modification percentages shown may be the same or different, but it is preferable that they be different.

[0265] Confirmation of the production of the target affinity-modified antibody or its salt can be performed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, depending on the specific molecular weight of the raw materials and product. Regioselectivity can be confirmed by peptide mapping. Peptide mapping can be performed by protease treatment and mass spectrometry, for example. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of affinity molecules can be confirmed by mass spectrometry (a DAR calculator (Agilent software) can be used in combination). The affinity-modified antibody or its salt can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0266] Affinity-modified antibodies or salts thereof may further contain additional modification moieties. Various methods are known for modifying antibodies. Therefore, in the present invention, affinity-modified antibodies or salts thereof can be modified to further contain additional modification moieties. The additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0267] In certain embodiments, the additional modification moiety may be an additional affinity substance comprising a fifth affinity moiety having affinity for a constant region in the heavy chain of the antibody. The "affinity moiety" in "fifth affinity moiety" and the "affinity substance" in "additional affinity substance" are the same as those described above. The fifth affinity moiety may be the same as or different from the first, second, third and / or fourth affinity moieties described above, but it is preferable that it be different.

[0268] In certain embodiments, an additional modification moiety, including a fifth affinity moiety having affinity for the constant region of the antibody heavy chain, may be introduced into the constant region of the two heavy chains via modification of amino groups in the side chains of lysine residues located at one or more positions in the constant region of the two heavy chains. The affinity-modified antibody or its salt is a constituent unit of the antibody (2 units). Additional modification moieties may be included via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of two heavy chains in an immunoglobulin unit (which includes the heavy chain and optionally two light chains). More specifically, the positions of the one or more (preferably one or two, more preferably one) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979). The position where the additional modification portion, which includes a fifth affinity moiety having affinity for the constant region of the antibody heavy chain, is introduced is preferably different from the position where the first modification portion, which includes a first affinity substance containing first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and the second modification portion, which includes a second affinity substance containing third and fourth affinity moieties having affinity for the constant region of the antibody heavy chain, are introduced. For example, if the position where both the first and second modification portions are introduced is the lysine residue at position 246 / 248, the position where the additional modification portion is introduced is preferably the lysine residue at position 288 / 290 or 317, with the lysine residue at position 288 / 290 being more preferred. If the position in which both the first and second modification portions are introduced is the lysine residue at position 317, the position in which the additional modification portion is introduced is preferably the lysine residue at positions 246 / 248 or 288 / 290.

[0269] 5. Antibodies or salts thereof that do not contain affinity substances. 5-1. Overview An antibody or salt thereof that does not contain affinity substances can be produced using an affinity substance-modified antibody or salt thereof that includes (A) an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of the antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and (C) further comprising a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

[0270] More specifically, a method for producing an antibody or a salt thereof that does not contain affinity substances may be the following methods 1-1 or 1-2.

[0271] (Method 1-1) A method for producing an antibody or salt that does not contain an affinity substance, comprising: (A) an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit); and (B) an antibody (immunoglobulin unit); and (C) an affinity substance-modified antibody or salt thereof comprising a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit); and cleaving the cleavable moiety of the affinity substance-modified antibody or salt thereof to produce an antibody or salt that does not contain an affinity substance.

[0272] (Method 1-2) A method for producing an antibody or a salt thereof that does not contain affinity substances, comprising (1) and (2) below: (1) Reacting an affinity substance or a salt thereof with an antibody containing two heavy chains and optionally two light chains, an immunoglobulin unit, to an affinity substance containing a cleavage moiety between the affinity substance and the antibody, with an affinity substance modified antibody or a salt thereof, comprising (A) an affinity substance containing first and second affinity moieties having affinity to the constant region of the heavy chain of an antibody (immunoglobulin unit), and (B) a reactive group for an antibody (immunoglobulin unit), and (C) a compound or salt thereof further comprising a cleavage moiety between (A) the affinity substance and (B) the reactive group; and (2) To produce an antibody or salt thereof that does not contain the affinity substance by cleaving an affinity substance-modified antibody or salt thereof that has a cleavable portion between the affinity substance and the antibody at the cleavable portion.

[0273] Cleavage 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 as described above, (b) treatment with physicochemical stimuli such as light, or (c) incubation when using a cleavage linker containing a self-degradable cleavage portion. For more information on these cleavage treatments, please refer to International Publication Nos. 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979.

[0274] Such cleavage reactions can be carried out under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). For example, such mild conditions are as described above. Also, if the cleavage site is an ester (e.g., a common ester, or other esters such as a thioester), the cleavage reaction can be carried out by incubation in a hydroxylamine hydrochloride solution (e.g., pH 4.0-8.0, 10 mM-10 M) for an appropriate time (e.g., 1 hour) (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160).

[0275] Confirmation of the production of antibodies or salts of antibodies or salts of antibodies obtained by the cleavage reaction that do not contain affinity substances can be performed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, depending on the specific molecular weight of the raw materials and products. Regioselectivity can be confirmed by peptide mapping as described above. The number of affinity substances introduced can be confirmed by mass spectrometry (a DAR calculator (Agilent software) can be used in conjunction). Affinity-modified antibodies or their salts can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0276] Incidentally, if an affinity substance-modified antibody or salt thereof, which includes a cleavable moiety between (A) an affinity substance having first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit) and (B) an antibody (immunoglobulin unit), is used, then (a) the cleavable moiety includes a cleavable moiety that can generate a bioorthothic functional group on the antibody (immunoglobulin unit) side by cleavage, or (b) the antibody (immunoglobulin unit) and the cleavable moiety include a bioorthothic functional group, then an antibody derivative or salt thereof containing a bioorthothic functional group can be produced as an antibody or salt thereof that does not contain an affinity substance.

[0277] Furthermore, by reacting an antibody derivative or salt containing a bioorthogonal functional group with a functional substance, an antibody-functional substance conjugate or salt can be produced as an antibody or salt that does not contain affinity substances.

[0278] Below, we will describe in detail antibodies or salts thereof that do not contain affinity substances, including (1) antibody derivatives or salts thereof that contain bioorthogonal functional groups, and (2) conjugates or salts thereof of antibodies and functional substances.

[0279] 5-2. Antibody derivatives or salts thereof containing at least one bioorthogonal functional group The present invention provides an antibody derivative or salt thereof comprising (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) a bioorthogonal functional group, wherein (c) the bioorthogonal functional group is introduced only in the constant region of one of the heavy chains in the immunoglobulin unit (i.e., the bioorthogonal functional group is introduced in the constant region of one heavy chain in the immunoglobulin unit, and not in the constant region of the other heavy chain). Definitions, examples, and preferred examples of the antibody, immunoglobulin unit, and bioorthogonal functional group, as well as the elements constituting them (e.g., constant regions), are as described above.

[0280] Antibody derivatives or salts thereof may contain bioorthogonal functional groups via modification of the functional groups in the side chains of one or more (e.g., two, three, or four) amino acid residues from the 14 amino acid residues consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are present in the constant region (preferably the Fc region or CH2 domain). Preferably, the antibody derivatives or salts thereof may contain bioorthogonal functional groups via modification of the functional group in the side chain of one amino acid from the lysine, tyrosine, tryptophan, or cysteine ​​present in the constant region (preferably the Fc region or CH2 domain), more preferably via modification of the functional group in the side chain of one amino acid from the lysine, tyrosine, or tryptophan, even more preferably via modification of the functional group in the side chain of lysine or tyrosine, and particularly preferably via modification of the amino group in the side chain of lysine. The positions of these amino acid residues in the constant region are as described above. The modification sites of antibodies or their salts by bioorthogonal functional groups can be confirmed by peptide mapping. As mentioned above, the modifications may be regioselective. Therefore, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modification unit via the functional group in the side chain of the above-mentioned amino acid residue.

[0281] Preferably, the antibody derivative may contain bioorthogonal functional groups via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of one of the antibody's constituent units (an immunoglobulin unit comprising two heavy chains and optionally two light chains). (In other words, it may contain bioorthogonal functional groups via amino groups in the side chains of lysine residues in the constant region of one heavy chain in the immunoglobulin unit, but may not contain bioorthogonal functional groups via amino groups in the side chains of lysine residues in the constant region of the other heavy chain.) More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979). The modification may be regioselective, as described above. Therefore, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modified unit via the amino group in the side chain of the lysine residue.

[0282] In certain embodiments, the antibody derivative or its salt is defined by the following formula (IIIa): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. L1 indicates the first linker. B represents a group containing a bioorthogonal functional group. The average modification percentage r of the above immunoglobulin unit by bioorthogonal functional groups is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, the first linker represented by L1, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the definition, examples, and preferred examples of the antibody are as described above. A particularly preferred bioorthogonal functional group is the thiol group.

[0283] In formula (IIIa), the molecular weight of the substructure represented by L1-B may be 700 or less. When the molecular weight of the substructure represented by L1-B is 700 or less, the antibody derivative or salt having a bioorthogonal functional group is relatively difficult to purify based on molecular weight differences because the ratio of the molecular weight of the substructure to the molecular weight of the entire antibody is very small. However, according to the present invention, which enables high-level control of DAR, an antibody derivative exhibiting the desired DAR can be obtained in high purity without necessarily requiring purification based on molecular weight differences. The molecular weight of the substructure represented by L1-B is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0284] In another specific embodiment, the antibody derivative or a salt thereof is given by the following formula (IIIa-1): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. W1 represents an oxygen atom or a sulfur atom. L3 indicates the third linker. SH indicates a thiol group. The average modification percentage r of the above immunoglobulin unit by bioorthogonal functional groups is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the immunoglobulin unit represented by Ig, the atom represented by W1, the third linker represented by L3, and the average modification percentage represented by r, as well as the antibody, are as described above.

[0285] In formula (IIIa-1), the molecular weight of the substructure represented by C(=W1)-L3-SH may be 700 or less. The molecular weight of the substructure represented by C(=W1)-L3-SH is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[0286] In equation (IIIa-1), the third linker indicated by L3 is (CH2) n1 It is also possible that n1 is an integer between 1 and 10. Preferably, n1 may be an integer greater than or equal to 2. n1 may also be an integer less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4, less than or equal to 3, or less than or equal to 2. Particularly preferably, n1 is 2.

[0287] In yet another specific embodiment, the antibody derivative or a salt thereof is defined by the following formula (IIIb): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. L5 indicates the fifth linker. B represents a group containing a bioorthogonal functional group. T1 indicates a monovalent group. The average modification percentage r of the above immunoglobulin unit by bioorthogonal functional groups is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, the fifth linker represented by L5, the group containing a bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the definition, examples, and preferred examples of the antibody are as described above. The particularly preferred bioorthogonal functional group is the azide group.

[0288] T1 is a monovalent group and can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of monovalent groups include those listed above. Examples of substituents when the monovalent group is substituted include those listed above.

[0289] In certain embodiments, the monovalent group represented by T1 may be a substituted hydroxyamino group. A substituted hydroxyamino group can be represented by the following formula (α). NR i -OR ii (α) [During the ceremony, R i , R ii Each of these independently represents either a hydrogen atom or a monovalent hydrocarbon group. Here, the monovalent hydrocarbon group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the monovalent hydrocarbon group and the substituents when the monovalent hydrocarbon group is substituted are as described above. Preferably, the hydroxyamino group which may be substituted is NH-OR ii (Here, R ii represents an alkyl group. ) It may also be substituted. More preferably, the substituted hydroxyamino group is NH-OR ii (Here, R ii This represents an alkyl group having 1 to 6 carbon atoms.

[0290] In formula (IIIb), the molecular weight of the substructure represented by L5(-B)-T1 may be 700 or less. The molecular weight of the substructure represented by L5(-B)-T1 is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0291] In yet another specific embodiment, the antibody derivative or its salt is defined by the following formula (IIIb-1): [ka] [During the ceremony, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains. W1 and W2 each independently represent either an oxygen atom or a sulfur atom. L7 indicates the seventh linker. B represents a group containing a bioorthogonal functional group. T2 indicates a monovalent group. The average modification percentage r of the above immunoglobulin unit by bioorthogonal functional groups is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The definition, examples, and preferred examples of the antibody are as described above: the immunoglobulin unit represented by Ig, the atoms represented by W1 and W2, the seventh linker represented by L7, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r. A particularly preferred bioorthogonal functional group is the azide group.

[0292] T2 is a monovalent group that can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of monovalent groups are those listed above. Examples of substituents when the monovalent group is substituted are those listed above. The monovalent group represented by T2 may be an unsubstituted hydroxyamino group. Details of the unsubstituted hydroxyamino group are the same as those described for T1.

[0293] In formula (IIIb-1), the molecular weight of the substructure represented by C(=W1)-L7(-B)-C(=W2)-T2 may be 700 or less. The molecular weight of the substructure represented by C(=W1)-L7(-B)-C(=W2)-T2 is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0294] In equation (IIIb-1), the seventh linker, indicated by L7, is (CH2) n2 It may also be the case that n2 is an integer between 1 and 10. Preferably, n2 may be an integer greater than or equal to 2, or greater than or equal to 3. n2 may also be an integer less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4, or less than or equal to 3. Particularly preferably, n2 may be 3.

[0295] In formula (IIIb-1), the group containing the bioorthogonal functional group is NH-C(=O)-(CH2) n3 -N3 may be present. n3 is an integer between 1 and 10. Preferably, n3 may be an integer greater than or equal to 2, 3 or greater, or 4 or greater. n3 may also be an integer less than or equal to 9, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. Particularly preferably, n3 may be 4.

[0296] The production of antibody derivatives or salts containing bioorthogonal functional groups is (A) antibody (immunoglobulin This can be done using an affinity substance comprising (B) an antibody (immunoglobulin unit) and (C) an affinity substance-modified antibody or a salt thereof, further comprising a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

[0297] In certain embodiments, if the affinity-modified antibody or a salt thereof contains a cleavable moiety that can generate a bioorthothic functional group on the antibody (immunoglobulin unit) side by cleavage, an antibody derivative or salt thereof containing a bioorthothic functional group can be produced.

[0298] More specifically, examples of such manufacturing methods include (2-1) to (2-6) below (Figures 2 to 6).

[0299] (Method 2-1) A method for producing an antibody derivative or salt containing a bioorthogonal functional group, comprising: (A) an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of an antibody (immunoglobulin unit); and (B) an antibody (immunoglobulin unit); and (C) an affinity substance-modified antibody or salt containing an affinity substance comprising a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit) (where the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage at the cleavable moiety.

[0300] (Method 2-2) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising (1) and (2) below: (1) Reacting a compound or salt thereof containing an affinity substance comprising (A) first and second affinity moieties having affinity for the constant region of the heavy chain of an antibody (immunoglobulin unit), and (B) a reactive group for the antibody (immunoglobulin unit), and further comprising (C) a cleavable moiety between (A) the affinity substance and (B) the reactive group (where the cleavable moiety is a cleavable moiety that can generate a bioorthoth functional group on the reactive group side for the antibody (immunoglobulin unit) upon cleavage), with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains, to produce an affinity substance-modified antibody or salt thereof containing a cleavable moiety between the affinity substance and the antibody (where the cleavable moiety is a cleavable moiety that can generate a bioorthoth functional group on the antibody side upon cleavage); and (2) To produce an antibody derivative or salt containing a bioorthogonal functional group by cleaving an affinity substance-modified antibody or a salt thereof, which has a cleavable moiety between the affinity substance and the antibody, at the cleavable moiety.

[0301] (Method 2-3) A method for producing an antibody derivative or salt containing a bioorthogonal functional group, comprising cleaving an antibody or a salt containing the structural unit represented by formula (IIa) above at a cleavable moiety to produce an antibody derivative or a salt containing the structural unit represented by formula (IIIa) above.

[0302] (Method 2-4) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising (1) and (2) below: (1) Reacting a compound represented by formula (Ia) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or a salt thereof containing a structural unit represented by formula (IIa); and (2) To produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa) by cleaving an antibody or salt thereof containing a structural unit represented by formula (IIIa) at a cleavable portion.

[0303] (Method 2-5) A method for producing an antibody derivative or salt containing a bioorthogonal functional group, comprising cleaving an antibody or a salt containing the structural unit represented by formula (IIa-1) at a cleavable portion to produce an antibody derivative or a salt containing the structural unit represented by formula (IIIa-1).

[0304] (Method 2-6) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising (1) and (2) below: (1) Reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or a salt thereof containing a structural unit represented by formula (IIa-1); and (2) Cutting an antibody or a salt thereof containing a structural unit represented by formula (IIa-1) at a cleavable portion to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIa-1).

[0305] Method 2-1 may be carried out by Method 2-3 or 2-5. Method 2-2 may be carried out by Method 2-4 or 2-6. Methods 2-2, 2-4, and 2-6 may further include reacting the affinity substance of the present invention with a partial compound containing a reactive group for an antibody to produce the compound of the present invention or a salt thereof (Figures 2-6).

[0306] In another specific embodiment, if the affinity-modified antibody or a salt thereof contains a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety, an antibody derivative containing a bioorthogonal functional group or a salt thereof can be produced.

[0307] More specifically, examples of such manufacturing methods include (2-7) to (2-12) below (Figures 2 to 6).

[0308] (Method 2-7) A method for producing an antibody derivative or salt containing a bioorthogonal functional group, comprising cleaving an affinity substance-modified antibody or salt containing an affinity for an antibody (immunoglobulin unit), comprising (A) an affinity substance having first and second affinity moieties having affinity for a constant region in the heavy chain of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and further comprising (C) a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit), and (D) a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety, at the cleavable moiety to produce an antibody derivative or salt containing a bioorthogonal functional group.

[0309] (Method 2-8) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising (1) and (2) below: (1) Reacting an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains with an antibody or salt thereof, wherein the antibody comprises an affinity substance comprising (A) first and second affinity moieties having affinity for the constant region of the heavy chain of an antibody (immunoglobulin unit), and (B) a reactive group for the antibody (immunoglobulin unit), and further comprising (C) a cleavage moiety between (A) the affinity substance and (B) the reactive group, and (D) a bioorthogonal functional group between the reactive group and the cleavage moiety; and (2) To produce an antibody derivative or salt containing a bioorthogonal functional group by cleaving an affinity substance-modified antibody or a salt thereof, which has a cleavable moiety between the affinity substance and the antibody, at the cleavable moiety.

[0310] (Method 2-9) An antibody or a salt containing the structural unit represented by the above formula (IIb) is subjected to cleavage A method for producing an antibody derivative or salt containing a bioorthogonal functional group, comprising performing a partial cleavage to produce an antibody derivative or salt containing a structural unit represented by the above formula (IIIb).

[0311] (Method 2-10) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising (1) and (2) below: (1) Reacting a compound represented by formula (Ib) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or a salt thereof containing a structural unit represented by formula (IIb); and (2) To produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb) by cleaving an antibody or salt thereof containing a structural unit represented by formula (IIIb) at a cleavable portion.

[0312] (Method 2-11) A method for producing an antibody derivative or salt containing a bioorthogonal functional group, comprising cleaving an antibody or salt containing the structural unit represented by formula (IIb-1) at a cleavable portion to produce an antibody derivative or salt containing the structural unit represented by formula (IIIb-1).

[0313] (Method 2-12) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising (1) and (2) below: (1) Reacting a compound represented by formula (Ib-1) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or a salt thereof containing a structural unit represented by formula (IIb-1); and (2) Cutting an antibody or a salt thereof containing a structural unit represented by formula (IIb-1) at a cleavable portion to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIb-1).

[0314] Method 2-7 above may be carried out by Method 2-9 or 2-11 above. Method 2-8 above may be carried out by Method 2-10 or 2-11 above. Methods 2-8, 2-10, and 2-12 above may further include reacting the affinity substance of the present invention with a moiety containing a reactive group for an antibody to produce the compound of the present invention or a salt thereof (Figures 2-6).

[0315] The antibody derivative or a salt thereof may further contain additional modification moieties. Various methods are known for modifying antibodies. Therefore, in the present invention, the antibody derivative or a salt thereof can be modified to further contain additional modification moieties. The additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0316] In certain embodiments, the additional modification portion may include a bioorthogonal functional group. The bioorthogonal functional group is the same as that described above. The bioorthogonal functional group included in the additional modification portion may be the same as or different from the bioorthogonal functional group in (b) above, but it is preferable that it be different.

[0317] In certain embodiments, additional modification moieties containing bioorthogonal functional groups may be introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues located at one or more positions in the constant regions of the two heavy chains. Antibody derivatives or salts thereof may contain additional modification moieties via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant regions (preferably the Fc region or CH2 domain) of the two heavy chains in an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979). The positions where additional modification moieties containing bioorthogonal functional groups are introduced are preferably different from the positions where the bioorthogonal functional groups are introduced in (b) above. For example, if the position in which the bioorthogonal functional group described in (b) above is introduced is the lysine residue at positions 246 / 248, the position in which the additional modification portion is introduced is preferably the lysine residue at positions 288 / 290 or 317, with the lysine residue at position 288 / 290 being more preferred. If the position in which the bioorthogonal functional group described in (b) above is introduced is the lysine residue at positions 288 / 290, the position in which the additional modification portion is introduced is preferably the lysine residue at positions 246 / 248 or 317, with the lysine residue at position 246 / 248 being more preferred. If the position in which the bioorthogonal functional group described in (b) above is introduced is the lysine residue at position 317, the position in which the additional modification portion is introduced is preferably the lysine residue at positions 246 / 248 or 288 / 290.

[0318] 5-3. Antibody derivatives or salts thereof containing at least two bioorthogonal functional groups The present invention also comprises (a) an immunoglobulin unit comprising two heavy chains consisting of first and second heavy chains and optionally two light chains, and (b) a first modified portion comprising a first bioorthogonal functional group and a second modified portion comprising a second bioorthogonal functional group. (c) The first modification portion is introduced into the steady region of the first heavy chain, (d) The second modification portion is introduced into the steady region of the second heavy chain, (e) Provide an antibody derivative or salt thereof comprising first and second modified portions, wherein the first and second modified portions are distinct from each other. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, as well as the elements constituting them (e.g., constant regions), are as described above.

[0319] The antibody derivative or a salt thereof may contain a first modified moiety containing a first bioorthogonal functional group and a second modified moiety containing a second bioorthogonal functional group, through modification of functional groups in the side chains of one or more (e.g., two, three, or four) amino acid residues from 14 amino acid residues consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are present in the constant region (preferably the Fc region or CH2 domain). The antibody derivative or its salt may preferably contain a first modified moiety containing a first bioorthogonal functional group and a second modified moiety containing a second bioorthogonal functional group, via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, tryptophan, or cysteine ​​present in the constant region (preferably the Fc region or CH2 domain), more preferably via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, or tryptophan, even more preferably via modification of a functional group in the side chain of lysine or tyrosine, and particularly preferably via modification of an amino group in the side chain of lysine. The positions of these amino acid residues in the constant region are as described above. The modification positions of the antibody or its salt by bioorthogonal functional groups can be confirmed by peptide mapping. The modification may be regioselective, as described above. Therefore, in the formula described later, the immunoglobulin unit may regioselectively have the corresponding modified unit via the functional group in the side chain of the amino acid residue.

[0320] Preferably, the antibody derivative has one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of one of the heavy chains in the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). The modification of the amino group in the side chain may include a first modified portion containing a first bioorthogonal functional group and a second modified portion containing a second bioorthogonal functional group, respectively. More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Therefore, in the formula described later, the immunoglobulin unit may regioselectively have the corresponding modified unit via the amino group in the side chain of the lysine residue.

[0321] In certain embodiments, the antibody derivative or its salt is defined by the following formula (VIa): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L1 and L R1 Each of these independently indicates the first linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0322] The production of an antibody or salt containing a structural unit represented by formula (VIa) is performed using CLE(B) in an affinity-modified antibody or salt containing a structural unit represented by formula (Va). L and CLE(B) RThis can be carried out by cleaving the two cleavable parts indicated by . If the two cleavable parts are the same, the cleavage reaction can be carried out in a single cleavage reaction. If the two cleavable parts are different, the cleavage reaction can be carried out in a single cleavage reaction (e.g., if two different cleavable parts can be cleaved with the same cleavage treatment or cleavage agent) or in two cleavage reactions (e.g., if two different cleavable parts can be cleaved with different cleavage treatments or cleavage agents). Details of the cleavage reaction are as described above.

[0323] In certain embodiments, the antibody derivative or its salt is defined by the following formula (VIa-1): [ka] [During the ceremony, Ig represents the immunoglobulin unit, W L1 and W R1 Each of these independently represents either an oxygen atom or a sulfur atom. L L3 and L R3 Each of these independently indicates the third linker. SH represents a thiol group, which is a bioorthogonal functional group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are 65-1 It is 35%. It may also be an antibody or a salt thereof containing the structural unit represented by ].

[0324] The production of antibodies or salts containing the structural unit represented by formula (VIa-1) can be carried out by cleaving the two cleavable moieties indicated by CS in the affinity-modified antibody or salt containing the structural unit represented by formula (Va-1). The cleavage reaction can be carried out in a single reaction. Details of the cleavage reaction are as described above.

[0325] In certain embodiments, the antibody derivative or its salt is defined by the following formula (VIb): [ka] [During the ceremony, Ig represents the immunoglobulin unit, L L5 and L R5 Each of these independently indicates the fifth linker. B L This represents a first group containing a first bioorthogonal functional group, B R This indicates a second group containing a second bioorthogonal functional group, T L1 and T R1 Each of these independently represents a monovalent group. The average modification percentage r of the immunoglobulin units by the first modification portion L , and the average modification percentage r of the immunoglobulin units by the second modification portion R These are each 65-135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof.

[0326] The production of an antibody or salt containing a structural unit represented by formula ...

Claims

1. A method for producing an antibody or a salt thereof that does not contain affinity polypeptides, (A) an affinity polypeptide comprising first and second affinity peptides having affinity for the constant region of the heavy chain of an antibody, and (B) an affinity polypeptide-modified antibody or a salt thereof comprising an antibody, are cleaved at a cleavable portion to produce an antibody or a salt thereof that does not contain an affinity polypeptide. The affinity polypeptide-modified antibody Formula (IIa) below: 【Chemistry 1】 [During the ceremony, Ig represents the antibody, L １ This indicates the first linker. L ２ This indicates the second linker. CLE(B) represents a cleavable portion that can generate bioorthogonal functional groups on the antibody side by cleavage. A represents the affinity polypeptide, The average modification percentage r of the antibody by the affinity polypeptide is 65 to 135%. The following equation (IIb): 【Chemistry 2】 [During the ceremony, Ig represents the antibody, L ５ This indicates the fifth linker. L ６ This indicates the sixth linker, B represents a group containing a bioorthogonal functional group. CLE indicates the cleavable portion. A represents the affinity polypeptide, The average modification percentage r of the antibody by the affinity polypeptide is 65 to 135%. It includes a structural unit represented by ] The antibody or salt thereof that does not contain the affinity polypeptide is an antibody derivative or salt thereof containing a bioorthogonal functional group. The antibody derivative or salt thereof containing the bioorthogonal functional group, The following formula (IIIa): 【Transformation 3】 [During the ceremony, Ig, L １ , and r are the same as those in formula (IIa) above, B represents a group containing the bioorthogonal functional group. The following formula (IIIb): 【Chemistry 4】 [During the ceremony, Ig, L ５ , B, and r are the same as those in formula (IIb) above, T １ A method comprising a structural unit represented by , where indicates a monovalent group.

2. The affinity polypeptide-modified antibody is of the following formula (IIa-1): 【Transformation 5】 [During the ceremony, Ig represents the antibody, W １ , W ２ , and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ This indicates the third linker. L ４ This indicates the fourth linker, S represents a sulfur atom. A represents the affinity polypeptide, The average modification percentage r of the antibody by the affinity polypeptide is 65 to 135%. It includes a structural unit represented by ] The antibody derivative or salt containing the bioorthogonal functional group is given by the following formula (IIIa-1): 【Transformation 6】 [During the ceremony, Ig, W １ , L ３ , and r are the same as those in formula (IIa-1) above, SH indicates a thiol group. It contains a structural unit represented by ] or The affinity polypeptide-modified antibody is given by the following formula (IIb-1): 【Transformation 7】 [During the ceremony, Ig represents the antibody, W １ , W ２ , and W ３ Each of these independently represents either an oxygen atom or a sulfur atom. L ７ This indicates the seventh linker, L ８ This indicates the eighth linker, B represents a group containing the bioorthogonal functional group, V represents an oxygen atom or a sulfur atom. A represents the affinity polypeptide, The average modification percentage r of the antibody by the affinity polypeptide is 65 to 135%. It includes a structural unit represented by ] The antibody derivative or salt containing the bioorthogonal functional group is given by the following formula (IIIb-1): 【Transformation 8】 [During the ceremony, Ig, W １ , W ２ , L ７ , B, and r are the same as those in formula (IIb-1) above, T ２ The method according to claim 1, comprising a structural unit represented by ], where represents a monovalent group.

3. The method according to claim 1 or 2, wherein the antibody is an Fc region protein, and the affinity polypeptide and the bioorthogonal functional group are introduced only in one of the heavy chain constant regions of the Fc region protein.

4. The method according to claim 3, wherein the affinity polypeptide and the bioorthogonal functional group are introduced only into the one heavy chain constant region by modification of amino groups in the side chains of lysine residues located at one or more positions in the one heavy chain constant region.

5. Claim 4, wherein one or more positions in the constant region of one of the heavy chains are the 246th / 248th, 288th / 290th, or 317th positions of the human IgG heavy chain according to EU numbering. The method.

6. A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, The method described in claim 1 is used to produce an antibody derivative containing a bioorthogonal functional group or a salt thereof, and The process involves reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance. The conjugate or a salt thereof The following formula (IVa): 【Chemistry 9】 [During the ceremony, Ig represents the antibody, L １ This indicates the first linker. Z represents the functional substance, The average modification percentage r of the antibody by the functional substance is 65 to 135%. The following formula (IVb): 【Chemistry 10】 [During the ceremony, Ig represents the antibody, L ５ This indicates the fifth linker. Z represents the functional substance, T １ This indicates a monovalent group, The average modification percentage r of the antibody by the functional substance is 65 to 135%. A method comprising a structural unit represented by [ ].

7. A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, The method described in claim 2 is to produce an antibody derivative containing a bioorthogonal functional group or a salt thereof, and The process involves reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance. The conjugate or a salt thereof The following formula (IVa-1): 【Chemistry 11】 [During the ceremony, Ig represents the antibody, W １ This indicates an oxygen atom or a sulfur atom. L ３ This indicates the third linker. Z represents the functional substance, The average modification percentage r of the antibody by the functional substance is 65 to 135%. The following formula (IVb-1): 【Chemistry 12】 [During the ceremony, Ig represents the antibody, W １ and W ２ Each of these independently represents either an oxygen atom or a sulfur atom. L ７ This indicates the seventh linker, Z represents the functional substance, T ２ This indicates a monovalent group, The average modification percentage r of the antibody by the functional substance is 65 to 135%. A method comprising a structural unit represented by [ ].

8. The method according to claim 6 or 7, wherein the antibody is an Fc region protein, and the bioorthogonal functional group and the functional substance are introduced only in one heavy chain constant region of the Fc region protein.

9. The method according to claim 8, wherein the bioorthogonal functional group and the functional substance are introduced only into the one heavy chain constant region by modification of amino groups in the side chains of lysine residues located at one or more positions in the one heavy chain constant region.

10. The method according to claim 9, wherein one or more positions in the one heavy chain constant region are the 246th / 248th, 288th / 290th, or 317th positions of a human IgG heavy chain according to EU numbering.

11. The functional substance is a drug, a labeling substance, an affinity substance, a transport substance, or a stabilizer. or the method according to claim 6 or 7.

12. The method according to claim 11, wherein the affinity substance is a full-length antibody or a fragment thereof.

13. A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, The process involves reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance. An antibody derivative or salt containing a bioorthogonal functional group is an antibody derivative or salt containing a bioorthogonal functional group, wherein (a) the antibody and (b) the bioorthogonal functional group are included, and (c) the bioorthogonal functional group is introduced only in the constant region of one of the heavy chains of the antibody. The antibody derivative or salt thereof containing the bioorthogonal functional group, The following formula (IIIa): 【Chemistry 13】 [During the ceremony, Ig represents the antibody, L １ This indicates the first linker. B represents a group containing the bioorthogonal functional group, The average modification percentage r of the antibody by the bioorthogonal functional group is 65 to 135%. The following formula (IIIb): 【Chemistry 14】 [During the ceremony, Ig represents the antibody, L ５ This indicates the fifth linker. B represents a group containing the bioorthogonal functional group, T １ This indicates a monovalent group, The average modification percentage r of the antibody by the bioorthogonal functional group is 65 to 135%. It includes a structural unit represented by ] A conjugate or salt thereof containing an antibody and a functional substance is a conjugate or salt thereof containing an antibody and a functional substance, wherein (a) the antibody and (b) the functional substance are included, and (c) the functional substance is introduced only into the constant region of one of the heavy chains of the antibody, The conjugate or a salt thereof The following formula (IVa): 【Chemistry 15】 [During the ceremony, Ig, L １ , and r are the same as those in formula (IIIa) above, Z represents the functional substance. The following formula (IVb): 【Chemistry 16】 [During the ceremony, Ig, L ５ , T １ , and r are the same as those in formula (IIIb) above. A method comprising a structural unit represented by ], where Z represents the functional substance.

14. The antibody derivative or salt containing the bioorthogonal functional group is given by the following formula (IIIa-1): 【Chemistry 17】 [During the ceremony, Ig represents the antibody, W １ This indicates an oxygen atom or a sulfur atom. L ３ This indicates the third linker. SH indicates a thiol group. The average modification percentage r of the antibody by the bioorthogonal functional group is 65 to 135%. It includes a structural unit represented by ] The conjugate or a salt thereof The following formula (IVa-1): [Chemistry 18] [During the ceremony, Ig, W １ , L ３ , and r are the same as those in formula (IIIa-1) above, Z represents the functional substance, which includes a structural unit represented by ] or The antibody derivative or salt containing the bioorthogonal functional group is given by the following formula (IIIb-1): 【Chemistry 19】 [During the ceremony, Ig represents the antibody, W １ and W ２ Each of these independently represents either an oxygen atom or a sulfur atom. L ７ This indicates the seventh linker, B represents a group containing the bioorthogonal functional group, T ２ This indicates a monovalent group, The average modification percentage r of the antibody by the bioorthogonal functional group is 65 to 135%. It includes a structural unit represented by ] The conjugate or a salt thereof The following formula (IVb-1): 【Chemistry 20】 [During the ceremony, Ig, W １ , W ２ , L ７ , T ２ , and r are the same as those in formula (IIIb-1) above, The method according to claim 13, comprising a structural unit represented by ] where Z represents the functional substance.

15. The method according to claim 13 or 14, wherein the antibody is an Fc region protein, and the bioorthogonal functional group and the functional substance are introduced only in one heavy chain constant region of the Fc region protein.

16. The method according to claim 15, wherein the bioorthogonal functional group and the functional substance are introduced only into the one heavy chain constant region by modification of amino groups in the side chains of lysine residues located at one or more positions in the one heavy chain constant region.

17. The method according to claim 16, wherein one or more positions in the one heavy chain constant region are the 246th / 248th, 288th / 290th, or 317th positions of the human IgG heavy chain according to EU numbering.

18. The method according to claim 13 or 14, wherein the functional substance is a drug, a labeling substance, an affinity substance, a transport substance, or a stabilizer.

19. The method according to claim 18, wherein the affinity substance is a full-length antibody or a fragment thereof.

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