Affinity substance, compound, and antibody and their salts

Abstract

The present invention relates to a technology for facile chemical modification of only one heavy chain in an antibody structural unit (immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the present invention relates to a compound that contains (A) an affinity substance that contains first and second affinity portions that have affinity for the constant region in the heavy chain of an antibody and (B) a group reactive with antibody, or to a salt of said compound.

Description

Affinity substances, compounds, antibodies and salts thereof

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

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

[0003] Heterogeneity has been a problem for ADCs, including T-DM1, since their initial development. For example, because small molecule drugs are randomly reacted with approximately 70 to 80 lysine residues in an antibody, the drug-antibody ratio (DAR) and conjugation position are not consistent. It is known that such random conjugation methods typically result in a DAR ranging from 0 to 8, resulting in multiple antibody drugs with different numbers of drugs attached. In recent years, it has been reported that changing the number and position of drugs attached to an ADC can affect pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs require control of the number and position of conjugated drugs. It is believed that a consistent number and position would achieve the expected efficacy, resolve variations in conjugated drugs, and resolve issues related to so-called regulation, such as lot-to-lot differences.

[0004] Methods for site-selective modification of antibodies have been studied worldwide, but most of these methods involve genetic engineering or enzyme-based modification. While genetic engineering modification methods can control site and number selectivity, they have been criticized for their reduced expression efficiency (reducing the overall yield when preparing ADCs). Another problem is the long time required to establish an antibody expression system.

[0005] Recently, the C-CAP (Chemical Conjugation by Affinity Peptide) method has been developed, which enables site-selective modification of antibodies using chemical synthesis techniques (Patent Document 1). This method successfully achieves site-selective modification of antibodies by reacting an antibody with a peptide reagent in which an NHS-activated ester and a drug are linked to an affinity peptide. However, the ADCs produced by this method conjugate the antibody and drug via a linker containing a peptide moiety. The peptide moiety has potential immunogenicity and is easily hydrolyzed in blood. Therefore, the ADCs produced by this method have room for improvement in terms of including a peptide moiety in the linker.

[0006] As an improvement over the C-CAP method, techniques have been reported that use a chemical synthesis technique using a specific compound containing an affinity peptide to prepare antibodies that regioselectively carry functional substances (e.g., drugs) without containing a peptide moiety as a linker (Patent Documents 2 to 5). Patent Document 6 also discloses a technique for easily mass-producing affinity peptides by utilizing an affinity peptide containing glutamine-glutamic acid-threonine (QET) at the N-terminus in the preparation of compounds containing affinity peptides. Avoiding the use of linkers containing peptide moieties is desirable for clinical applications. These techniques propose several positions in an antibody that can be regioselectively modified with drugs, corresponding to various amino acid residues (e.g., lysine, tyrosine, serine, and threonine residues) in the CH2 and CH3 domains. However, it is not always easy to regioselectively modify an antibody with a functional substance and control the binding ratio between the antibody and the functional substance within a desired range. In particular, it is not easy to control the modification of only one of the heavy chains in the antibody building block (immunoglobulin unit containing two heavy chains).

[0007] Incidentally, Patent Document 6 discloses that (i) a peptide molecule comprising a first and a second physiologically active peptide moiety for a protein such as various receptors (e.g., fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR / c-Met), erythropoietin receptor, thrombopoietin receptor), and a peptide linker, can regulate the activity of the protein by non-covalently binding to the protein, and (ii) that a peptide linker of a predetermined length comprising an amino acid sequence consisting of amino acid residues of proline (P), alanine (A), and serine (S) can be used as the peptide linker.

[0008] International Publication No. 2016 / 186206 International Publication No. 2018 / 199337 International Publication No. 2019 / 240287 International Publication No. 2019 / 240288 International Publication No. 2020 / 090979 International Publication No. 2021 / 112249

[0009] An object of the present invention is to develop a technique that enables easy chemical modification of only one of the heavy chains in the constituent unit of an antibody (in other words, an immunoglobulin unit containing two heavy chains and, if necessary, two light chains).

[0010] A further object of the present invention is to develop a site-selectively modified antibody, in which only one of the heavy chains in the antibody building block can be easily chemically modified.

[0011] As a result of extensive research, the present inventors have found that it is possible to easily chemically modify only one of the heavy chains in the antibody structural unit by using (A) an affinity substance comprising first and second affinity moieties having affinity for the constant region in the heavy chain of the antibody, and (B) a compound or its salt comprising a group reactive to the antibody.

[0012] The compound of the present invention or a salt thereof can associate with the two heavy chains in an antibody building block via an affinity substance (A) comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and then specifically reacts with the side chain of a specific amino acid residue in one of the heavy chains in the antibody building block via the antibody-reactive group (R), thereby producing an affinity substance-modified antibody or a salt thereof in which only one of the two heavy chains in the antibody building block is modified (Figure 1). Without wishing to be bound by theory, the mechanism by which the compound of the present invention or a salt thereof modifies only one heavy chain in the antibody building block is as follows: The affinity substance (comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain) contained in the compound of the present invention or a salt thereof can stably associate with the constant regions of the two heavy chains in the antibody building block, and therefore the reactive group contained in the compound of the present invention or a salt thereof can modify only one heavy chain (Figure 1). In this case, the constant region of the other heavy chain (the constant region of the unmodified heavy chain) is associated with the affinity moiety, causing steric hindrance, so other molecules (the compound of the present invention or a salt thereof) cannot associate with the constant region of the other heavy chain via the affinity substance contained therein. The compound of the present invention or a salt thereof can associate with the constant regions of two heavy chains via the two affinity moieties contained in the affinity substance, and therefore can stably associate with the antibody building block, thereby highly inhibiting the association of other molecules with the antibody building block. Thus, the compound of the present invention or a salt thereof can highly inhibit modification of the constant region of the other heavy chain, and therefore can modify only the constant region of one heavy chain (Figure 1).

[0013] Patent Documents 1 to 5 disclose that an antibody can be regioselectively modified with a functional substance by using an affinity substance and a compound containing a group reactive to the antibody. However, they do not describe or suggest (1) the problem of developing a technology that can easily chemically modify only one of the heavy chains in the building blocks of an antibody, or (2) the use of a substance containing first and second affinity moieties that have affinity for the constant region in the heavy chain of an antibody as an affinity substance (in particular, the technical idea of ​​chemically modifying only one of the heavy chains in the building blocks of an antibody by using the affinity substance and a compound containing a group reactive to the antibody or a salt thereof).

[0014] Patent Document 6 discloses that a peptide molecule comprising the above-mentioned first and second physiologically active peptide moieties and a specific peptide linker for a protein can regulate the activity of the protein by non-covalently binding to the protein, but it neither describes nor suggests (1) the problem of developing a technology that can easily chemically modify only one of the heavy chains in the building blocks of an antibody, nor (2) the use of a substance comprising first and second affinity moieties that have affinity for the constant region of the heavy chain of an antibody as an affinity substance (in particular, the technical idea of ​​chemically modifying only one of the heavy chains in the building blocks of an antibody by using the affinity substance and a compound or its salt that comprises a reactive group for an antibody).

[0015] The present inventors have also succeeded in producing antibodies in which only one heavy chain of a constituent unit of the antibody (an immunoglobulin unit containing two heavy chains and, optionally, two light chains) has been chemically modified by using the compound of the present invention or a salt thereof. Such antibodies are characterized by comprising (a) an immunoglobulin unit containing two heavy chains and, optionally, two light chains, and (b) a modifying unit (e.g., the above-mentioned affinity substance, bioorthogonal functional group, or functional substance), and (c) the modifying unit has been introduced only into the constant region of one heavy chain of the above-mentioned immunoglobulin unit.

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

[10] Any of the compounds of [1] to [8], or a salt thereof, wherein the affinity polypeptide is represented by the following formula (A'): AP1-PL Ａ -AP2 (A') (wherein, AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and located on the N-terminal side of the affinity polypeptide, AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain and located on the C-terminal side of the affinity polypeptide, and PL Ａindicates a peptide linker.

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

[10] , wherein the affinity polypeptide (i) (i-1) contains only one amino acid residue having an amino group in its side chain, and (ii-2) is linked to a group reactive to an antibody via the amino group, or (ii) is linked to a group reactive to an antibody via the N-terminal amino group in the first affinity peptide.

[12] A compound or a salt thereof according to

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

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

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

[14] A compound or a salt thereof according to

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

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

[14] , or a salt thereof, wherein one of the first and second affinity peptides has affinity for the constant region of an antibody heavy chain and has one lysine residue, and the other of the first and second affinity peptides has affinity for the constant region of an antibody heavy chain and has no lysine residue.

[16] An affinity peptide having affinity for the constant region of an antibody heavy chain and having one lysine residue is selected from the group consisting of the following (1) to (4): (1) an affinity peptide comprising the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38) (Fc3K); (2) an affinity peptide comprising an amino acid sequence in which one or two amino acid residues other than lysine residues and cysteine ​​residues in the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38) are substituted with other amino acid residues other than lysine residues and cysteine ​​residues, and having affinity for the constant region of an antibody heavy chain; (3) an affinity peptide comprising the amino acid sequence of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) (Z34CK);and (4) an affinity peptide having affinity for the constant region of an antibody heavy chain (wherein the amino acid sequence of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) is substituted with another amino acid residue other than a lysine residue or a cysteine ​​residue, and having affinity for the constant region of an antibody heavy chain); (wherein the two cysteine ​​residues contained in the amino acid sequence may be cross-linked by a disulfide bond), and / or an affinity peptide having affinity for the constant region of an antibody heavy chain and having no lysine residues is any one of the following (5) to (10): (5) an affinity peptide comprising the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40) (Z34CM); (6) 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 FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40) are substituted with a lysine residue and another amino acid residue other than cysteine ​​residues, and having affinity for the constant region of the heavy chain of an antibody; (7) An affinity peptide comprising 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 lysine residues and other amino acid residues other than cysteine ​​residues, and having affinity for the constant region of 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 lysine residues and other amino acid residues other than cysteine ​​residues, and having affinity for the constant region of the heavy chain of an antibody;

[17] The compound of

[15] , or a salt thereof, wherein the compound is any one of the following formula (I): [wherein R represents the reactive group, L represents a linker, and A represents the affinity substance], or a salt thereof.

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

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

[19] The compound or salt thereof according to

[18] , wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side upon cleavage.

[20] The compound or salt thereof, wherein the compound is represented by the following formula (Ia): [wherein R represents the reactive group; L １ represents a first linker, L ２ represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, and A represents the affinity substance.

[21] The compound according to

[19] or a salt thereof, wherein the compound is represented by the following formula (Ia-1): [wherein X represents a leaving group; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ represents a third linker, and L ４ (a) R represents a fourth linker, S represents a sulfur atom, and A represents the affinity substance.

[22] The compound or a salt thereof according to

[21] , wherein the leaving group is selected from the following: (a) R Ａ -S (where 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 (where R Ａrepresents 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 (where R Ａ and R Ｂ each independently represent a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom; or (d) a halogen atom.

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

[17] , further comprising (iv) a bioorthogonal functional group between (ii) the reactive group and (iii) the cleavable moiety. [24 ... the following formula (Ib): [wherein R represents the reactive group; L ５ represents a fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, and A represents the affinity substance.

[25] The compound of

[23] or a salt thereof, wherein the compound is represented by the following formula (Ib-1): [wherein X represents a leaving group; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ represents the seventh linker, L ８ represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, and A represents the affinity substance.

[26] A compound or a salt thereof 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 thiol residue, a furan residue, or a halocarbonyl residue.

[0017]

[27] A reagent for antibody derivatization, comprising any one of the compounds according to [1] to

[26] or a salt thereof.

[0018]

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

[29] An affinity substance-modified antibody or a salt thereof according to

[28] , comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) the affinity substance, and (c) the affinity substance is introduced only into the constant region of one of the heavy chains in the immunoglobulin unit.

[30] An affinity substance-modified antibody or a salt thereof according to

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

[31] An affinity substance-modified antibody or a salt thereof according to

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

[32] The affinity substance-modified antibody is represented by the following formula (II): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and optionally two light chains, L represents a linker, A represents the affinity substance, and the average percentage modification r of the immunoglobulin unit with the affinity substance is 65 to 135%.]

[33] The affinity substance-modified antibody or salt thereof of any of

[28] to

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

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

[33] , wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side upon cleavage.

[35] The affinity substance-modified antibody or salt thereof of

[35] , wherein the affinity substance-modified antibody is a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side upon cleavage. [wherein Ig represents the immunoglobulin unit; L １ represents a first linker, L ２represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, A represents the affinity substance, and the average modification percentage r of the immunoglobulin unit with the affinity substance is 65 to 135%.

[36] The affinity substance-modified antibody or a salt thereof according to

[34] , wherein the affinity substance-modified antibody comprises a structural unit represented by the following formula (IIa-1): [wherein Ig represents the immunoglobulin unit; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ represents a third linker, and L ４

[37] The affinity substance-modified antibody or a salt thereof according to

[33] , wherein (ii') the antibody or its salt further comprises (iv') a bioorthogonal functional group between the antibody and the cleavable moiety.

[38] The affinity substance-modified antibody or a salt thereof according to

[34] , wherein the affinity substance-modified antibody comprises a structural unit represented by the following formula (IIb): [wherein Ig represents the immunoglobulin unit; L ５ represents a fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, A represents the affinity substance, and the average modification percentage r of the immunoglobulin units with the affinity substance is 65 to 135%.

[39] The affinity substance-modified antibody or a salt thereof according to

[37] , wherein the affinity substance-modified antibody comprises a structural unit represented by the following formula (IIb-1): [wherein Ig represents the immunoglobulin unit; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ represents the seventh linker, L ８

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

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

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

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

[43] The affinity substance-modified antibody or salt thereof of

[42] , wherein the additional affinity substance is introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains.

[43] The affinity substance-modified antibody or a salt thereof according to

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

[0019]

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

[45] The method of

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

[46] The method of

[45] , wherein the cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage.

[47] The method of

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

[48] ​​The method according to

[44] , wherein the affinity substance-modified antibody or a salt thereof is any one of the affinity substance-modified antibodies or salts thereof according to

[29] to

[43] .

[0020]

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

[50] The antibody derivative or salt thereof according to

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

[51] The antibody derivative or salt thereof according to

[49] or

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

[52] An antibody derivative or salt thereof comprising a bioorthogonal functional group represented by the following formula (IIIa): [wherein Ig represents the immunoglobulin unit; L １represents a first linker, B represents a group containing a bioorthogonal functional group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.

[53] The antibody derivative or salt thereof according to any of

[49] to

[51] , comprising a structural unit represented by the following formula (IIIa-1): [wherein Ig represents the immunoglobulin unit; W １ represents an oxygen atom or a sulfur atom, L ３ represents a third linker, SH represents a thiol group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.

[54] The antibody derivative or salt thereof according to any of

[49] to

[51] , comprising a structural unit represented by the following formula (IIIb): [wherein Ig represents the immunoglobulin unit; L ５ represents a fifth linker, B represents a group containing a bioorthogonal functional group, and T １ represents a monovalent group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.

[55] The antibody derivative or salt thereof according to any of

[49] to

[51] , comprising a structural unit represented by the following formula (IIIb-1): [wherein Ig represents the immunoglobulin unit; W １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, B represents a group containing a bioorthogonal functional group, and T ２represents a monovalent group, and the modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%.

[56] The antibody derivative or salt thereof of any of

[49] to

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

[57] The antibody derivative or salt thereof of

[56] , wherein the additional modifying moiety is an additional modifying moiety comprising a bioorthogonal functional group, and the additional affinity substance comprising the bioorthogonal functional group is contained in the constant region of the heavy chain of the antibody.

[58] The antibody derivative or salt thereof of

[57] , wherein the additional modifying moiety comprising a bioorthogonal functional group is introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains.

[59] The antibody derivative or salt thereof according to

[58] , wherein one or more positions in the constant regions 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, and (c) the functional substance is introduced only into the constant region of one of the heavy chains in the immunoglobulin unit.

[61] The conjugate of

[60] or a salt thereof, wherein the functional substance is introduced only into the constant region of one of the heavy chains via modification of an amino group in the side chain of a lysine residue present at one or more positions in the constant region of the one heavy chain.

[62] The conjugate of

[60] or

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

[63] The conjugate or a salt thereof, wherein the conjugate or a salt thereof is represented by the following formula (IVa): [wherein Ig represents the immunoglobulin unit; L １represents a first linker, Z represents a functional substance, and the average modification percentage r of the immunoglobulin units with the functional substance is 65 to 135%.

[64] The conjugate or salt thereof according to any of

[60] to

[62] , comprising a structural unit represented by the following formula (IVa-1): [wherein Ig represents the immunoglobulin unit; W １ represents an oxygen atom or a sulfur atom, L ３ represents a third linker, Z represents a functional substance, and the average modification percentage r of the immunoglobulin units with the functional substance is 65 to 135%.

[65] The conjugate or salt thereof according to any of

[60] to

[62] , comprising a structural unit represented by the following formula (IVb): [wherein Ig represents the immunoglobulin unit; L ５ represents a fifth linker, Z represents a functional substance, and T １ represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%.

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

[60] to

[62] , comprising a structural unit represented by the following formula (IVb-1): [wherein Ig represents the immunoglobulin unit; W １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, Z represents a functional substance, and T ２represents a monovalent group, and the percentage modification r of the immunoglobulin unit with the functional substance is 65 to 135%.

[67] The conjugate or salt thereof of any of

[60] to

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

[68] The conjugate or salt thereof of

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

[69] The conjugate or salt thereof of any of

[60] to

[68] , wherein the conjugate further comprises an additional modifying moiety.

[70] The conjugate or salt thereof of

[69] , wherein the additional modifying moiety is an additional modifying moiety comprising a functional substance, and the additional modifying moiety comprising a functional substance is contained in the constant region of the heavy chain of the antibody.

[71] The conjugate or a salt thereof according to

[70] , wherein the additional modifying moiety comprising the functional substance is introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains.

[72] The conjugate or a salt thereof according to

[71] , wherein the one or more positions in the constant regions 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 affinity substance-free antibody or its salt, comprising: (A) an affinity substance comprising first and second affinity moieties having affinity for a constant region in the heavy chain of the antibody; and (B) an antibody; and (C) an affinity substance-modified antibody or its salt further comprising a cleavable moiety between (A) the affinity substance and (B) the antibody, by cleaving the antibody or its salt with the cleavable moiety to produce an affinity substance-free antibody or its salt.

[74] The method of

[73] , wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody upon cleavage, and the affinity substance-free antibody or its salt is an antibody derivative or its salt comprising a bioorthogonal functional group.

[75] The method of

[74] , wherein the antibody derivative or its salt comprising a bioorthogonal functional group is any of the antibody derivatives or salts described above.

[76] The method of

[73] , wherein the affinity substance-free antibody or its salt further comprises a bioorthogonal functional group between the antibody and the cleavable moiety, and the affinity substance-free antibody or its salt is an antibody derivative or its salt comprising a bioorthogonal functional group.

[77] The method of

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

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

[74] ; and (2) reacting the antibody derivative or salt thereof comprising a bioorthogonal functional group with a functional substance to produce a conjugate or salt thereof comprising an antibody and a functional substance.

[79] The method of

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

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

[76] ; and (2) reacting the antibody derivative or salt thereof comprising a bioorthogonal functional group with a functional substance to produce a conjugate or salt thereof comprising an antibody and a functional substance.

[81] The method according to

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

[0023]

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

[83] The method of

[80] , wherein the antibody derivative or salt thereof comprising a bioorthogonal functional group is any one of the antibody derivatives or salts thereof described above.

[84] The method of

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

[0024]

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

[86] The affinity substance is a compound represented by the following formula (A): AP1-L Ａ -AP2 (A) (wherein, AP1 represents a first affinity peptide having affinity for the constant region of the heavy chain of an antibody, AP2 represents a second affinity peptide having affinity for the constant region of the heavy chain of an antibody, and L Ａindicates a linker.].

[87] The affinity substance of

[85] or

[86] , or a salt thereof, wherein the affinity substance contains only one specific reactive group.

[88] The affinity substance of any of

[85] to

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

[89] The affinity polypeptide is represented by the following formula (A'): AP1-PL Ａ -AP2 (A') (wherein, AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and located on the N-terminal side of the affinity polypeptide, AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain and located on the C-terminal side of the affinity polypeptide, and PL Ａ indicates a peptide linker.

[90] An affinity substance or a salt thereof according to any one of

[85] to

[89] , wherein the affinity polypeptide (i) contains only one amino acid residue having an amino group in the side chain, or (ii) contains an unprotected N-terminal amino group.

[91] An affinity substance or a salt thereof according to

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

[92] An affinity substance or a salt thereof according to any one of

[85] to

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

[93] An affinity substance or a salt thereof according to any one of

[89] to

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

[94] The affinity substance or salt thereof according to any one of

[85] to

[93] , wherein one of the first and second affinity peptides is an affinity peptide that has affinity for the constant region of an antibody heavy chain and has one lysine residue, and the other of the first and second affinity peptides is an affinity peptide that has affinity for the constant region of an antibody heavy chain and has no lysine residue.

[0025]

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

[0026]

[96] An expression vector comprising the polynucleotide of

[95] and a promoter operably linked thereto.

[0027]

[97] A host cell comprising an expression unit comprising the polynucleotide of

[95] and a promoter operably linked thereto.

[0028]

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

[99] An affinity substance-modified antibody or a salt thereof according to

[98] , comprising: (a) an immunoglobulin unit comprising two heavy chains consisting of first and second heavy chains and optionally two light chains, and (b) the first and second modifying portions, (c) the first modifying portion has been introduced into the constant region of the first heavy chain in the immunoglobulin unit, and (d) the second modifying portion has been introduced into the constant region of the second heavy chain in the immunoglobulin unit.

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

[98] or

[99] , wherein the first modifying moiety 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 present at one or more positions in the constant region of the first heavy chain, and the second modifying moiety 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 present at one or more positions in the constant region of the second heavy chain.

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

[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 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] The affinity substance-modified antibody or salt thereof, wherein the affinity substance-modified antibody comprising first and second modifying moieties is a compound represented by the following formula (V): where Ig represents an immunoglobulin unit comprising two heavy chains, consisting of a first and a second heavy chain, and optionally two light chains; L Ｌ and L Ｒ each independently represents a linker; Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ、 and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ are 65 to 135%, respectively.

[103] The affinity substance-modified antibody or salt thereof of any of

[98] to

[102] , which comprises a structural unit represented by the following formula (Va):

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

[103] , wherein the first and second cleavable moieties are cleavable moieties capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage.

[105] The affinity substance-modified antibody or salt thereof of any of

[98] to

[101] , which comprises a structural unit represented by the following formula (Va): [wherein Ig represents the immunoglobulin unit; L Ｌ１ and L Ｒ１ each independently represents a first linker; L Ｌ２ and L Ｒ２ each independently represents a second linker, CLE(B) Ｌ and CLE(B) Ｒ each independently represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit upon cleavage, and A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌand the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the affinity substance-modified antibody or a salt thereof according to any one of

[98] to

[104] , which comprises a structural unit represented by the following formula (Va-1): [wherein 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 Ｌ３ and L Ｒ３ each independently represents a third linker; L Ｌ４ and L Ｒ４ each independently represents a fourth linker; S represents a sulfur atom; A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ are 65 to 135%, respectively.

[107] The affinity substance-modified antibody or salt thereof of any of

[98] to

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

[108] The affinity substance-modified antibody or salt thereof of any of

[98] to

[104] , wherein the affinity substance-modified antibody comprising first and second modifying moieties is represented by the following formula (Vb): [wherein Ig represents the immunoglobulin unit; L Ｌ５ and L Ｒ５ each independently represents a fifth linker; L Ｌ６and L Ｒ６ each independently represents a sixth linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group, and CLE Ｌ and CLE Ｒ each independently represents a cleavable moiety; A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the affinity substance-modified antibody or a salt thereof according to any one of

[98] to

[107] , which comprises a structural unit represented by the following formula (Vb-1): [wherein 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 Ｌ７ and L Ｒ７ each independently represents a seventh linker; L Ｌ８ and L Ｒ８ each independently represents an eighth linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; V Ｌ and V Ｒ each independently represents an oxygen atom or a sulfur atom, Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌand the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ are 65 to 135%.

[110] The affinity substance-modified antibody or salt thereof of any of

[98] to

[109] , comprising a structural unit represented by the following formula (Vc):

[111] The affinity substance-modified antibody or salt thereof of any of

[98] to

[108] , comprising a structural unit represented by the following formula (Vc):

[112] The affinity substance-modified antibody or salt thereof of any of

[98] to

[109] , further comprising a (iii') first cleavable moiety between (i') the first affinity substance and (ii') the immunoglobulin unit, and further comprising a (iv'') first bioorthogonal functional group between (ii'') the immunoglobulin unit and (iii'') the second cleavable moiety, wherein the first cleavable moiety is a cleavable moiety capable of generating a second bioorthogonal functional group on the immunoglobulin unit side upon cleavage.

[113] The affinity substance-modified antibody or salt thereof comprising a first and second modifying moiety, comprising a structural unit represented by the following formula (Vc): [wherein Ig represents the immunoglobulin unit; L Ｒ１ represents a first linker, L Ｒ２ represents a second linker, and L Ｌ５ represents a fifth linker, L Ｌ６ represents the sixth linker, B Ｌ represents a group containing a first bioorthogonal functional group, and CLE Ｌ represents the first cleavable moiety, CLE(B) Ｒ represents a second cleavable moiety that can be cleaved to generate a second bioorthogonal functional group on the immunoglobulin unit; A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the affinity substance-modified antibody or a salt thereof according to any one of

[98] to

[110] , which comprises a structural unit represented by the following formula (Vc-1): [wherein 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 each independently represents a third linker; L Ｒ４ each independently represents a fourth linker; L Ｌ７ each independently represents a seventh linker; L Ｌ８ each independently represents an eighth linker; B Ｌ represents a group containing a first bioorthogonal functional group, and V Ｌ represents an oxygen atom or a sulfur atom, Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ are 65 to 135%, respectively.

[113] The affinity substance-modified antibody or salt thereof of any of

[98] to

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

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

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

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

[114] , wherein the additional affinity substance has been introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains.

[116] The affinity substance-modified antibody or a salt thereof according to

[115] , wherein one or more positions in the constant regions 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] A method for producing an affinity substance-modified antibody or a salt thereof comprising first and second modifying moieties, the method comprising: (1) reacting (A) a first 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 first reactive group for the antibody, with an antibody comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains, to produce an affinity substance-modified antibody or a salt thereof comprising a first modifying moiety comprising the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit; and (2) reacting an affinity substance-modified antibody or a salt thereof comprising the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with a second affinity substance comprising third and fourth affinity moieties having affinity for the constant region of the antibody heavy chain, and (B) a compound or salt thereof comprising a second reactive group for the antibody, to produce an affinity substance-modified antibody or a salt thereof comprising the first and second modifying moieties in the constant region of the heavy chain of the immunoglobulin unit.

[118] The method of

[117] , wherein the affinity substance-modified antibody or its salt comprising the first and second modifying moieties is any of the affinity substance-modified antibodies or their salts described above.

[0030]

[119] An antibody derivative or a salt thereof comprising: (a) an immunoglobulin unit comprising two heavy chains, a first and a second heavy chain, and optionally two light chains; and (b) a first modifying moiety comprising a first bioorthogonal functional group, and a second modifying moiety comprising a second bioorthogonal functional group; (c) the first modifying moiety has been introduced into the constant region of the first heavy chain; (d) the second modifying moiety has been introduced into the constant region of the second heavy chain; and (e) a first and second modifying moiety, wherein the first and second modifying moieties are different from each other.

[120] The antibody derivative or salt thereof according to

[119] , wherein the first modifying moiety 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 present at one or more positions in the constant region of the first heavy chain, and the second modifying moiety 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 present at one or more positions in the constant region of the second heavy chain.

[121] The antibody derivative or salt thereof according to

[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 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] The antibody derivative or salt thereof comprising first and second modifying moieties is an antibody derivative or salt thereof represented by the following formula (VIa): [wherein Ig represents the immunoglobulin unit; L Ｌ１ and L Ｒ１ each independently represents a first linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group, and the average percentage modification of said immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ

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

[119] to

[121] , which comprises a structural unit represented by the following formula (VIa-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｒ１ each independently represents an oxygen atom or a sulfur atom, L Ｌ３ and L Ｒ３ each independently represents a third linker, SH represents a thiol group that is a bioorthogonal functional group, and the average modification percentage r of the immunoglobulin unit with the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the respective values ​​of the modified antibody derivative or salt thereof according to any one of

[119] to

[122] , which comprises a structural unit represented by the following formula (VIb): [wherein Ig represents the immunoglobulin unit; L Ｌ５ and L Ｒ５ each independently represents a fifth linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; T Ｌ１ and T Ｒ１ each independently represents a monovalent group, and the average modification percentage r of the immunoglobulin units by the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the respective values ​​of the modified antibody derivative or salt thereof according to any one of

[119] to

[123] , which comprises a structural unit represented by the following formula (VIb-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｌ２ , and W Ｒ１and W Ｒ２ each independently represents an oxygen atom or a sulfur atom, L Ｌ７ and L Ｒ７ each independently represents a seventh linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; T Ｌ２ and T Ｒ２ each independently represents a monovalent group, and the average modification percentage r of the immunoglobulin units by the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the respective values ​​of the modified antibody derivative or salt thereof according to any one of

[119] to

[124] , which comprises a structural unit represented by the following formula (VIc): [wherein Ig represents the immunoglobulin unit; L Ｒ１ represents a first linker, L Ｌ５ represents the fifth linker, B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; T Ｌ１ represents a monovalent group, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the respective values ​​of the modified antibody derivative or salt thereof according to any one of

[119] to

[125] , which comprises a structural unit represented by the following formula (VIc-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｌ２ , and W Ｒ１ each independently represents an oxygen atom or a sulfur atom, L Ｒ３ represents a third linker, L Ｌ７represents the seventh linker, B Ｌ represents a first group containing a first bioorthogonal functional group; SH represents a thiol group, which is a second bioorthogonal functional group; T Ｌ２ represents a monovalent group, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ are 65 to 135%, respectively.

[128] The antibody derivative or salt thereof of any of

[119] to

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

[129] The antibody derivative or salt thereof of

[128] , wherein the additional modifying moiety is an additional modifying moiety comprising a bioorthogonal functional group, and the additional modifying moiety comprising the bioorthogonal functional group is contained in the constant region of the heavy chain of the antibody.

[130] The antibody derivative or salt thereof of

[129] , wherein the additional modifying moiety comprising the bioorthogonal functional group is introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains.

[131] The antibody derivative or salt thereof according to

[130] , wherein one or more positions in the constant regions 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 conjugate or salt thereof of an antibody and a first and second modifying moiety, comprising: (a) an immunoglobulin unit comprising two heavy chains, each consisting of a first and a second heavy chain, and optionally two light chains; and (b) a first modifying moiety comprising a first functional substance and a second modifying moiety comprising a second functional substance, (c) the first modifying moiety has been introduced into the constant region of the first heavy chain, (d) the second modifying moiety has been introduced into the constant region of the second heavy chain, and (e) the first and second modifying moieties are different from each other.

[133] The conjugate or salt thereof of

[132] , wherein the first modifying moiety has been introduced into the constant region of the first heavy chain via modification of an amino group in the side chain of a lysine residue present at one or more positions in the constant region of the second heavy chain, and the second modifying moiety has been introduced into the constant region of the second heavy chain via modification of an amino group in the side chain of a lysine residue present at one or more positions in the constant region of the second heavy chain.

[134] The conjugate or salt thereof according to

[132] or

[133] , 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 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 salt thereof is a conjugate represented by the following formula (VIIa): [wherein Ig represents the immunoglobulin unit; L Ｌ１ and L Ｒ１ each independently represents a first linker; Z Ｌ represents a first functional substance, and Z Ｒ represents a second functional substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and each of them is 65 to 135%.

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

[132] to

[134] , which contains a structural unit represented by the following formula (VIIa-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｒ１ each independently represents an oxygen atom or a sulfur atom, L Ｌ３ and L Ｒ３ each independently represents a third linker; Z Ｌ represents a first functional substance, and Z Ｒ represents a second functional substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the ratios of the conjugate or salt thereof to the structural unit represented by the following formula (VIIb): [wherein Ig represents the immunoglobulin unit; L Ｌ５ and L Ｒ５ each independently represents a fifth linker; Z Ｌ represents a first functional substance, and Z Ｒ represents a second functional substance, and T Ｌ１ and T Ｒ１ each independently represents a monovalent group, and the average modification percentage r of the immunoglobulin units by the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the ratios of the conjugate or salt thereof to the structural unit represented by the following formula (VIIb-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｌ２ , and W Ｒ１ and W Ｒ２ each independently represents an oxygen atom or a sulfur atom, L Ｌ７ and L Ｒ７ each independently represents a seventh linker; Z Ｌ represents a first functional substance, and ZＲ represents a second functional substance, and T Ｌ２ and T Ｒ２ each independently represents a monovalent group, and the average modification percentage r of the immunoglobulin units by the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the ratios of the conjugate or salt thereof to the structural unit represented by the following formula (VIIc): [wherein Ig represents the immunoglobulin unit; L Ｒ１ represents a first linker, L Ｌ５ represents a fifth linker, Z Ｌ represents a first functional substance, and Z Ｒ represents a second functional substance, and T Ｌ１ represents a monovalent group, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the ratios of the conjugate or salt thereof to the structural unit represented by the following formula (VIIc-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｌ２ , and W Ｒ１ each independently represents an oxygen atom or a sulfur atom, L Ｒ３ represents a third linker, L Ｌ７ represents the seventh linker, Z Ｌ represents a first functional substance, and Z Ｒ represents a second functional substance, and T Ｌ２ represents a monovalent group, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒare 65 to 135%, respectively.

[141] The conjugates or salts thereof according to

[132] to

[139] , comprising a structural unit represented by the following formula:

[142] .

[143] The conjugates or salts thereof according to

[132] to

[142] , wherein 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 a full-length antibody or a fragment thereof.

[144] The conjugates or salts thereof according to

[143] , wherein the additional modifying moiety is an additional modifying moiety comprising a functional substance, and wherein the additional modifying moiety comprising a functional substance is contained in the constant region of the heavy chain of the antibody.

[145] The conjugate or a salt thereof according to

[144] , wherein the additional modifying moiety comprising the functional substance is introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains.

[146] The conjugate or a salt thereof according to

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

[0032] According to the present invention, only one of the heavy chains in an antibody structural 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 antibody structural unit.

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

[0034] 1. Definitions of General Terms In this specification, terms and expressions used to describe a particular invention or item may also be used to describe other inventions or items. Therefore, the definitions, examples, and preferred examples of terms and expressions used to describe a particular invention or item may also be the same for other inventions or items described using such terms and expressions.

[0035] In the present invention, the term "antibody" is as follows. Furthermore, the term "immunoglobulin unit" corresponds to a bivalent monomer unit that is a constituent unit of such an antibody, and is an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains. Therefore, the definitions, examples, and preferred examples of terms and expressions for the immunoglobulin unit, such as its origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of amino acid residues (e.g., lysine residues), and regioselectivity, are the same as those for antibodies described below, and are used interchangeably with the term "antibody."

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

[0037] The antibody may be a polyclonal or monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent 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 with specific glycosylation (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide-reduced antibodies. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, full-length antibodies or antibody fragments containing the variable region and CH1 and CH2 domains can be used as monoclonal antibodies, with full-length antibodies being preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0038] Any antigen can be used as the antigen for the antibody. Examples of such antigens include proteins (including oligopeptides and polypeptides, and may also be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low-molecular-weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

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

[0040] (1) Cancer area: 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, mesotheli n, CD138, c-Met, Ang2, VEGF- A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, maccrophase inhibitory factor, CD74, Notch1, Notch 2. Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM- CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEG F, RSPO, LIV-1, SLITRK6, Nect in-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissu 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, C D166, ROR1, GITR, BCMA, TBA, LAG- 3. EphA2, TIM-3, CD-200, EGFRvIII , CD16A, CD32B, PIGF, Axl, MICA / B , Thomsen-Friedenreich, CD39, CD 37、CD73、CLEC12A、Lgr3、トランスフェリンReceptor, TGFβ, IL-17, 5T4, RTK, Immune Supplier Protein, NaPi2b, ルイス blood type B antigen, A34, Lysil-Oxidase , DLK-1, TROP-2, α9インテグリン, TAG-72 (CA72-4), CD70 ,

[0041] (2) Autoimmune diseases / 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 diseases: Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0044] (5) Genetic 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 / orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0047] (8) Blood diseases 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, Edoglin, 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, and ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, and daratumumab). , ikesekizumab (IgG4), reslizumab (IgG4), atezolizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (if no IgG subtype is mentioned, IgG1 is implied).

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

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

[0052] Preferably, the specific amino acid residues in the heavy chain constant region to be regioselectively modified can be lysine residues (e.g., lysine residues at positions 246 / 248 or 288 / 290). As used herein, "regioselectivity" or "regioselectivity" refers to the fact that a specific structural unit capable of binding to a specific amino acid residue in an antibody is preferentially distributed in a specific region of the antibody, even though the specific amino acid residue is not preferentially distributed in a specific region of the antibody. Therefore, expressions related to regioselectivity, such as "regioselectively possessed," "regioselective binding," and "regioselective binding," mean that the retention or binding rate of a specific structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues that are homologous to the specific amino acid residues in the target region. Such regioselectivity may be 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 even 100%. According to the present invention, specific lysine residues in the heavy chain of an antibody can be regioselectively modified without using a peptide-containing linker. Peptide moieties have potential immunogenicity and are easily hydrolyzed in blood. Therefore, avoiding the use of linkers containing peptide moieties is desirable in clinical applications.

[0053] In the present invention, as long as a specific amino acid residue (e.g., a lysine residue at a specific position) in the heavy chain constant region is regioselectively modified, specific amino acid residues at other positions may also be regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in WO 2018 / 199337, WO 2019 / 240288, WO 2019 / 240287, and WO 2020 / 090979. Such specific amino acid residues may be amino acid residues (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine ​​residues) having a side chain that is easily modified (e.g., amino group, carboxy group, amide group, hydroxy group, thiol group), but are preferably lysine residues having a side chain containing an amino group, tyrosine residues, serine residues, and threonine residues having a side chain containing a hydroxy group, or cysteine ​​residues having a side chain containing a thiol group, and more preferably lysine residues (i.e., of the lysine residues at positions 246 / 248, 288 / 290, and 317, two lysine residues may be regioselectively doubly modified, or three lysine residues may be regioselectively triple modified).

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

[0055] (Monovalent Group) Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

[0056] The monovalent group may be substituted with one or more (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) substituents described below.

[0057] (Monovalent Hydrocarbon Group and Related Terms) Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

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

[0059] The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms. When the alkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0060] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. When the alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0061] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. When the alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0062] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0063] The monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not have to be composed only of alicyclic hydrocarbons, and may contain a chain structure as part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0064] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. When the cycloalkyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0065] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 or 6 carbon atoms. When the cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0066] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 or 6 carbon atoms. When the cycloalkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0067] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0068] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed of only aromatic rings, and it may contain a chain structure or an alicyclic hydrocarbon as part of the ring, and the aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, an aryl group having 6 to 12 carbon atoms is preferred, an aryl group having 6 to 10 carbon atoms is more preferred, and an aryl group having 6 carbon atoms is even more preferred. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms mentioned above. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0069] The monovalent aromatic hydrocarbon group is preferably phenyl.

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

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

[0072] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0073] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

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

[0075] (Divalent Group) The divalent group is a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, —C(═O)—, —C(═S)—, —NR １ -, -C(=O)-NR １ -, -NR １ -C(=O)-, -C(=S)-NR １ -, -NR １ -C(=S)-, -O-, -S-, -(OR ２ ) ｎ -, and -(S-R ２ ) ｍ -, or a group having a main chain structure containing 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). １ represents a hydrogen atom or a substituent to be described later. ２represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. n and m are each an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, and particularly preferably an integer of 1 to 3.

[0076] The divalent linear hydrocarbon group is a linear alkylene, linear alkenylene, or linear alkynylene. The linear alkylene is a linear alkylene having 1 to 6 carbon atoms, preferably a linear alkylene having 1 to 4 carbon atoms. Examples of linear alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of the straight-chain alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. As the divalent straight-chain hydrocarbon group, a straight-chain alkylene is preferred.

[0077] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene.

[0078] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocycle preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As 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, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, 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)—, —NR １ -, -C(=O)-NR １ -, -NR １ -C(=O)-, -O-, and -(O-R ２ ) ｎ or a divalent group having a main chain structure containing one group selected from the group consisting of alkylene, arylene, —C(═O)—, —NR １ -, -C(=O)-NR １ -, -NR １ -C(=O)-, -O-, and -(O-R ２ ) ｎ - is a divalent group having a main chain structure containing two or more groups selected from the group consisting of １ is a hydrogen atom or alkyl, R ２ is alkylene or arylene, and n may be an integer of 1 to 5 (i.e., 1, 2, 3, 4, or 5). Alkylene, arylene, and alkyl are as defined above.

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

[0081] (Substituents) Examples of the substituents include: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) an aralkyl; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ 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 are the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, and a carboxyl group.

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

[0083] Aralkyl refers to arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0084] Preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) an aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 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 are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (vii) the same groups as those enumerated in (vii) above.

[0085] More preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 12 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 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 are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0086] Even more preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 6 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 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 ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0087] Particularly preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 4 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 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 are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0088] (Bio-orthogonal functional group) Bio-orthogonal 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. Bio-orthogonal functional groups are well known in the art (e.g., Sharpless K.B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

[0089] In the present invention, a bioorthogonal functional group for a protein is used as the bioorthogonal functional group. This is because the antibody to be derivatized with the reagent of the present invention is a protein. The bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 naturally occurring amino acid residues that make up proteins, or that reacts slowly with the side chains, but reacts with the desired functional group. The 20 naturally occurring amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Among these 20 naturally occurring amino acids, glycine has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine have hydrocarbon side chains (i.e., do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms). These amino acids are inert to normal reactions. Therefore, the bioorthogonal functional group for proteins is a group that does not react or reacts slowly with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, in addition to the side chains of these amino acids that have side chains that are inert to normal reactions, but reacts with the desired functional group.

[0090] Examples of such bioorthogonal functional groups include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, it is sufficient that they have a vinylene (ethenylene) moiety, which is the smallest unit having a double bond between carbon atoms; the same applies below), alkyne residues (in other words, it is sufficient that they have an ethynylene moiety, which is the smallest unit having a triple bond between carbon atoms; the same applies below), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position; the same applies below), isonitrile residues, sydnone residues, and selenium residues.

[0091] The bioorthogonal functional group may be protected or unprotected. A bioorthogonal functional group refers to an unprotected bioorthogonal functional group or a 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 cleavage of the protecting group. Cleavage of the protecting group can be carried out by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli selected from the group consisting of light, or (c) leaving the linker when using a cleavable linker containing a self-cleaving cleavable moiety. Such protecting groups and their cleavage conditions are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, J.M., Journal of American Chemical Society. Society. 132, 17928 (2010); Thompson, D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004)). Reaction conditions under mild conditions (e.g., reaction temperature, reaction time, reaction solution) are as described below.

[0092] Protected bioorthogonal functional groups include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol 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., reactivity 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 the substituents described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues. Combinations of two bioorthogonal functional groups that can react with each other include, for example, 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 (substitution reaction in which the halogen is replaced by thiol), and combinations of thiol residues and other thiol residues (formation of a disulfide bond).

[0095] (Functional Substance) The functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and examples thereof include drugs, labeling substances, affinity substances, transport substances, and stabilizers, but drugs, labeling substances, affinity substances, or transport substances are preferred. 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 a drug for any disease, such as 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 (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infection, viral infection), genetic / rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone / orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0097] More specifically, the drug may be an anticancer drug. Examples of anticancer drugs include chemotherapeutic agents, toxins, radioisotopes, and substances containing the same. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving 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., ricin). Examples of radioisotopes include radioactive isotopes of hydrogen atoms (e.g., ３ H), radioactive isotopes of carbon atoms (e.g., １４ C), radioactive isotopes of phosphorus atoms (e.g., ３２ P), radioactive isotopes of sulfur atoms (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 iodine atoms (e.g., １２３ I, １２５ I, １２９ I, １３１ I), radioactive isotopes of samarium (e.g., １５３Sm), radioactive isotopes of rhenium (e.g., １８６ Re), radioactive isotopes of astatine (e.g., ２１１ At), radioactive isotopes of bismuth (e.g., ２１２ More specifically, examples of the drug include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0098] A labeling substance is a substance that enables detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2′-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), and substances containing the same.

[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. The affinity substance is preferably an affinity protein or affinity peptide, and more preferably an antibody. The species of animals from which antibodies used as functional substances are derived are the same as those described above.

[0100] The type of antibody used as the 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 tetravalent 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 with specific glycosylation (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide-reduced antibodies. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Examples of antibodies used as the functional substance include full-length antibodies and fragments thereof (fragment antibodies). The antibody fragment may be any fragment that maintains binding to a desired antigen, and may be, for example, Fab, Fab', F(ab') ２ , scFv, and VHH antibodies.

[0101] The antigenicity of the antibody used as the 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, preferably different. Furthermore, the origin of the antibody used as the functional substance may be the same as or different from the origin of the immunoglobulin unit, preferably different. Therefore, the antibody used as the functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom. The antibody used as the functional substance may also be an IgG1, IgG2, IgG3, or IgG4 antibody mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom.

[0102] The transporter is a substance capable of transporting a compound. Preferred transporters are substances capable of encapsulating a compound in a protein shell (e.g., multimers) (e.g., ferritin, virus particles, virus-like particles).

[0103] Stabilizers are substances that enable antibody stabilization, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0104] The functional substance may also be a peptide, a protein, a nucleic acid, an organic compound, an inorganic compound, a sugar chain, a lipid, a high molecular weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. 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. Examples of nucleic acids include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of organic compounds include low-molecular-weight organic compounds such as proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds. Examples of inorganic compounds include silica, talc, and alumina.

[0105] (Salts) 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. Salts with inorganic acids include, for example, salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Salts with organic acids include, for example, 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. Salts with inorganic bases include, for example, 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. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0106] 2. Affinity Substances or Salts Thereof, and Related Inventions 2-1. Affinity Substances or Salts Thereof The present invention provides affinity substances or salts thereof comprising first and second affinity moieties that have affinity for the constant region of an antibody heavy chain.

[0107] The affinity substance used in the present invention comprises first and second affinity moieties having affinity for the heavy chain constant region of an antibody structural unit. The antibody structural unit is an immunoglobulin unit comprising two heavy chains and, optionally, two light chains. Thus, the antibody structural 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), tetravalent or higher antibodies (e.g., IgA antibodies, IgM antibodies), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and disulfide-reduced antibodies. 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-reduced antibodies. This is because the Fc region contains the CH2 and CH3 domains of the heavy chain but does not contain the light chain.

[0108] The first and second affinity moieties may be the same or different. When 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., an affinity substance in which one of the first and second affinity moieties has affinity for the CH2 domain and the other has 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., an affinity substance in which both the first and second affinity moieties have affinity for the CH2 domain or the CH3 domain), preferably different affinity moieties having affinity for the same region in the constant region of the antibody heavy chain. Furthermore, the first and second affinity moieties are preferably in a relationship in which the first binding site of the first affinity moiety to the constant region of the heavy chain and the second binding 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 inhibit the association of the other, for example, to inhibit the association of the first and second affinity moieties to the constant region of a single heavy chain. In this relationship, in an antibody structural unit (an immunoglobulin unit containing two heavy chains and, if necessary, two light chains), the affinity substance and two molecules (the first molecule and the second molecule) of a compound containing an antibody-reactive group can be prevented from competitively associating within a single antibody structural unit (the above-mentioned immunoglobulin unit), thereby promoting 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 predetermined structural units (e.g., peptides (including oligopeptides, polypeptides, and proteins), nucleic acids (including oligonucleic acids and polysaccharides), and sugars (including oligosaccharides and polysaccharides)), as well as non-polymeric substances (e.g., low-molecular-weight compounds).Many substances, such as peptides, nucleic acids, sugars, and low molecular weight compounds, have been reported as usable substances for the first and second affinity moieties having affinity for the constant region of an antibody heavy chain (see, for example, WO 2007 / 004748, WO 2008 / 054030, WO 2013 / 027796, WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979; Nomura Y et al., Nucleic Acids Res., 2010 Nov; 38(21):7822-9; Miyakawa S et al., J. Immunol., 2014, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, al., RNA., 2008 Jun;14(6):1154-63, and the scientific articles below.) Preferably, the first and second affinity moieties are identical.

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

[0110] The first and second affinity moieties can 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 can have affinity for the CH1 domain, CH2 domain, or CH3 domain as the heavy chain constant region, or a region spanning these (e.g., the adjacent region between the CH1 domain and the CH2 domain, or the adjacent region between the CH2 domain and the CH3 domain). The first and second affinity moieties may have affinity for the same or different CHX domains (X is 1, 2, or 3), and preferably 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 partial region in the CHX domain, and may also have affinity for a partial region in the CHX domain, or a region spanning the CHX domain and another CHX domain (e.g., an adjacent region). Therefore, the first and second affinity moieties having affinity for the CH2 domain may have affinity for only a partial region in the CH2 domain, or for a region spanning the CH2 domain and the CH1 domain or the CH3 domain (e.g., the adjacent region of the CH1 domain and the CH2 domain, or the adjacent region of the CH2 domain and the CH3 domain). The first and second affinity moieties having affinity for the CH2 domain may preferably have affinity for only a partial region in the CH2 domain, or for a region spanning the CH2 domain and the CH3 domain (e.g., the adjacent region of the CH2 domain and the CH3 domain), and more preferably may have affinity for only a partial region in the CH2 domain.

[0111] In the affinity substance or salt thereof of the present invention, the first and second affinity moieties may be directly linked, or a linker may be inserted between the first affinity substance and the second affinity moiety. When the binding sites of the first and second affinity moieties to the constant region of the antibody heavy chain are close to each other and the sizes of the first affinity substance and the second affinity moiety are sufficiently large, and therefore it is not necessary to adjust the distance between them, the first and second affinity moieties can be directly linked. On the other hand, when the binding sites of the first and second affinity moieties to the constant region of the antibody heavy chain are not close to each other and the sizes of the first affinity substance and the second affinity moiety are not sufficiently large, and therefore it is necessary to adjust the distance between them, 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 unsubstituted. Examples of divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above. Furthermore, as the linker, for example, a substance such as a peptide, a nucleic acid, a sugar, another polymeric substance (e.g., polyethylene glycol), or a divalent hydrocarbon group (e.g., an alkyl chain) 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 the 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 region in the heavy chain of the antibody to which the first and second affinity moieties have affinity may be derived from an animal (e.g., a mammal, a bird) as described above. The constant region in the heavy chain of the antibody may be preferably a mammalian constant region, more preferably a primate constant region or a rodent constant region, and even more preferably a human constant region.

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

[0114] Preferably, the first and second affinity moieties may be affinity peptides having affinity for the constant region of the antibody heavy chain. Many peptides have been reported as such affinity peptides. Examples of such affinity peptides are as follows: (1) various IgG-binding peptides having affinity for a specific region (CH2 domain) of human IgG in general (i.e., human IgG1, IgG2, IgG3, and IgG4; the same applies below) (see, for example, WO 2008 / 054030, WO 2013 / 027796, and WO 2016 / 186206); (2) Protein A Mimetic (PAM) peptides having affinity for a specific region (CH2 domain) of human IgG in general (see, for example, Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, VOL. 6, 564-569); (3) EPIHRSTLTALL (SEQ ID NO: 27) having affinity for a specific region (CH2 domain) of human IgG in general (see, for example, Ehrlich G.K et al., J. Biochem. Biophys. Methods, 2001, VOL. 49, 443-454); (4) having affinity for a specific region (Fc region) of human IgG in general (NH ２ -Cys1-X1-X2-X3-X4) ２-Lys-Gly-OH (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) having affinity for a specific region (Fc region) of human IgG in general (see, for example, Krook M et al., Journal of Immunological Methods, 1998, VOL. 221, 151-157); (6) QSYP (SEQ ID NO: 31) having affinity for a specific region of human IgG in general (see, for example, Jacobs J.M. et al., 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) having affinity for a specific region (Fc region) of human IgG in general (see, for example, Carbonell R.G. et al., Journal of Chromatography A, 2009, VOL. 1216, 910-918); (8) DAAG (SEQ ID NO: 35) having affinity for a specific region (Fc region) of human IgG in general (see, for example, Lund L.N. et al., Journal of Chromatography A, 2009, VOL. 1216, 910-918); A, 2012, VOL. 1225, 158-167); (9) Fc-I, Fc-II, and Fc-III having affinity for a specific region (Fc region) of human IgG in general (see, e.g., Warren L. Delano et al., Science, 2000, VOL. 287, 1279-1283; WO 2001 / 045746); and (10) NARKFYKG (SEQ ID NO: 36) and NKFRGKYK (SEQ ID NO: 37) having affinity for a specific region (Fc region) of human IgG in general (see, e.g., Biochemical Engineering Journal, 2013, VOL. 79, 33-40); (11) Protein A, Protein G, Protein L, or Protein Z, or fragments thereof, having 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 Mel 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 having affinity for specific regions (Fc region or CH2 domain) of human IgG in general (see, for example, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979);

[0115] Furthermore, affinity peptides can be obtained by the screening methods described above (eg, the methods using the libraries described above or the display methods described above).

[0116] The amino acid residues constituting the affinity peptide can be any of the 20 naturally occurring amino acids commonly constituting proteins, or unnatural amino acid residues, such as L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (hereinafter, the abbreviation "L" will be omitted).

[0117] In certain embodiments, one of the first and second affinity peptides may be an affinity peptide having one lysine residue, and the other may be an affinity peptide having no lysine residues.

[0118] Many peptides have been reported as affinity peptides that have affinity for the constant region of an antibody heavy chain and contain one lysine residue (see, for example, WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 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 in the heavy chain of an antibody and have one lysine residue: (1) affinity peptides comprising the amino acid sequences of SEQ ID NOs: 39 to 72 in WO 2018 / 199337; (2) affinity peptides comprising the amino acid sequences of SEQ ID NOs: 5, 6, 37 to 100 in WO 2019 / 240288; (3) affinity peptides comprising the amino acid sequences of SEQ ID NOs: 5, 8 to 57, 68 to 92 in WO 2019 / 240287; (4) affinity peptides having QET at the N-terminus (SEQ ID NOs: 7 to 10, 22 to 25, 52, 53 in WO 2020 / 090979); and (5) affinity peptides having one lysine residue from the affinity peptides (1) to (12) listed above as examples of affinity peptides.

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

[0121] Many peptides have been reported as affinity peptides that have affinity for the constant region of the antibody heavy chain and do not have a lysine residue. In addition, in the affinity peptides that have affinity for the constant region of the antibody heavy chain and have 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 another moiety (e.g., a moiety containing a reactive group) (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 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, an affinity peptide that has affinity for the constant region in the heavy chain of an antibody and does not have a lysine residue can be an affinity peptide that has affinity for the constant region in the heavy chain of an antibody and has one lysine residue, in which the lysine residue is replaced with another amino acid residue (preferably a normal natural amino acid residue that constitutes proteins other than lysine residues and cysteine ​​residues), and that has affinity for the constant region in the heavy chain of an antibody.

[0122] More specifically, the following may be used as affinity peptides that have affinity for the constant region in the heavy chain of an antibody and do not have a lysine residue: (1) SEQ ID NOs: 20 to 38, 73 to 75 (when Xaa1 is other than a lysine residue), and SEQ ID NO: 92 in WO 2018 / 199337; (2) SEQ ID NOs: 7, 11 to 14, and 108 in WO 2019 / 240288; (3) Of the affinity peptides (1) to (4) listed above as examples of affinity peptides that have affinity for the constant region in the heavy chain of an antibody and have one lysine residue, 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) Of the affinity peptides (1) to (12) listed above as examples of affinity peptides, affinity peptides that do not have a lysine residue.

[0123] In certain embodiments, examples of affinity peptides that do not have lysine residues include the following (5) to (10): (5) an affinity peptide comprising the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40); (6) 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 FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40) are substituted with a lysine residue and another amino acid residue other than a cysteine ​​residue, and having affinity for the constant region of an antibody heavy chain; (7) an affinity peptide comprising the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41); (8) an affinity peptide comprising an amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41) 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, and having affinity for the heavy chain constant region 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 of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78) 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, and having affinity for the heavy chain constant region of an antibody. Here, the two cysteine ​​residues contained in the amino acid sequence may be bridged by a disulfide bond.

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

[0125] When the first and second affinity moieties are affinity peptides, the affinity substance or its salt of the present invention may have the first and second affinity peptides directly linked to each other, or may have a linker 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 be inserted. When the first and second affinity moieties are affinity peptides, the linker is preferably a peptide linker.

[0126] The number of amino acid residues constituting the peptide linker can be appropriately set depending on 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 less than 50, less than 49, less than 48, less than 47, less than 46, or less than 45 amino acid residues.

[0127] The amino acid residues constituting the peptide linker can be the above-mentioned natural amino acid residues or unnatural amino acid residues.Preferably, the amino acid residues constituting the peptide linker may only contain the above-mentioned natural amino acid residues.Suitable amino acid residues for the peptide linker include, but are not limited to, alanine, proline, serine, and glycine.The peptide linker can also be disclosed in WO2021 / 112249 and WO2011 / 144756.

[0128] Preferably, the affinity substance or salt thereof of the present invention may be an affinity substance or salt thereof in which the first and second affinity moieties are affinity peptides and which includes a linker between the first affinity peptide and the second affinity peptide. Such an affinity substance or salt thereof may be represented by the following formula (A): AP1-L Ａ -AP2 (A) (wherein, AP1 represents a first affinity peptide having affinity for the constant region of the heavy chain of an antibody, AP2 represents a second affinity peptide having affinity for the constant region of the heavy chain of an antibody, and L Ａ represents a linker.

[0129] In formula (A) and other formulae presented in connection with this invention, a hyphen (-) indicates that the two units on either side of it are covalently bonded. Thus, in formula (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, denoted AP1 and AP2, respectively, are as described above.

[0131] L Ａ The linker represented by the formula (I) is a divalent group. The divalent group may be substituted or unsubstituted. Examples of the divalent group include those described above. When the divalent group is substituted, examples of the substituent include those described above. Preferably, the linker may be, for example, a substance such as a peptide, a nucleic acid, a sugar, another polymeric substance (e.g., polyethylene glycol), or a divalent hydrocarbon group (e.g., an alkyl chain).

[0132] More preferably, the affinity substance or its salt of the present invention may be an affinity polypeptide or its salt, in which the first and second affinity moieties are affinity peptides and a peptide linker is included between the first affinity peptide and the second affinity peptide. Such an affinity polypeptide has the following formula (A'): AP1-PL Ａ -AP2 (A') (wherein, AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and located on the N-terminal side of the affinity polypeptide, AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain and located on the C-terminal side of the affinity polypeptide, and PL Ａ represents a peptide linker.

[0133] Affinity substances such as affinity polypeptides or salts thereof may contain the above-mentioned natural amino acid residues or unnatural amino acid residues as the amino acid residues that constitute them. When the affinity substance contains only the above-mentioned 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). When the affinity substance contains unnatural 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, which enables large-scale production of the affinity substance 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 and carboxyl groups at the termini 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., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). Preferably, the N-terminal amino group may be alkylated, formylated, or acetylated. Examples of protecting groups for the C-terminal carboxy group include groups capable of forming esters or amides. Examples of groups capable of forming esters or amides include alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups.

[0135] Furthermore, when the N-terminal amino acid of the affinity substance is glutamic acid (E) or glutamine (Q), the N-terminus can be protected using the side chain. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid can have a cyclic structure of pyroglutamic acid. When the N-terminal amino acid is glutamine, the protected N-terminal glutamine ... ２ ) can react with the amide group present in its side chain (pyroglutamylation) to form a pyroglutamic acid-type cyclic structure. Therefore, the N-terminal amino acid may preferably be glutamic acid or glutamine.

[0136] When the affinity substance is an affinity polypeptide, the affinity polypeptide may further contain a tripeptide consisting of Gln-Glu-Thr (QET) at the N-terminus. In this case, a polypeptide expression system using a host cell enables the protection of the N-terminal amino group by pyroglutamylation of Q, as well as the simple and large-scale secretion production of the affinity polypeptide (see Examples, WO 2013 / 062029, WO 2020 / 090979). In this case, a signal peptide such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 42) can be added to the N-terminus of QET (see Examples, WO 2013 / 062029, WO 2020 / 090979).

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

[0138] Examples of the specific reactive groups include the following: (1) an amino group (NH ２ , N.H.R. ３ , N.R. ３ R ４ .R ３ and R ４are 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) a residue capable of reacting with an amino group, such as an activated ester residue (e.g., an N-hydroxysuccinimide residue), a vinyl sulfone residue, a sulfonyl chloride residue, an isocyanate residue, an isothiocyanate residue, an aldehyde residue, a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue, a 2-imino-2-methoxyethyl residue, or a diazonium terephthalate residue; (3) a carboxyl group (COOH); (4) a residue capable of reacting with a carboxyl group, such as an amino group as described above; (5) a hydroxyl group (OH) (including alcoholic and phenolic hydroxyl groups); and (6) a residue capable of reacting with a hydroxyl group, such as a diazonium residue, a diazodicarboxyl residue, or a 2,3-dihydro-1H-pyrazin-6-one residue. The specific reactive group may preferably be any of (1) to (4). More preferably, the specific reactive group may be any of (1) or (2), or any of (3) or (4). Alternatively, more preferably, the specific reactive group may be any of (1) or (3). The specific reactive group is even more preferably (1), and particularly preferably an amino group (NH ２ ) may also be used.

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

[0140] Alternatively, the affinity polypeptide may be a polypeptide that does not contain any amino acid residues having an amino group in the side chain (e.g., lysine residues) and has an amino group at the N-terminus, thereby containing only one amino group as a specific reactive group. The affinity polypeptide may also be a polypeptide that does not contain any amino acid residues having a carboxyl group in the side chain (e.g., aspartic acid residues, glutamic acid residues) and has a carboxyl group at the C-terminus, thereby containing only one carboxyl group as a specific reactive group.

[0141] Preferably, the 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 residue, a natural amino acid constituting proteins, but also other amino acid residues having an amino group in their side chain (e.g., ornithine) can be used. More preferably, the affinity polypeptide containing only one specific reactive group contains an amino group (NH ２The lysine residue may be a polypeptide containing only one lysine residue having a carboxyl group (C-C) in its side chain. In this case, the lysine residue may be contained in either the first affinity peptide, the second affinity peptide, or the peptide linker. When the lysine residue is contained in the peptide linker, it may be contained in a position close to the first affinity peptide or the second affinity peptide (e.g., 10 or less, 5 or less, or 1 to 3 amino acid residues from the first affinity peptide or the 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 the above formula (A) or a salt thereof includes a first and a second affinity peptide represented by AP1 and AP2, respectively, and L Ａ The linker represented by the formula (I) may further have the following characteristic (1) or (2), preferably characteristic (1):

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

[0144] (2) An affinity substance or a salt thereof containing only one carboxy group as a specific reactive group: (2-1) (i) an affinity substance or a salt thereof, in which the first affinity peptide is an affinity peptide containing only one amino acid residue having a carboxy group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid), (ii) the second affinity peptide is an affinity peptide containing no amino acid residue having a carboxy group in its side chain, and the linker is a linker containing no carboxy group; (2-2) (i) an affinity substance or a salt thereof, in which the first affinity peptide is an affinity peptide containing no amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide is an affinity peptide containing only one amino acid residue having a carboxy group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) and having a protected C-terminus, and the linker is a linker containing no carboxy group; (2-3) An affinity substance or a salt thereof, wherein (i) the first affinity peptide does not contain an amino acid residue having a carboxy group in its side chain, (ii) the second affinity peptide does not contain an amino acid residue having a carboxy group in its side chain and has an unprotected C-terminus, and the linker does not contain a carboxy group; (2-4) An affinity substance or a salt thereof, wherein (i) the first affinity peptide does not contain an amino acid residue having a carboxy group in its side chain and (ii) the second affinity peptide does not contain an amino acid residue having a carboxy group in its side chain and has a protected C-terminus, and the linker is a linker containing only one carboxy group. An affinity substance or a salt thereof containing only one carboxy group as a specific reactive group may also be an affinity substance or a salt thereof that does not contain a group capable of reacting with a carboxy group (e.g., an amino group) in order to suppress undesired reactions (e.g., intramolecular or intermolecular reactions). Therefore, in the above (2-1) to (2-4), 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 not containing an amino group.

[0145] Preferably, the affinity substance represented by the above formula (A') or its salt comprises a first affinity peptide represented by AP1 and a second affinity peptide represented by AP2, respectively, and a PL Ａ The peptide linker represented by the formula (I) may further have the following characteristic (1') or (2'), preferably characteristic (1'):

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

[0147] (2') An affinity substance or a salt thereof containing only one carboxy group as a specific reactive group; (2-1') An affinity substance or a salt thereof, in which (i) the first affinity peptide is an affinity peptide containing only one amino acid residue having a carboxy group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid), (ii) the second affinity peptide is an affinity peptide containing no amino acid residue having a carboxy group in its side chain, and the peptide linker is a peptide linker containing no amino acid residue having a carboxy group in its side chain; (2-2') An affinity substance or a salt thereof, in which (i) the first affinity peptide is an affinity peptide containing no amino acid residue having a carboxy group in its side chain, and (ii) the second affinity peptide is an affinity peptide containing only one amino acid residue having a carboxy group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) and having a protected C-terminus, and the peptide linker is a peptide linker containing no amino acid residue having a carboxy group in its side chain; (2-3') An affinity substance or a salt thereof, wherein (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain, (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy group in its side chain and has an unprotected C-terminus, and the peptide linker is a peptide linker that does not contain an amino acid residue having a carboxy group in its side chain; (2-4') An affinity substance or a salt thereof, wherein (i) the first affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxy 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 carboxy group in its side chain and has a protected C-terminus, and the peptide linker is a linker that contains only one amino acid residue having a carboxy group in its side chain. An affinity substance or its salt containing only one carboxy group as a specific reactive group may be an affinity substance or its salt that does not contain a group (e.g., an amino group) that can react with the carboxy group, in order to suppress undesired reactions (e.g., intramolecular reactions or intermolecular reactions).Therefore, in the above (2-1') to (2-4'), the first affinity peptide may be one that does not contain an amino acid residue having an amino group in its side chain and / or one that has a protected N-terminus, the second affinity peptide may be one that does 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. Inventions Related to Affinity Substances or Their Salts When the affinity substance or its salt of the present invention is an affinity polypeptide comprising first and second affinity peptides that have affinity for the constant region of an antibody heavy chain, such affinity polypeptides can be prepared using a host cell comprising an expression unit comprising 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 polynucleotides and host cells, as well as expression vectors that can be used to produce the host cells.

[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 is preferably DNA.

[0150] The host cell of the present invention can be produced, for example, by a method using an expression vector containing a polynucleotide of the present invention (e.g., a competent cell method, an electroporation method), or by genome modification technology. When the expression vector is an integrative vector that undergoes homologous recombination with the genomic DNA of the host cell, the expression unit can be integrated into the genomic DNA of the host cell by transformation. On the other hand, when the expression vector is a non-integrative vector that does not undergo homologous recombination with the genomic DNA of the host cell, the expression unit is not integrated into the genomic DNA of the host cell by transformation, and can exist in the host cell as an expression vector, independent of the genomic DNA. Alternatively, genome editing technology (e.g., the CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)) can be used to integrate the expression unit into the genomic DNA of the host cell and modify the expression unit inherently contained in the host cell.

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

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

[0153] The expression vector may be a plasmid, a viral vector, a phage, or an artificial chromosome. The expression vector may also be an integrative vector or a non-integrative vector. An integrative vector may be a vector that is integrated in its entirety into the genome of a host cell. Alternatively, an integrative vector may be a vector that is integrated only in part (e.g., an expression unit) into the genome of a host cell. The expression vector may further 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 derivatives thereof.

[0154] Host cells for expressing the affinity polypeptide of the present invention include various prokaryotic cells such as Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (e.g., Corynebacterium glutamicum), and Bacillus bacteria (e.g., Bacillus subtilis), as well as Saccharomyces bacteria (e.g., Saccharomyces cerevisiae), Pichia bacteria (e.g., Pichia stipitis), and Aspergillus bacteria (e.g., Aspergillus oryzae). Various eukaryotic cells, including E. oryzae, can be used as hosts. Alternatively, insect cells, plant cells, and animal cells (e.g., mammalian cells such as Chinese hamster ovary (CHO) cells) can be used as hosts. A strain lacking a specific gene may also be used as a host. Examples of host cells include host cells that carry an expression vector in the cytoplasm and host cells into which a 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 the N-terminus, it is preferable to use an affinity polypeptide that can be prepared by a polypeptide secretion production method using coryneform bacteria as a host (WO 2013 / 062029). This method can add the N-terminal three residues of the Csp mature protein, Gln-Glu-Thr (QET), to the N-terminus of the target polypeptide, and can easily prepare large amounts of polypeptides containing a glutamine residue (Q) at the N-terminus, making it suitable for preparing affinity polypeptides. 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-terminus of QET (see Examples, WO 2013 / 062029, WO 2020 / 090979). Examples of coryneform bacteria that can be used in this method include bacteria of the genus Corynebacterium (eg, Corynebacterium glutamicum, Corynebacterium stationis) and bacteria of the genus Brevibacterium.

[0156] The host cells of the present invention can be cultured in a medium having the composition described below using a predetermined culture device (e.g., test tube, flask, or jar fermenter). Culture conditions can be set appropriately. 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 1 hour to 100 hours. 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 index. Aeration and agitation conditions can be controlled so that the relative dissolved oxygen concentration (DO value) when the atmospheric oxygen concentration is 21% does not fall below, for example, 1% to 10%, preferably 3% to 8%. Culture may be performed by batch or fed-batch culture. In fed-batch culture, the culture can be continued by sequentially adding a solution serving as a sugar source or a solution containing phosphate to the culture medium, either continuously or discontinuously.

[0157] As a promoter for expressing the polynucleotide of the present invention, a promoter typically used for heterologous protein production in E. coli can be used, and examples thereof include strong promoters such as 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. Furthermore, examples of vectors that may be used 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 derivatives thereof.

[0158] Furthermore, a terminator, which is a transcription termination sequence, may be ligated downstream of the polynucleotide of the present invention. Examples of such terminators include the T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and Escherichia coli trpA gene terminator.

[0159] The medium may be a medium typically used for culturing E. coli, such as M9-casamino acids medium or LB medium. The medium may contain a predetermined carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc. may be used. The culture conditions and production induction conditions are appropriately selected depending on the type of marker, promoter, host bacterium, etc. of the vector 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 disruptant or lysate by recovering the transformed cells of the present invention and then disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the cells. If the affinity polypeptide is secreted or leaked outside the cells, a sterilized 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 disruptant, lysate, or sterilized solution to techniques such as extraction, precipitation, filtration, and column chromatography.

[0161] 3. Compound or Salt Thereof The compound or salt thereof of the present invention comprises (A) an affinity substance comprising first and second affinity moieties having affinity for the constant region of an antibody heavy chain, and (B) a group reactive with an antibody. The definitions, examples, and preferred examples of the affinity substance and its 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 the antibody-reactive group, a group reactive to an amino acid residue having a reactive side chain among the amino acid residues constituting an antibody (protein) can be used. Of the 20 naturally occurring amino acids constituting proteins as described above, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, which have hydrocarbon side chains, are inactive in normal reactions. Therefore, the antibody-reactive group is a group capable of reacting with the side chains of one or more (e.g., two, three, or four) of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Depending on conditions such as the amino acid composition of the antibody, one or more (e.g., two, three, or four) reactive groups may be contained in the compound of the present invention or a salt thereof. Preferably, the compound of the present invention or a salt thereof contains only one reactive group.

[0163] Preferably, the antibody-reactive group is a group capable of reacting with the side chain of any one of the 14 amino acids constituting proteins. The antibody-reactive group is more preferably a group specifically reactive with the side chain of any one of the amino acids lysine, tyrosine, tryptophan, or cysteine, even more preferably a group specifically reactive with the side chain of any one of the amino acids lysine, tyrosine, or tryptophan, and particularly preferably a group specifically reactive with the side chain of lysine or tyrosine, especially the side chain of lysine. For details of such reactive groups, see, for example, WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979.

[0164] The reactive group specific to the side chain of a lysine residue is the amino group (NH ２ ), and examples thereof include activated ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethyl residues, diazonium terephthalic acid residues, α-halogen-substituted acetamides, and α-halogen-substituted methyl ketones. ２ ) can produce, for example, an amide residue, a urea residue, a pyridine residue, a carbamate residue, or a sulfonamide residue as a linking moiety.

[0165] In certain embodiments, the compound of the present invention or a salt thereof has the following formula (I): [wherein R represents a group reactive with an antibody, L represents a linker, and A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain.] The definitions, examples, and preferred examples of the antibody-reactive group 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 the divalent group include those described above. When the divalent group is substituted, examples of the substituent include those described above.

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

[0168] A cleavable moiety is a site that can be cleaved by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of a protein (e.g., cleavage of an amide bond). Therefore, a cleavable moiety can be said to be a site (a bond other than an amide bond) that can be cleaved by a specific cleavage treatment under mild conditions. Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli such as light, or (c) incubation when a cleavable linker containing a self-degrading cleavable moiety is used. Such cleavable linkers and the cleavage conditions thereof are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, J.M., Journal of American Chemical Society. Society. 132, 17928 (2010); Thompson, D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004)). Reaction conditions under mild conditions (e.g., reaction temperature, reaction time, reaction solution) are as described below.Examples of the cleavable moiety include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues (e.g., tert-butyloxycarbamate residues), silane residues, hydrazone-containing residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0169] The aromatic ring group having an electron-withdrawing group is preferably one having an aromatic ring group selected from the group consisting of aryl, aralkyl, aromatic heterocyclic group, and alkyl having an aromatic heterocyclic group, and more preferably aralkyl or alkyl having an aromatic heterocyclic group. The electron-withdrawing group is preferably bonded to the 2-position of the ring. Even more preferably, the aromatic ring-containing residue having an electron-withdrawing group is, for example, an aralkyl (e.g., benzyl) having an electron-withdrawing group at the 2-position. Examples of the electron-withdrawing group include halogen atoms, alkyl substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl group, and keto group (e.g., acyl).

[0170] The definitions, examples, and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, aralkyl, etc. found as prefixes, suffixes, etc. in connection with the names of residues as cleavable moieties are the same as those described above.

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

[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) and diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane).

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

[0175] The unsaturated bond-containing chain residue is a residue containing an unsaturated bond moiety consisting of only carbon atoms (e.g., vinyl (ethenyl), the smallest unit having a double bond between carbon atoms, or acetylenyl (ethynyl), the smallest unit having a triple bond between carbon atoms), or a residue containing an unsaturated bond moiety (e.g., aldehyde, cyano) consisting of carbon atoms and heteroatoms (e.g., nitrogen atom, sulfur atom, oxygen atom). Examples of the unsaturated bond-containing chain residue include vinyl ether residue, cyanoethyl residue, ethylene residue, and malondialdehyde residue.

[0176] Examples of acidic substances (also referred to as electrophiles) include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acidic substances such as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3-morpholinopropanesulfonic acid, sodium dihydrogenphosphate, citric acid, dodecylsulfuric acid, N-dodecanoylsarcosinic acid, and trifluoroacetic acid. Examples of sites that can be cleaved by acidic substances include alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, β-thiopropionate residues, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, and 2-(diphenylphosphino)benzoate residues.

[0177] Examples of basic substances (also referred to as nucleophiles) include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, ammonium acetate, etc., and organic basic substances such as hydroxylamine, triethylamine, N,N'-diisopropylamine, etc. Examples of sites that can be cleaved by basic substances include silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyl disuccinate residues, α-thiophenyl ester residues, unsaturated vinyl sulfide residues, malondialdehyde residues, acylhydrazone residues, and alkylthioester residues.

[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 an oxidizing agent include vicinal diol residues and selenium residues.

[0180] Examples of the enzyme include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloprotease, lipase, endoglycosidase, and PN gauze F. Examples of the site cleavable by the enzyme include an ester residue, a phosphodiester residue, and a glycosyl residue.

[0181] Examples of the photocleavable moiety include a 2-nitrobenzyl residue, a phenacyl ester residue, an 8-quinolinebenzenesulfonate residue, a coumarin residue, a phosphotriester residue, a bisarylhydrazone residue, and a bimandithiopropionic acid residue.

[0182] Autolytic cleavable moieties include, for example, activated ester residues (eg, N-hydroxysuccinimide residues).

[0183] When the compound of the present invention or a salt thereof 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 conventional ester residues and other ester residues such as thioester residues), acetal residues (including conventional ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.

[0184] When the compound of the present invention or a salt thereof contains a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, it can be represented by the following formula (Ia): [wherein R represents a group reactive to an antibody, L １ represents a first linker, L ２represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, and A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain. 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(B) capable of generating a bioorthogonal functional group on the reactive group side upon cleavage are as described above.

[0185] L １ a first linker represented by ２ The second linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0186] In certain embodiments, the total number of atoms constituting the main chain of the first linker and the second linker may be 2 to 10. This total number of atoms may be 3 or more, or 4 or more. This total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, this 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 of each of the first linker and the second linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0188] The main chains of the first linker and the second linker are composed of a chain structure, a cyclic structure, or a structure including a combination thereof. When the main chain is a chain 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 structure. On the other hand, when the main chain includes a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms constituting the cyclic structure as the number of atoms in the main chain. 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). When the main chain includes a combination of a chain structure and a cyclic structure, the number of atoms in the main chain can be determined by adding the number of atoms in the chain 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 of counting the number of atoms in the main chain is similar for other linkers. - is a bond. In the case of (a), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 2. In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 3. In the case of (c), both paths are the shortest paths (equidistant), so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4. In the case of (d), the path of the condensation site is the shortest path, so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4.

[0189] In certain embodiments, the compound of the present invention or a salt thereof has the following formula (Ia-1): [wherein X represents a leaving group; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ represents a third linker, and L ４represents a fourth linker, S represents a sulfur atom, and A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region of an antibody heavy chain. 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 C═W group adjacent to X. １ A leaving group is a group that can be eliminated by a reaction between a carbon atom in R and an amino group. Those skilled in the art can appropriately design such a leaving group. Examples of such leaving groups include the following: (a) R Ａ -S (where 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 (where 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 (where R Ａ and R Ｂ each independently represent a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom; or (d) a halogen atom.

[0191] Preferably, the leaving group represented by X may be: (a) R Ａ -S (where 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 (where 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; or (c) R Ａ - (R Ｂ -) N (where R Ａ and RＢ 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.

[0192] More preferably, the leaving group represented by X may be: (a) R Ａ -S (where 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; or (b) R Ａ -O (where 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.

[0193] Even more preferably, the leaving group represented by X may be: (a) R Ａ -S (where 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.

[0194] Particularly preferably, the leaving group represented by X may be: (a') R Ａ -S (where R Ａ represents an optionally substituted monovalent aromatic hydrocarbon group (e.g., phenyl), and S represents a sulfur atom.

[0195] W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom. １ , W ２ and W ３ may be an oxygen atom.

[0196] L ３ a third linker represented by ４The fourth linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0197] In certain embodiments, the total number of atoms constituting the main chain of the third linker and the fourth linker may be 2 to 10. This total number of atoms may be 3 or more, or 4 or more. This total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, this 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 of each of the third linker and the fourth linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0199] When the compound of the present invention or a salt thereof contains a cleavable moiety, it may further contain a bioorthogonal functional group between the antibody-reactive group 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 a substituent as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue.

[0200] When the compound of the present invention or a salt thereof further comprises a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety, it can be represented by the following formula (Ib): [wherein R represents a group reactive to an antibody, L ５ represents a fifth linker, L ６represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, and A represents an affinity substance containing first and second affinity moieties having affinity for the constant region of the antibody heavy chain. 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] L ５ a fifth linker represented by L ６ The sixth linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

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

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

[0204] The group containing a bioorthogonal functional group represented by B may be a group consisting of a bioorthogonal functional group, or may be a group containing a bioorthogonal functional group and another moiety. Examples of other moieties include the linking moiety between the bioorthogonal functional group and the linker. The linking moiety 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 substituent when the divalent group is substituted are as described above.

[0205] In certain embodiments, the compound of the present invention or a salt thereof has the following formula (Ib-1): [wherein X represents a leaving group; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ represents the seventh linker, L ８ represents an eighth 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 having affinity for the constant region of an antibody heavy chain. The compound may be a compound represented by the formula (I) or a salt thereof. The definitions, examples, and preferred examples of the leaving group represented by X, the group containing a bioorthogonal functional group represented by B, and the affinity substance represented by A are as described above.

[0206] W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom. １ , W ２ and W ３ may be an oxygen atom.

[0207] L ７ a seventh linker represented by L ８ The eighth linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent groups are substituted, examples of the substituents include those described above.

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

[0209] The number of atoms constituting the main chain of each of the seventh linker and the eighth linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such 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, and preferably, V may be a sulfur atom.

[0211] The compound of the present invention or a salt thereof can easily modify only one of the heavy chains in the antibody structural unit, and can also provide a regioselectively modified antibody while easily modifying only one of the heavy chains in the antibody structural unit.

[0212] The above series of compounds or salts thereof can be prepared by reacting the affinity substance of the present invention with a moiety containing a reactive group for an antibody. For example, such a reaction can be carried out in a suitable organic solvent (e.g., CH ２ Cl ２ The reaction can be carried out in an organic solvent containing an alkyl halide (e.g., methyl halide) such as methyl amine, and an amine such as triethylamine, at an appropriate temperature (e.g., about −10 to 30° C.). 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 production of the above-mentioned series of compounds or salts thereof can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Such compounds or salts thereof can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0214] 4. Affinity Substance-Modified Antibodies or Salts Thereof 4-1. Affinity Substance-Modified Antibodies or Salts Thereof Comprising At Least One Affinity Substance (Comprising First and Second Affinity Moieties) The present invention provides affinity substance-modified antibodies or salts thereof, which comprise an affinity substance in the constant region of the antibody heavy chain, the affinity substance comprising first and second affinity moieties having affinity for 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.

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

[0216] The affinity substance-modified antibody or its salt can contain an affinity substance via modification of a functional group in the side chain of one or more (e.g., two, three, or four) of 14 amino acid residues present in the constant region (preferably the Fc region or CH2 domain): asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. The affinity substance-modified antibody or its salt can contain an 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 position of an antibody or its salt with an affinity substance can be confirmed by peptide mapping. As described above, the modification may be site-selective. Therefore, in the formulae (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin unit may site-selectively have the corresponding modifying unit via a functional group in the side chain of the amino acid residue.

[0217] Preferably, the affinity substance-modified antibody can contain the above-mentioned affinity substance through modification of the amino group in the side chain 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 heavy chains in the antibody constituent unit (an immunoglobulin unit containing two heavy chains and, if necessary, two light chains) (in other words, it contains the affinity substance through the amino group in the side chain of a lysine residue in the constant region of one heavy chain in the immunoglobulin unit, but does not contain the affinity substance through the amino group in the side chain of a lysine residue in the constant region of the other heavy chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, and WO 2020 / 090979). The modification may be regioselective, as described above. Thus, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin unit may have the corresponding modifying unit regioselectively via the amino group in the side chain of the lysine residue.

[0218] An affinity substance-modified antibody or a salt thereof can be produced by reacting a compound of the present invention or a salt thereof with an antibody or a salt thereof comprising an immunoglobulin unit containing two heavy chains and, if necessary, two light chains. The amount of the compound of the present invention or a salt thereof relative to the antibody in the reaction (compound of the present invention or a salt thereof / antibody) is not particularly limited because it varies depending on factors such as the type of compound of the present invention or a 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 a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, a reaction under such mild conditions can be carried out in an appropriate reaction system, such as a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer solution may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such a reaction, see, for example, G. J. L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G. J. L. Bernardes et al., Chem. Asian. J. , 4, 630 (2009); B. G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0220] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (II): [wherein Ig represents an immunoglobulin unit comprising two heavy chains and optionally two light chains, L represents a linker, A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and the average percentage modification r of the immunoglobulin unit with the affinity substance is 65 to 135%.] The definitions, examples, and preferred examples of the immunoglobulin unit represented by Ig, the linker represented by L, the affinity substance represented by A, and the antibody are as described above (for example, with regard to the linker represented by L, see the linker represented by L in the compound of formula (I)).

[0221] The average percentage modification r of the immunoglobulin units with the affinity substance is 65-135%. The average percentage modification 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 percentage modification 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 can be determined by mass spectrometry (which can be performed in conjunction with a DAR calculator (software from Agilent Corporation); see Examples).

[0222] The average modification percentage r may also be preferably 65 to 100%, more preferably 70 to 100%, even more preferably 75 to 100%, and particularly preferably 80 to 100%, 85 to 100%, 90 to 100%, or 95 to 100%. The upper limit of these average modification percentages may be a value equal to or less than the aforementioned percentages, such as 98% or less, or 96% or less. Alternatively, the average modification percentage r may be 96 to 100%, 97 to 100%, 98 to 100%, 99 to 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 described below with an affinity substance, but also to the average modification percentage r described below with any modification (e.g., a bioorthogonal functional group, a functional substance).

[0224] An antibody comprising a structural unit represented by formula (II) or a salt thereof can be produced by reacting a compound represented by formula (I) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains or a salt thereof.

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

[0226] When the affinity substance-modified antibody or its salt contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthogonal functional group on the antibody (immunoglobulin unit) upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including conventional ester residues and other ester residues such as thioester residues), acetal residues (including conventional ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.

[0227] When the affinity substance-modified antibody or a salt thereof contains a cleavable moiety capable of generating a bioorthogonal functional group on the antibody (immunoglobulin unit) side upon cleavage, the affinity substance-modified antibody or a salt thereof can be represented by the following formula (IIa): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; L １ represents a first linker, L ２ represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region of an antibody heavy chain, and the average modification percentage r of the immunoglobulin unit with the affinity substance is 65 to 135%. １ a first linker represented by L ２ The definitions, examples, and preferred examples of the second linker represented by , the cleavable moiety represented by CLE(B), the affinity substance represented by A, and the average modification percentage represented by r, as well as the antibody, are as described above.

[0228] An antibody comprising a structural unit represented by formula (IIa) or a salt thereof can be produced by reacting a compound represented by formula (Ia) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains or a salt thereof.

[0229] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (IIa-1): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ represents a third linker, L ４ represents a fourth linker, S represents a sulfur atom, A represents an affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and the average percentage modification r of the immunoglobulin unit with the affinity substance is 65 to 135%. The immunoglobulin unit represented by Ig, W １ , W ２ and W ３ An atom represented by L ３ a third linker represented by L ４ The definitions, examples, and preferred examples of the fourth linker represented by , the affinity substance represented by A, and the average modification percentage represented by r, as well as the antibody, are as described above.

[0230] An antibody comprising a structural unit represented by formula (IIa-1) or a salt thereof can be produced by reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains or a salt thereof.

[0231] When the affinity substance-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. Preferred bioorthogonal functional groups include azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with the substituents described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues.

[0232] When the affinity substance-modified antibody or a salt thereof further comprises a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety, the affinity substance-modified antibody or a salt thereof may be represented by the following formula (IIb): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; L ５ represents a fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, A represents an affinity substance containing first and second affinity moieties having affinity for a constant region in an antibody heavy chain, and the average percentage modification r of the immunoglobulin unit with the affinity substance is 65 to 135%. ５ a fifth linker represented by L ６ The definitions, examples, and preferred examples of the sixth linker represented by (I), the group containing a bioorthogonal functional group represented by (B), the cleavable site represented by (CLE), the affinity substance represented by (A), and the average modification percentage represented by (r), as well as the antibody, are as described above.

[0233] An antibody comprising a structural unit represented by formula (IIb) or a salt thereof can be produced by reacting a compound represented by formula (Ib) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains or a salt thereof.

[0234] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (IIb-1): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ represents the seventh linker, L ８ represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A represents an affinity substance containing first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and the percentage modification r of the immunoglobulin unit by the affinity substance is 65 to 135%. The immunoglobulin unit represented by Ig, W １ , W ２ and W ３ An atom represented by L ７ A seventh linker represented by L ８ The definitions, examples, and preferred examples of the eighth linker represented by (I), the group containing a bioorthogonal functional group represented by (B), the atom represented by (V), the affinity substance represented by (A), and the average modification percentage represented by (r), as well as the antibody, are as described above.

[0235] An antibody comprising a structural unit represented by formula (IIb-1) or a salt thereof can be produced by reacting a compound represented by formula (Ib-1) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains or a salt thereof.

[0236] The affinity substance-modified antibody or its salt may further comprise an additional modifying moiety. Various methods are known for modifying antibodies. Thus, in the present invention, the affinity substance-modified antibody or its salt may be modified to further comprise an additional modifying moiety. The additional modifying moiety may be introduced into the heavy chain or light chain of the antibody, preferably into the heavy chain of the antibody (particularly into the constant region of the heavy chain).

[0237] In certain embodiments, the additional modifying moiety may be an additional affinity substance comprising a third affinity moiety having affinity for the constant region of the antibody heavy chain. The "affinity moiety" in the "third affinity moiety" and the "affinity substance" in the "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 moieties described above, but is preferably different.

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

[0239] 4-2. Affinity-Substance-Modified Antibodies or Salts Thereof Comprising At Least Two Affinity Substances (including First, Second, Third, and Fourth Affinity Moieties) The present invention also provides affinity-substance-modified antibodies or salts thereof comprising first and second modifying moieties, wherein the first modifying moiety comprises a first affinity substance comprising first and second affinity moieties having affinity for the constant region of the antibody heavy chain, and the second modifying moiety comprises a second affinity substance comprising third and fourth affinity moieties having affinity for the constant region of the antibody heavy chain. The 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 modifying moieties may be the same or different, and are preferably different.

[0240] Preferably, such an affinity substance-modified antibody or a salt thereof comprises (a) an antibody structural unit (an immunoglobulin unit containing two heavy chains and, if necessary, two light chains), and (b) the above-mentioned first and second modifying moieties, wherein (c) the first modifying moiety is introduced into the constant region of the first heavy chain in the immunoglobulin unit, and (d) the second modifying moiety is introduced into the constant region of the second heavy chain in the immunoglobulin unit. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, and affinity substances, as well as their constituent elements (e.g., constant regions), are as described above.

[0241] The affinity substance-modified antibody or its salt can comprise a first modified portion containing a first affinity substance and a second modified portion containing a second affinity substance, through modification of functional groups in the side chains of one or more (e.g., two, three, or four) of 14 amino acid residues consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine present in the constant region (preferably the Fc region or CH2 domain). The affinity substance-modified antibody or its salt can comprise a first modifying portion containing a first affinity substance and a second modifying portion containing a second affinity substance, preferably 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 with the affinity substance can be confirmed by peptide mapping. As described above, the modification may be site-selective. Therefore, in the formula described below, the immunoglobulin unit may have the corresponding modifying unit site-selectively via a functional group in the side chain of the amino acid residue.

[0242] Preferably, the affinity substance-modified antibody can comprise a first modifying moiety containing the first affinity substance 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 constant region of the first heavy chain (preferably the Fc region or CH2 domain) in the antibody building block (an immunoglobulin unit containing two heavy chains and, optionally, two light chains). The affinity substance-modified antibody can also comprise a second modifying moiety containing the second affinity substance 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 constant region of the second heavy chain (preferably the Fc region or CH2 domain). More specifically, the position 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 site-selective, as described above. Therefore, in the formula below, the immunoglobulin unit may have the corresponding modifying unit regioselectively via the amino group in the side chain of the lysine residue.

[0243] The affinity substance-modified antibody or its salt can be produced by the following steps: (1) reacting the compound of the present invention or its salt with an antibody comprising an immunoglobulin unit containing two heavy chains and, optionally, two light chains, to produce an affinity substance-modified antibody or its salt comprising a first modifying moiety comprising the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit; and (2) reacting the affinity substance-modified antibody or its salt comprising the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with the compound of the present invention or its salt to produce an affinity substance-modified antibody or its salt comprising the first and second modifying moieties in the constant region of the heavy chain of the immunoglobulin unit. The compound of the present invention or its salt used in step (1) and the compound of the present invention or its salt used in step (2) may be the same or different, and are preferably different. The amount of the compound of the present invention or its salt relative to the antibody in the reaction (compound of the present invention or its salt / antibody) is not particularly limited, as it varies depending on factors such as the type of the compound of the present invention or its salt 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.

[0244] Such a reaction can be suitably carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such a reaction under mild conditions is similar to that described above.

[0245] In certain embodiments, such an affinity substance-modified antibody or salt thereof has the following formula (V): where Ig represents an immunoglobulin unit comprising two heavy chains, consisting of a first and a second heavy chain, and optionally two light chains; L Ｌ and L Ｒ each independently represents a linker; Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ、and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

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

[0247] When the affinity substance-modified antibody or its salt contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthogonal functional group on the immunoglobulin unit upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including conventional ester residues and other ester residues such as thioester residues), acetal residues (including conventional ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.

[0248] When the affinity substance-modified antibody or a salt thereof contains a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, the affinity substance-modified antibody or a salt thereof may be represented by the following formula (Va): [wherein Ig represents the immunoglobulin unit; L Ｌ１ and L Ｒ１ each independently represents a first linker; L Ｌ２ and L Ｒ２ each independently represents a second linker, CLE(B) Ｌ and CLE(B) Ｒ each independently represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit upon cleavage, and A Ｌ represents the first affinity substance, and A Ｒrepresents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0249] An antibody comprising a structural unit represented by formula (Va) or a salt thereof can be produced by reacting a compound represented by formula (Ia) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains or a salt thereof.

[0250] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (Va-1): [wherein 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 Ｌ３ and L Ｒ３ each independently represents a third linker; L Ｌ４ and L Ｒ４ each independently represents a fourth linker; S represents a sulfur atom; A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0251] An antibody comprising a structural unit represented by formula (Va-1) or a salt thereof can be produced by reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains or a salt thereof.

[0252] The affinity substance-modified antibody or salt thereof may also further comprise (iv') a first bioorthogonal functional group between the (ii') immunoglobulin unit and the (iii') first cleavable moiety, and / or (iv'') a second bioorthogonal functional group between the (ii'') immunoglobulin unit and the (iii'') second 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 a substituent as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue.

[0253] When the affinity substance-modified antibody or a salt thereof further comprises a bioorthogonal functional group between the immunoglobulin unit and the cleavable moiety, it has the following formula (Vb): [wherein Ig represents the immunoglobulin unit; L Ｌ５ and L Ｒ５ each independently represents a fifth linker; L Ｌ６ and L Ｒ６ each independently represents a sixth linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group, and CLE Ｌ and CLE Ｒ each independently represents a cleavable moiety; A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒand the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0254] An antibody comprising a structural unit represented by formula (Vb) or a salt thereof can be produced by reacting a compound represented by formula (Ib) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains or a salt thereof.

[0255] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (Vb-1): [wherein 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 Ｌ７ and L Ｒ７ each independently represents a seventh linker; L Ｌ８ and L Ｒ８ each independently represents an eighth linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; V Ｌ and V Ｒ each independently represents an oxygen atom or a sulfur atom, Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0256] An antibody comprising a structural unit represented by formula (Vb-1) or a salt thereof can be produced by reacting a compound represented by formula (Ib-1) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains or a salt thereof.

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

[0258] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (Vc): [wherein Ig represents the immunoglobulin unit; L Ｒ１ represents a first linker, L Ｒ２ represents a second linker, L Ｌ５ represents a fifth linker, L Ｌ６ represents the sixth linker, B Ｌ represents a group containing a first bioorthogonal functional group, and CLE Ｌ represents the first cleavable moiety, CLE(B) Ｒ represents a second cleavable moiety that can be cleaved to generate a second bioorthogonal functional group on the immunoglobulin unit; A Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0259] An antibody or a salt thereof comprising a structural unit represented by formula (Vc) can be produced by reacting a compound represented by formula (Ia) or a salt thereof, and a compound represented by formula (Ib) or a salt thereof with an antibody or a salt thereof comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains.

[0260] In certain embodiments, the affinity substance-modified antibody or salt thereof has the following formula (Vc-1): [wherein 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 each independently represents a third linker; L Ｒ４ each independently represents a fourth linker; L Ｌ７ each independently represents a seventh linker; L Ｌ８ each independently represents an eighth linker; B Ｌ represents a group containing a first bioorthogonal functional group, and V Ｌ represents an oxygen atom or a sulfur atom, Ｌ represents the first affinity substance, and A Ｒ represents the second affinity substance, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification r of said immunoglobulin units by said second modifying moiety Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0261] An antibody comprising a structural unit represented by formula (Vc-1) or a salt thereof can be produced by reacting a compound represented by formula (Ia-1) or a salt thereof, and a compound represented by formula (Ib-1) or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains or a salt thereof.

[0262] In the above formulas (V), (Va), (Va-1), (Vb), (Vb-1), (Vc), and (Vc-1), the definitions, 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" to any symbol is used for convenience to refer to the symbol positioned to the left of the immunoglobulin unit (Ig), while the subscript "R" to any symbol is used for convenience to refer to the symbol 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 the symbol 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 Is W １ and an oxygen atom is preferred. Ｌ２ and W Ｒ２ Is W ２ and an oxygen atom is preferred. Ｌ３ and W Ｒ３ Is W ３ and an oxygen atom is preferred. Ｌ and L Ｒ The linker represented by is the same as the linker represented by L. Ｌ and L Ｒ The linkers represented by may be the same or different, and are preferably different. Ｌ１ and L Ｒ１ The first linker represented by L １ The first linker is the same as the first linker represented by L Ｌ１ and L Ｒ１ The first linkers represented by L may be the same or different, and are preferably different. Ｌ２ and L Ｒ２ The second linker, represented by L ２The second linker is the same as the second linker represented by L Ｌ２ and L Ｒ２ The second linkers represented by L may be the same or different, and are preferably different. Ｌ３ and L Ｒ３ The third linker represented by L ３ The third linker is the same as that shown in L Ｌ３ and L Ｒ３ The third linkers represented by may be the same or different, and are preferably different. Ｌ４ and L Ｒ４ The fourth linker represented by L ４ The fourth linker is the same as that shown in L Ｌ４ and L Ｒ４ The fourth linkers represented by may be the same or different, and are preferably different. Ｌ５ and L Ｒ５ The fifth linker represented by L ５ The fifth linker is the same as that shown in L Ｌ５ and L Ｒ５ The first linkers represented by L may be the same or different, and are preferably different. Ｌ６ and L Ｒ６ The sixth linker represented by L ６ The sixth linker is the same as that shown in L Ｌ６ and L Ｒ６ The sixth linkers represented by L may be the same or different, and are preferably different. Ｌ７ and L Ｒ７ The seventh linker, represented by L ７ The seventh linker is the same as that shown in L Ｌ７ and L Ｒ７ The seventh linkers represented by may be the same or different, and are preferably different. Ｌ８ and L Ｒ８ The eighth linker, represented by L ８ The same as the eighth linker shown in L Ｌ８ and L Ｒ８ The eighth linkers represented by may be the same or different, and are preferably different.Ｌ and CLE(B) Ｒ The cleaving moiety represented by is the same as the cleaving moiety represented by CLE(B). Ｌ and CLE(B) Ｒ The cleavable moieties represented by may be the same or different, preferably different. Ｌ a first group comprising a first bioorthogonal functional group represented by Ｒ The second group containing the second bioorthogonal functional group represented by is the same as the group containing the bioorthogonal functional group represented by B. Ｌ a first group comprising a first bioorthogonal functional group represented by Ｒ The second groups comprising the second bioorthogonal functional group represented by CLE may be the same or different, and are preferably different. Ｌ and CLE Ｒ The cleaving moiety represented by is the same as the cleaving moiety represented by CLE. Ｌ and CLE Ｒ The cleavable moieties represented by may be the same or different, and are preferably different. Ｌ and A Ｒ The affinity substance represented by is the same as the affinity substance represented by A. Ｌ and A Ｒ The affinity substances represented by may be the same or different. Ｌ and r Ｒ The degree of average percent modification, denoted by r, and the method for determining it are similar to the average percent modification, denoted by r. Ｌ and r Ｒ The average percentages of modification indicated by may be the same or different, preferably different.

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

[0266] The affinity substance-modified antibody or its salt may further comprise an additional modifying moiety. Various methods are known for modifying antibodies. Thus, in the present invention, the affinity substance-modified antibody or its salt may be modified to further comprise an additional modifying moiety. The additional modifying moiety may be introduced into the heavy chain or light chain of the antibody, preferably into the heavy chain of the antibody (particularly into the constant region of the heavy chain).

[0267] In certain embodiments, the additional modifying moiety may be an additional affinity substance comprising a fifth affinity moiety having affinity for the constant region of the antibody heavy chain. The "affinity moiety" in the "fifth affinity moiety" and the "affinity substance" in the "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 is preferably different.

[0268] In certain embodiments, additional modifying moieties, including a fifth affinity moiety having affinity for the constant region of the antibody heavy chain, may be introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the two heavy chains. The affinity substance-modified antibody or a salt thereof can contain additional modifying 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 Fc regions or CH2 domains) of the two heavy chains in the antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, optionally, two light chains). More specifically, the position 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., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, WO 2020 / 090979). The position at which the additional modifying moiety, including the fifth affinity moiety having affinity for the antibody heavy chain constant region, is introduced is preferably different from the position at which the first modifying moiety, including the first affinity substance, including the first and second affinity moieties having affinity for the antibody heavy chain constant region, and the second modifying moiety, including the second affinity substance, including the third and fourth affinity moieties having affinity for the antibody heavy chain constant region, are introduced. For example, when the positions at which both the first modifying moiety and the second modifying moiety are introduced are lysine residues at positions 246 / 248, the position at which the additional modifying moiety is introduced is preferably lysine residues at positions 288 / 290 or 317, more preferably lysine residues at positions 288 / 290. When the positions at which both the first modifying moiety and the second modifying moiety are introduced are lysine residues at positions 288 / 290, the position at which the additional modifying moiety is introduced is preferably lysine residues at positions 246 / 248 or 317, more preferably lysine residues at positions 246 / 248.When the position at which both the first modifying moiety and the second modifying moiety are introduced is the lysine residue at position 317, the position at which the additional modifying moiety is introduced is preferably the lysine residue at positions 246 / 248 or 288 / 290.

[0269] 5. Affinity Substance-Free Antibodies or Salts Thereof 5-1. Overview An affinity substance-free antibody or salt thereof can be produced using an affinity substance-modified antibody or salt thereof that includes (A) an affinity substance containing 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 further includes (C) a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

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

[0271] (Method 1-1) A method for producing an affinity substance-free antibody or its salt, 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 its salt, which further comprises a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit), with the cleavable moiety, to produce an affinity substance-free antibody or its salt.

[0272] (Method 1-2) A method for producing an affinity substance-free antibody or a salt thereof, comprising the following steps (1) and (2): (1) reacting (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) a compound or salt thereof comprising a reactive group for an antibody (immunoglobulin unit) and further comprising (C) a cleavable moiety between (A) the affinity substance and (B) the reactive group, with an antibody or salt thereof comprising an immunoglobulin unit comprising two heavy chains and, optionally, two light chains, to produce an affinity substance-modified antibody or salt thereof comprising a cleavable moiety between the affinity substance and the antibody; and (2) cleaving the affinity substance-modified antibody or salt thereof comprising a cleavable moiety between the affinity substance and the antibody with the cleavable moiety to produce an affinity substance-free antibody or salt thereof.

[0273] Examples of 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 cleavable linker containing a self-degrading cleavable moiety. For these cleavage treatments, see International Publication Nos. WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, and WO 2020 / 090979.

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

[0275] The production of an affinity substance-free antibody or a salt thereof obtained by the cleavage reaction can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Regioselectivity can be confirmed by peptide mapping as described above. The number of affinity substances introduced can be confirmed by mass spectrometry (which can be performed in combination with DAR Calculator (Agilent software)). The affinity substance-modified antibody or a salt thereof can be appropriately purified by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

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

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

[0278] As antibodies or salts thereof that do not contain an affinity substance, (1) antibody derivatives or salts thereof that contain a bioorthogonal functional group, and (2) conjugates or salts thereof of an antibody and a functional substance will be described in detail below.

[0279] 5-2. Antibody Derivatives or Salts Comprising at Least One Bioorthogonal Functional Group The present invention provides antibody derivatives or salts thereof comprising (a) an antibody building block (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 into the constant region of one heavy chain in the immunoglobulin unit (i.e., the bioorthogonal functional group is introduced into the constant region of one heavy chain in the immunoglobulin unit, and the bioorthogonal functional group is not introduced into the constant region of the other heavy chain). The definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, as well as the components (e.g., constant regions) that make them up, are as described above.

[0280] The antibody derivative or salt thereof can contain a bioorthogonal functional group via modification of a functional group in the side chain of one or more (e.g., two, three, or four) of the 14 amino acid residues present in the constant region (preferably the Fc region or CH2 domain): asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. The antibody derivative or salt thereof can contain a 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 position of an antibody or salt thereof with a bioorthogonal functional group can be confirmed by peptide mapping. The modification may be site-selective, as described above. Therefore, in the formulae (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit may have the corresponding modifying unit site-selectively via a functional group in the side chain of the amino acid residue.

[0281] Preferably, the antibody derivative can contain a bioorthogonal functional group 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 constant region (preferably the Fc region or CH2 domain) of one of the heavy chains in the antibody building block (an immunoglobulin unit comprising two heavy chains and, optionally, two light chains) (in other words, the antibody derivative contains a bioorthogonal functional group via the amino group in the side chain of a lysine residue in the constant region of one of the heavy chains in the immunoglobulin unit, but does not contain a bioorthogonal functional group via the amino group in the side chain of a lysine residue in the constant region of the other heavy chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979). The modification may be regioselective, as described above. Thus, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit may regioselectively have the corresponding modifying unit via the amino group in the side chain of the lysine residue.

[0282] In certain embodiments, the antibody derivative or salt thereof has the following formula (IIIa): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; L １ represents a first linker, B represents a group containing a bioorthogonal functional group, and the average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%. １ The definitions, examples, and preferred examples of the first linker represented by B, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the antibody, are as described above. A particularly preferred bioorthogonal functional group is a thiol group.

[0283] In formula (IIIa), L １ The molecular weight of the partial structure represented by -B may be 700 or less. １ When the molecular weight of the partial structure represented by -B is 700 or less, the ratio of the molecular weight of the partial structure to the molecular weight of the entire antibody is very small, making purification based on differences in molecular weight relatively difficult for antibody derivatives or salts thereof having a bioorthogonal functional group. However, according to the present invention, which enables advanced control of DAR, it is possible to obtain highly purified antibody derivatives exhibiting the desired DAR without necessarily requiring purification based on differences in molecular weight. １ The molecular weight of the partial structure represented by -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 salt thereof has the following formula (IIIa-1): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; W １ represents an oxygen atom or a sulfur atom, L ３ represents a third linker, SH represents a thiol group, and the average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%. １ An atom represented by L ３ The definitions, examples, and preferred examples of the third linker, denoted by , and the average modification percentage, denoted by r, and the antibody are as described above.

[0285] In formula (IIIa-1), C(=W １ )-L ３ The molecular weight of the partial structure represented by -SH may be 700 or less. C(=W １ )-L ３The molecular weight of the partial structure represented by —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 formula (IIIa-1), L ３ The third linker represented by (CH ２ ) ｎ１ n1 may be an integer of 1 to 10. Preferably, n1 may be an integer of 2 or greater. n1 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Particularly preferably, n1 is 2.

[0287] In yet another specific embodiment, the antibody derivative or salt thereof has the following formula (IIIb): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; L ５ represents a fifth linker, B represents a group containing a bioorthogonal functional group, and T １ represents a monovalent group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.]. The immunoglobulin unit represented by Ig, L ５ The definitions, examples, and preferred examples of the fifth linker represented by B, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the antibody, are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[0288] T １ is a monovalent group and can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of the monovalent group include those described above. When the monovalent group is substituted, examples of the substituent include those described above.

[0289] In certain embodiments, T １The monovalent group represented by the formula (α) may be an optionally substituted hydroxyamino group. The optionally substituted hydroxyamino group can be represented by the following formula (α): NR ｉ -OR ｉｉ (α) [wherein, R ｉ , R ｉｉ are each independently 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 optionally substituted hydroxyamino group is NH-OR ｉｉ (where R ｉｉ represents an alkyl group.) More preferably, the optionally substituted hydroxyamino group is ｉｉ (where R ｉｉ represents an alkyl group having 1 to 6 carbon atoms.

[0290] In formula (IIIb), L ５ (-B)-T １ The molecular weight of the partial structure represented by may be 700 or less. ５ (-B)-T １ The molecular weight of the partial structure represented by the formula (I) 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 salt thereof has the following formula (IIIb-1): where Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains; W １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, B represents a group containing a bioorthogonal functional group, and T ２represents a monovalent group, and the percentage modification r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%. The immunoglobulin unit may be an antibody or a salt thereof comprising a structural unit represented by the following formula: １ and W ２ An atom represented by L ７ The definitions, examples, and preferred examples of the seventh linker represented by B, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the antibody, are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[0292] T ２ is a monovalent group that can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of the monovalent group include those described above. When the monovalent group is substituted, examples of the substituent include those described above. T ２ The monovalent group represented by the formula may be an optionally substituted hydroxyamino group. Details of the optionally substituted hydroxyamino group are given in T １ It is similar to what was stated above.

[0293] In formula (IIIb-1), C(=W １ )-L ７ (-B)-C (=W ２ )-T ２ The molecular weight of the partial structure represented by C(=W １ )-L ７ (-B)-C (=W ２ )-T ２ The molecular weight of the partial structure represented by the formula (I) 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 formula (IIIb-1), L ７ The seventh linker represented by (CH ２ ) ｎ２n2 may be an integer of 1 to 10. Preferably, n2 may be an integer of 2 or greater, or 3 or greater. n2 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less. Particularly preferably, n2 may be 3.

[0295] In formula (IIIb-1), the group containing a bioorthogonal functional group is NH—C(═O)—(CH ２ ) ｎ３ -N ３ n3 may be an integer of 1 to 10. Preferably, n3 may be an integer of 2 or more, 3 or more, or 4 or more. n3 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. Particularly preferably, n3 may be 4.

[0296] The antibody derivative or salt thereof containing a bioorthogonal functional group can be produced using an affinity substance-modified antibody or salt thereof that contains (A) an affinity substance containing 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 further contains (C) a cleavable moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

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

[0298] More specifically, such production methods include, for example, the following (2-1) to (2-6) (FIGS. 2 to 6).

[0299] (Method 2-1) A method for producing an antibody derivative or salt thereof 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 thereof further comprising a cleavable moiety (here, the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side upon cleavage) between (A) the affinity substance and (B) the antibody (immunoglobulin unit), with the cleavable moiety to produce an antibody derivative or salt thereof containing a bioorthogonal functional group.

[0300] (Method 2-2) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) reacting (A) an affinity substance containing first and second affinity moieties having affinity for a constant region in the heavy chain of an antibody (immunoglobulin unit), and (B) a group reactive with the antibody (immunoglobulin unit), and (C) a compound or salt thereof further containing a cleavable moiety (wherein the cleavable moiety is capable of generating a bioorthogonal functional group on the antibody (immunoglobulin unit) reactive group side upon cleavage) between (A) the affinity substance and (B) the reactive group, with an antibody or salt thereof containing immunoglobulin units including two heavy chains and, if necessary, two light chains, to produce an affinity substance-modified antibody or a salt thereof containing a cleavable moiety (wherein the cleavable moiety is capable of generating a bioorthogonal functional group on the antibody side upon cleavage) between the affinity substance and the antibody; and (2) An affinity substance-modified antibody or its salt containing a cleavable moiety between the affinity substance and the antibody is cleaved with the cleavable moiety to produce an antibody derivative or its salt containing a bioorthogonal functional group.

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

[0302] (Method 2-4) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following steps (1) and (2): (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, if necessary, two light chains, to produce an antibody or a salt thereof containing a structural unit represented by formula (IIa); and (2) cleaving the antibody or a salt thereof containing a structural unit represented by formula (IIa) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIa).

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

[0304] (Method 2-6) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following steps (1) and (2): (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, if necessary, two light chains, to produce an antibody or a salt thereof containing a structural unit represented by formula (IIa-1); and (2) cleaving the antibody or a salt thereof containing a structural unit represented by formula (IIa-1) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIa-1).

[0305] The above method 2-1 may be carried out by the above method 2-3 or 2-5. The above method 2-2 may be carried out by the above method 2-4 or 2-6. The above methods 2-2, 2-4, and 2-6 may further comprise reacting the affinity substance of the present invention with a moiety containing a group reactive to an antibody to produce the compound of the present invention or a salt thereof (FIGS. 2 to 6).

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

[0307] More specifically, examples of such production methods include the following (2-7) to (2-12) (FIGS. 2 to 6).

[0308] (Method 2-7) A method for producing an antibody derivative or salt thereof 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 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; and (C) an affinity substance-modified antibody or salt thereof, with the cleavable moiety to produce an antibody derivative or salt thereof containing a bioorthogonal functional group.

[0309] (Method 2-8) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) reacting an affinity substance containing first and second affinity moieties having affinity for the constant region in 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, and (D) a bioorthogonal functional group between the reactive group and the cleavable moiety, or a salt thereof, with an antibody or a salt thereof containing immunoglobulin units containing two heavy chains and, optionally, two light chains, to produce an affinity substance-modified antibody or a salt thereof containing a cleavable moiety between the affinity substance and the antibody, and (D) a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety; and (2) cleaving the affinity substance-modified antibody or a salt thereof containing a cleavable moiety between the affinity substance and the antibody with the cleavable moiety, to produce an antibody derivative or a salt thereof containing a bioorthogonal functional group.

[0310] (Method 2-9) A method for producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIb) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb) above.

[0311] (Method 2-10) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following steps (1) and (2): (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, if necessary, two light chains, to produce an antibody or a salt thereof containing a structural unit represented by formula (IIb); and (2) cleaving the antibody or a salt thereof containing a structural unit represented by formula (IIb) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIb).

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

[0313] (Method 2-12) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following steps (1) and (2): (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, if necessary, two light chains, to produce an antibody or a salt thereof containing a structural unit represented by formula (IIb-1); and (2) cleaving the antibody or a salt thereof containing a structural unit represented by formula (IIb-1) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIb-1).

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

[0315] The antibody derivative or its salt may further comprise an additional modifying moiety. Various methods are known for modifying antibodies. Thus, in the present invention, the antibody derivative or its salt may be modified to further comprise an additional modifying moiety. The additional modifying moiety may be introduced into the heavy chain or light chain of the antibody, preferably into the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0316] In certain embodiments, the additional modifying moiety may be an additional modifying moiety that includes a bioorthogonal functional group. The bioorthogonal functional group is the same as that described above. The bioorthogonal functional group contained in the additional modifying moiety may be the same as or different from the bioorthogonal functional group described in (b) above, but is preferably different.

[0317] In certain embodiments, additional modifying 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 present at one or more positions in the constant regions of the two heavy chains. The antibody derivative or its salt can include additional modifying 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 Fc regions or CH2 domains) of the two heavy chains in an antibody building block (an immunoglobulin unit containing two heavy chains and, optionally, two light chains). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, WO 2020 / 090979). The position at which the additional modifying moiety containing the bioorthogonal functional group is introduced is preferably different from the position at which the bioorthogonal functional group in (b) above is introduced. For example, when the position where the bioorthogonal functional group (b) is introduced is a lysine residue at position 246 / 248, the position where the additional modifying moiety is introduced is preferably a lysine residue at position 288 / 290 or 317, more preferably a lysine residue at position 288 / 290. When the position where the bioorthogonal functional group (b) is introduced is a lysine residue at position 288 / 290, the position where the additional modifying moiety is introduced is preferably a lysine residue at position 246 / 248 or 317, more preferably a lysine residue at position 246 / 248. When the position where the bioorthogonal functional group (b) is introduced is a lysine residue at position 317, the position where the additional modifying moiety is introduced is preferably a lysine residue at position 246 / 248 or 288 / 290.

[0318] 5-3. Antibody Derivatives or Salts Comprising At Least Two Bioorthogonal Functional Groups The present invention also provides antibody derivatives or salts thereof comprising: (a) an immunoglobulin unit comprising two heavy chains, a first and a second heavy chain, and optionally two light chains; and (b) a first modifying moiety comprising a first bioorthogonal functional group and a second modifying moiety comprising a second bioorthogonal functional group; (c) the first modifying moiety is introduced into the constant region of the first heavy chain; (d) the second modifying moiety is introduced into the constant region of the second heavy chain; and (e) the first and second modifying moieties are different from each other. The definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, as well as the components (e.g., constant regions) that constitute them, are as described above.

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

[0320] Preferably, the antibody derivative can comprise a first modifying moiety containing a first bioorthogonal functional group and a second modifying moiety containing a second bioorthogonal functional group, respectively, 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 constant region (preferably the Fc region or CH2 domain) of one of the heavy chains in the antibody building block (an immunoglobulin unit comprising two heavy chains and, optionally, two light chains). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be 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 below, the immunoglobulin unit may have the corresponding modifying unit regioselectively via the amino group in the side chain of the lysine residue.

[0321] In certain embodiments, the antibody derivative or salt thereof has the following formula (VIa): [wherein Ig represents the immunoglobulin unit; L Ｌ１ and L Ｒ１ each independently represents a first linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group, and the average percentage modification of said immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0322] The antibody or its salt containing a structural unit represented by formula (VIa) can be produced by the following method: Ｌ and CLE(B) ＲThe cleavage reaction can be carried out by cleaving two cleavable moieties represented by the formula: When the two cleavable moieties are the same, the cleavage reaction can be carried out in a single cleavage reaction. When the two cleavable moieties are different, the cleavage reaction can be carried out in a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable by the same cleavage treatment or cleavage agent), or in two cleavage reactions (e.g., when the two different cleavable moieties are cleavable by different cleavage treatments or cleavage agents). Details of the cleavage reaction are as described above.

[0323] In certain embodiments, the antibody derivative or salt thereof has the following formula (VIa-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｒ１ each independently represents an oxygen atom or a sulfur atom, L Ｌ３ and L Ｒ３ each independently represents a third linker, SH represents a thiol group that is a bioorthogonal functional group, and the average modification percentage r of the immunoglobulin unit with the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0324] An antibody or a salt thereof comprising a structural unit represented by formula (VIa-1) can be produced by cleaving the two cleavable moieties represented by C-S in an affinity substance-modified antibody or a salt thereof comprising a structural unit represented by formula (Va-1). The cleavage reaction can be carried out in a single cleavage reaction. Details of the cleavage reaction are as described above.

[0325] In certain embodiments, the antibody derivative or salt thereof has the following formula (VIb): [wherein Ig represents the immunoglobulin unit; L Ｌ５ and L Ｒ５ each independently represents a fifth linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒrepresents a second group comprising a second bioorthogonal functional group; T Ｌ１ and T Ｒ１ each independently represents a monovalent group, and the average modification percentage r of the immunoglobulin units by the first modifying moiety Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0326] The antibody or its salt containing a structural unit represented by formula (VIb) can be produced by the following method: Ｌ and CLE Ｒ The cleavage reaction can be carried out by cleaving two cleavable moieties represented by the formula: When the two cleavable moieties are the same, the cleavage reaction can be carried out in a single cleavage reaction. When the two cleavable moieties are different, the cleavage reaction can be carried out in a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable by the same cleavage treatment or cleavage agent), or in two cleavage reactions (e.g., when the two different cleavable moieties are cleavable by different cleavage treatments or cleavage agents). Details of the cleavage reaction are as described above.

[0327] In certain embodiments, the antibody derivative or salt thereof has the following formula (VIb-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｌ２ , and W Ｒ１ and W Ｒ２ each independently represents an oxygen atom or a sulfur atom, L Ｌ７ and L Ｒ７ each independently represents a seventh linker; B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; T Ｌ２ and T Ｒ２ each independently represents a monovalent group, and the average modification percentage r of the immunoglobulin units by the first modifying moiety Ｌand the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0328] The antibody or salt thereof containing the structural unit represented by formula (VIb-1) can be produced by: Ｌ and C.V. Ｒ The cleavage reaction can be carried out by cleaving two cleavable moieties represented by the formula: When the two cleavable moieties are the same, the cleavage reaction can be carried out in a single cleavage reaction. When the two cleavable moieties are different, the cleavage reaction can be carried out in a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable by the same cleavage treatment or cleavage agent), or in two cleavage reactions (e.g., when the two different cleavable moieties are cleavable by different cleavage treatments or cleavage agents). Details of the cleavage reaction are as described above.

[0329] In certain embodiments, the antibody derivative or salt thereof has the following formula (VIc): [wherein Ig represents the immunoglobulin unit; L Ｒ１ represents a first linker, L Ｌ５ represents the fifth linker, B Ｌ represents a first group comprising a first bioorthogonal functional group; B Ｒ represents a second group comprising a second bioorthogonal functional group; T Ｌ１ represents a monovalent group, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0330] The antibody or salt thereof comprising a structural unit represented by formula (VIc) can be produced by the following method: Ｌ and CLE(B) ＲThe cleavage reaction can be carried out by cleaving two cleavable moieties represented by the formula: When the two cleavable moieties are the same, the cleavage reaction can be carried out in a single cleavage reaction. When the two cleavable moieties are different, the cleavage reaction can be carried out in a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable by the same cleavage treatment or cleavage agent), or in two cleavage reactions (e.g., when the two different cleavable moieties are cleavable by different cleavage treatments or cleavage agents). Details of the cleavage reaction are as described above.

[0331] In certain embodiments, the antibody derivative or salt thereof has the following formula (VIc-1): [wherein Ig represents the immunoglobulin unit; W Ｌ１ and W Ｌ２ , and W Ｒ１ each independently represents an oxygen atom or a sulfur atom, L Ｒ３ represents a third linker, and L Ｌ７ represents the seventh linker, B Ｌ represents a first group containing a first bioorthogonal functional group; SH represents a thiol group, which is a second bioorthogonal functional group; T Ｌ２ represents a monovalent group, and the average percentage modification of the immunoglobulin units by the first modifying moiety, r Ｌ and the average percentage modification of said immunoglobulin units by the second modifying moiety, r Ｒ and the β-glucan bond strength is 65 to 135%, respectively.] may also be an antibody or a salt thereof.

[0332] The antibody or salt thereof containing the structural unit represented by formula (VIc-1) can be produced by: Ｌand C-S. When the two cleavable moieties are the same, the cleavage reaction can be carried out in a single cleavage reaction. When the two cleavable moieties are different, the cleavage reaction can be carried out in a single cleavage reaction (e.g., when the two different cleavable moieties are cleavable by the same cleavage treatment or cleavage agent), or in two cleavage reactions (e.g., when the two different cleavable moieties are cleavable by different cleavage treatments or cleavage agents). Details of the cleavage reaction are as described above.

[0333] In the above formulas (VIa), (VIa-1), (VIb), (VIb-1), (VIc), and (VIc-1), the definitions, examples, and preferred examples of the immunoglobulin unit represented by Ig and the antibody are as described above.

[0334] In the above formulas (VIa), (VIa-1), (VIb), (VIb-1), (VIc), and (VIc-1), the symbols with the subscripts "L" and "R" are as follows: W Ｌ１ and W Ｒ１ Is W １ and an oxygen atom is preferred. Ｌ２ and W Ｒ２ Is W ２ and an oxygen atom is preferred. Ｌ１ and L Ｒ１ The first linker represented by L １ The first linker is the same as the first linker represented by L Ｌ１ and L Ｒ１ The first linkers represented by L may be the same or different, and are preferably different. Ｌ３ and L Ｒ３ The third linker represented by L ３ The third linker is the same as that shown in L Ｌ３ and L Ｒ３ The third linkers represented by may be the same or different, and are preferably different. Ｌ５ and L Ｒ５ The fifth linker represented by L ５ The fifth linker is the same as that shown in L Ｌ５ and LＲ５ The first linkers represented by L may be the same or different, and are preferably different. Ｌ７ and L Ｒ７ The seventh linker, represented by L ７ The seventh linker is the same as that shown in L Ｌ７ and L Ｒ７ The seventh linkers represented by may be the same or different, and are preferably different. Ｌ a first group comprising a first bioorthogonal functional group represented by Ｒ The second group containing the second bioorthogonal functional group represented by is the same as the group containing the bioorthogonal functional group represented by B. Ｌ a first group comprising a first bioorthogonal functional group represented by Ｒ The second groups comprising the second bioorthogonal functional group represented by T may be the same or different, and are preferably different. Ｌ１ and T Ｒ１ The monovalent group represented by １ It is the same as the monovalent group represented by the formula: T Ｌ１ and T Ｒ１ The monovalent groups represented by may be the same or different. Ｌ２ and T Ｒ２ The monovalent group represented by ２ It is the same as the monovalent group represented by the formula: T Ｌ２ and T Ｒ２ The monovalent groups represented by may be the same or different. Ｌ and r Ｒ The degree of average percent modification, denoted by r, and the method for determining it are similar to the average percent modification, denoted by r. Ｌ and r Ｒ The average percentages of modification indicated by may be the same or different, preferably different.

[0335] In certain embodiments, in formulas (VIa) and (VIc), L Ｌ１ -B Ｌ , and / or L Ｒ１ -B Ｒ The molecular weight of the partial structure represented by １It may be the same as the molecular weight of the partial structure represented by -B.

[0336] In certain embodiments, in formulas (VIa-1) and (VIc-1), C(=W Ｌ１ )-L Ｌ３ -SH, and / or C(=W Ｒ１ )-L Ｒ３ The molecular weight of the partial structure represented by —SH is determined by the C(═W) in formula (IIIa-1). １ )-L ３ It may be the same as the above molecular weight of the partial structure represented by —SH.

[0337] In certain embodiments, in formulas (VIa-1) and (VIc-1), L Ｌ３ , and / or L Ｒ３ The third linker represented by the formula (I...

Claims

1. A compound or a salt thereof, comprising: (A) an affinity substance containing first and second affinity moieties having an affinity for a constant region in a heavy chain of an antibody; and (B) a reactive group for an antibody.

2. The compound or a salt thereof according to claim 1, wherein the constant region is an Fc region.

3. The compound or a salt thereof according to claim 1, wherein the constant region is a CH2 domain.

4. The compound or a salt thereof according to claim 1, wherein the constant region is a human constant region.

5. The compound or a salt thereof according to claim 1, wherein the antibody is IgG.

6. The compound or a salt thereof according to claim 1, wherein the first and second affinity moieties are different affinity moieties.

7. The affinity substance is represented by the following formula (A): AP1-L Ａ -AP2 (A) 〔wherein, AP1 represents a first affinity peptide having an affinity for a constant region in a heavy chain of an antibody, AP2 represents a second affinity peptide having an affinity for a constant region in a heavy chain of an antibody. L Ａ represents a linker.〕 The compound according to claim 1 or a salt thereof, represented by

8. The compound or a salt thereof according to claim 7, wherein the affinity substance (i) contains only one specific reactive group, and (ii) is linked to a reactive group for an antibody via the specific reactive group.

9. The compound or a salt thereof according to claim 1, wherein the affinity substance is an affinity polypeptide containing first and second affinity peptides having an affinity for a constant region in a heavy chain of an antibody.

10. The affinity polypeptide is represented by the following formula (A'): AP1-PL Ａ -AP2 (A') 〔wherein, AP1 represents a first affinity peptide having an affinity for a constant region in a heavy chain of an antibody and present on the N-terminal side of the affinity polypeptide, AP2 represents a second affinity peptide having an affinity for a constant region in a heavy chain of an antibody and present on the C-terminal side of the affinity polypeptide. PL Ａ represents a peptide linker.〕 The compound according to claim 9 or a salt thereof, represented by

11. The compound or a salt thereof according to claim 10, wherein the affinity polypeptide (i) contains only one amino acid residue having an amino group in a side chain, and (ii) is linked to a reactive group for an antibody via the amino group, or (ii) is linked to a reactive group for an antibody via an N-terminal amino group in the first affinity peptide.

12. The compound or a salt thereof according to claim 11, wherein the amino acid residue having an amino group in a side chain is a lysine residue.

13. The compound or a salt thereof according to claim 10, wherein the affinity polypeptide further contains, at the N-terminus, a tripeptide consisting of Gln-Glu-Thr (QET).

14. The compound or a salt thereof according to claim 10, wherein the peptide linker has a length consisting of 20 or more amino acid residues.

15. One of the first and second affinity polypeptides is an affinity polypeptide having an affinity for the constant region in the heavy chain of an antibody and having one lysine residue, and The other of the first and second affinity polypeptides is an affinity polypeptide having an affinity for the constant region in the heavy chain of an antibody and having no lysine residue, the compound or a salt thereof according to claim 1.

16. The affinity polypeptide having an affinity for the constant region in the heavy chain of an antibody and having one lysine residue is as follows (1) to (4): (1) An affinity polypeptide containing the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (2) An affinity polypeptide containing an amino acid sequence in which one or two amino acid residues other than the lysine residue and the cysteine residue in the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38) are substituted with another amino acid residue other than the lysine residue and the cysteine residue, and having an affinity for the constant region in the heavy chain of an antibody; (3) An affinity polypeptide containing the amino acid sequence (Z34CK) of FNKQCQRRFYERALHDPNLNEEQRNARIRSIREEEC (SEQ ID NO: 39); and (4) An affinity polypeptide containing an amino acid sequence in which one or two amino acid residues other than the lysine residue and the cysteine residue in the amino acid sequence of FNKQCQRRFYERALHDPNLNEEQRNARIRSIREEEC (SEQ ID NO: 39) are substituted with another amino acid residue other than the lysine residue and the cysteine residue, and having an affinity for the constant region in the heavy chain of an antibody) (wherein the two cysteine residues contained in the amino acid sequence may be cross-linked by a disulfide bond), and / or The affinity polypeptide having an affinity for the constant region in the heavy chain of an antibody and having no lysine residue is as follows (5) to (10): (5) An affinity polypeptide containing the amino acid sequence (Z34CM) of FNMQCQRRFYERALHDPNLNEEQRNARIRSIREEEC (SEQ ID NO: 40); ​ (6) 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 FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEEC (SEQ ID NO: 40) 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; (7) An affinity peptide comprising the amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSQLRDDPSQSANLLAEA (SEQ ID NO: 41) (ProAR); (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 FNREQQNAFYEILHLPNLNEEQRNGFIQSQLRDDPSQSANLLAEA (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; The compound or a salt thereof according to claim 15, which is any one of the above (wherein the two cysteine residues contained in the amino acid sequence may be cross-linked by a disulfide bond).

17. The compound is represented by the following formula (I): 【Chemical 1】 [In the formula, R represents the reactive group, L represents a linker, A represents the affinity substance. ] The compound or a salt thereof according to claim 1.

18. The compound or a salt thereof according to claim 1, further comprising (iii) a cleavable moiety between (i) the affinity substance and (ii) the reactive group.

19. The compound or a salt thereof according to claim 18, wherein the cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side by cleavage.

20. The compound is represented by the following formula (Ia): 【Chemical 2】 [In the formula, R represents the reactive group, L １ represents the first linker, L ２ represents the second linker, CLE(B) is a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side by cleavage indicating minutes, A represents the affinity substance.〕 The compound according to claim 19 or a salt thereof.

21. The compound is represented by the following formula (Ia-1): [Chemical Formula 3] 〔In the formula, X represents a leaving group, W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ represents the third linker, L ４ represents the fourth linker, S represents a sulfur atom, A represents the affinity substance.〕 The compound according to claim 19 or a salt thereof.

22. The leaving group is selected from the following, the compound according to claim 21 or a salt thereof: (a) R Ａ - S (wherein 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 (wherein 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 (wherein R Ａ and R Ｂ each independently represents 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.

23. The compound or a salt thereof further comprises a bioorthogonal functional group (iv) between (ii) the reactive group and (iii) the cleavable moiety, the compound according to claim 1 or a salt thereof.

24. The compound is represented by the following formula (Ib): 【Chemical 4】 〔In the formula, R represents the reactive group, L ５ represents the fifth linker, L ６ represents the sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, A represents the affinity substance.〕 The compound according to claim 23 or a salt thereof.

25. The compound is represented by the following formula (Ib-1): 【Chemical Formula 5】 〔In the formula, X represents a leaving group, W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ represents the seventh linker, L ８ represents the eighth 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 compound according to claim 23 or a salt thereof.

26. The bioorthogonal functional group is an azide residue, an alkyne residue, a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue, the compound according to claim 23 or a salt thereof.

27. A reagent for antibody derivatization, comprising the compound according to any one of claims 1 to 26 or a salt thereof.

Images