Affinity substance, compound, antibody, and salts thereof

Abstract

The present invention relates to a technique for easily chemically modifying only one heavy chain of constituent units of an antibody (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains). More specifically, the present invention relates to a compound or a salt thereof, comprising (A) an affinity substance comprising a first affinity moiety and a second affinity moiety having an affinity for a constant region of an antibody heavy chain, and (B) an antibody-reactive group.

Description

Technical Field

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

[0002] In recent years, research and development of antibody-drug conjugates (ADCs) has been actively progressing. As the name suggests, ADCs are pharmaceutical agents that combine a drug (e.g., an anticancer drug) with an antibody, and they exhibit direct cytotoxic activity against cancer cells. A representative ADC is T-DM1 (trade name: Kadcyla (registered trademark)), jointly developed by Immunogene and Roche.

[0003] ADCs, such as T-DM1, have faced heterogeneity issues since their early stages of development. For example, because small molecule drugs are randomly reacted with the approximately 70 to 80 lysine residues present in antibodies, the drug-antibody ratio (DAR) and conjugation sites are not constant. It is known that using this random conjugation method typically results in a DAR range of 0 to 8, resulting in a variety of antibody agents with varying numbers of drug binding. In recent years, it has been reported that altering the number and binding sites of drugs in an ADC can alter in vivo kinetics, drug release rate, and efficacy. Based on these findings, next-generation ADCs require control over the number and sites of conjugated drugs. It is believed that maintaining a constant number and sites will address the issues of desired efficacy, drug diversity, and batch variability, often referred to as regulation.

[0004] Research into site-selective antibody modification is underway globally, but most approaches rely on genetic engineering or enzyme-based modification. While genetic engineering allows for control of site and number selectivity, it has been noted to pose challenges such as reduced antibody expression efficiency (reduced overall yield when formulating ADCs). Furthermore, the construction of antibody expression systems can be time-consuming.

[0005] Recently, a C-CAP (Chemical conjugation by Affinity Peptide, based on chemical coupling of affinity peptides) method has been developed (Patent Document 1), which can be used to selectively modify the site of antibodies by chemical synthesis. This method successfully performed site-selective modification of antibodies by reacting a peptide reagent formed by linking an NHS activated ester and a drug to an affinity peptide (Affinity Peptide). However, in the case of ADCs produced by this method, the antibody and the drug are combined via a linker containing a peptide portion. The peptide portion has potential immunogenicity and is easily hydrolyzed in the blood. Therefore, there is room for improvement in the inclusion of a peptide portion in the linker of the ADC produced by this method.

[0006] As an improved method of the above-mentioned C-CAP method, the following technology has been reported: by using a chemical synthesis method of a predetermined compound containing an affinity peptide, an antibody that does not contain a peptide portion as a linker and site-selectively has a functional substance (e.g., a drug) can be prepared (Patent Documents 2 to 5). In addition, Patent Document 6 discloses the following technology: in the preparation of a compound containing an affinity peptide, by utilizing an affinity peptide containing glutamine-glutamic acid-threonine (QET) at the N-terminus, affinity peptides can be produced in large quantities and simply. In clinical applications, it is desirable to avoid the use of linkers containing peptide portions. Among these technologies, multiple sites corresponding to various amino acid residues (e.g., lysine residues, tyrosine residues, serine residues, and threonine residues) in the CH2 and CH3 domains have been proposed as sites of amino acid residues in antibodies that can be site-selectively modified by drugs. However, it is not necessarily easy to site-selectively modify an antibody with a functional substance and control the binding ratio of the antibody to the functional substance within the desired range. In particular, it is not easy to control so that only one heavy chain in the constituent unit of the antibody (an immunoglobulin unit containing two heavy chains) is specifically modified.

[0007] However, Patent Document 6 discloses: (i) a peptide molecule comprising a first physiologically active peptide site and a second physiologically active peptide site for proteins 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, which can regulate the activity of the protein by non-covalently binding to the protein; and (ii) as a peptide linker, a peptide linker of a specified length comprising an amino acid sequence consisting of amino acid residues of proline (P), alanine (A) and serine (S) can be used.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. 2016 / 186206;

[0011] Patent Document 2: International Publication No. 2018 / 199337;

[0012] Patent Document 3: International Publication No. 2019 / 240287;

[0013] Patent Document 4: International Publication No. 2019 / 240288;

[0014] Patent Document 5: International Publication No. 2020 / 090979;

[0015] Patent document 6: International Publication No. 2021 / 112249. Summary of the Invention

[0016] Problems to be solved by the invention

[0017] An object of the present invention is to develop a technology that can easily chemically modify only one heavy chain of an antibody constituent unit (in other words, an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains).

[0018] Another object of the present invention is to develop an antibody that can be easily and site-selectively modified by chemically modifying only one heavy chain of an antibody's constituent units.

[0019] Means for solving problems

[0020] The present inventors conducted intensive studies and found that chemical modification of only one heavy chain of an antibody constituent unit can be easily performed by using a compound or a salt thereof, wherein the compound or salt comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain, and (B) an antibody-reactive group.

[0021] The compound of the present invention or its salt can associate with two heavy chains in the constituent unit of the antibody via an affinity substance (A) comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain, and then specifically react with the side chain of a specific amino acid residue in one of the heavy chains in the constituent unit of the antibody via an antibody reactive group (R), thereby generating an affinity substance-modified antibody or its salt ( Figure 1Although the present invention does not wish to be bound by theory, the mechanism by which the compound of the present invention or its salt modifies only one heavy chain in the constituent unit of the antibody is as follows. Since the affinity substance contained in the compound of the present invention or its salt (comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain) can stably associate with the constant regions of the two heavy chains in the constituent unit of the antibody, the reactive group contained in the compound of the present invention or its salt can modify only one heavy chain ( Figure 1 ). At this time, since the constant region of the other heavy chain (the constant region of the unmodified heavy chain) is associated with the affinity portion and produces steric hindrance, other molecules (the compound of the present invention or its salt) cannot associate with the constant region of the other heavy chain via the affinity substance contained therein. The compound of the present invention or its salt can associate with the constant region of the two heavy chains via the two affinity portions contained in the affinity substance, and thus can stably associate with the constituent units of the antibody, thereby highly inhibiting the association of other molecules with the constituent units of the antibody. Therefore, the compound of the present invention or its salt can highly inhibit the modification of the constant region of the other heavy chain, so only the constant region of one heavy chain can be modified ( Figure 1 ).

[0022] Patent Documents 1 to 5 disclose that antibodies can be site-selectively modified with functional substances by using a compound containing an affinity substance and an antibody-reactive group. However, they neither describe nor suggest: (1) the problem of developing a technology that can easily chemically modify only one heavy chain in the constituent units of an antibody, and (2) the use of a substance containing a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain as an affinity substance (particularly, the technical concept of chemically modifying only one heavy chain in the constituent units of an antibody by using a compound containing the affinity substance and an antibody-reactive group or a salt thereof).

[0023] Patent Document 6 discloses that a peptide molecule comprising the above-mentioned first physiologically active peptide site and second physiologically active peptide site for a protein and a predetermined peptide linker can regulate the activity of the protein by non-covalently binding to the protein, but does not disclose or suggest: (1) the problem of developing a technology that can easily chemically modify only one heavy chain of an antibody constituent unit, and (2) the use of a substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain as an affinity substance (in particular, the technical idea of ​​chemically modifying only one heavy chain of an antibody constituent unit by using a compound comprising the affinity substance and an antibody reactive group or a salt thereof).

[0024] The present inventors have also successfully produced antibodies in which only one of the heavy chains of an antibody's constituent units (an immunoglobulin unit comprising two heavy chains and, if desired, two light chains) has been chemically modified using the compounds of the present invention or their salts. Such antibodies have the following characteristics: they comprise: (a) an immunoglobulin unit comprising two heavy chains and, if desired, two light chains, and (b) a modifying unit (e.g., the affinity substance, bioorthogonal functional group, or functional substance described above), and (c) the modifying unit is introduced only into the constant region of one of the heavy chains of the immunoglobulin unit.

[0025] That is, the present invention is as follows.

[0026] [1] A compound or a salt thereof comprising: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of an antibody heavy chain, and (B) an antibody-reactive group.

[0027] [2] The compound or salt thereof according to [1], wherein the constant region is an Fc region.

[0028] [3] The compound or salt thereof according to [1] or [2], wherein the constant region is a CH2 domain.

[0029] [4] The compound or salt thereof according to any one of [1] to [3], wherein the constant region is a human constant region.

[0030] [5] The compound or salt thereof according to any one of [1] to [4], wherein the antibody is IgG.

[0031] [6] The compound or salt thereof according to any one of [1] to [5], wherein the first affinity portion and the second affinity portion are different affinity portions.

[0032] [7] The compound or salt thereof according to any one of [1] to [6], wherein the affinity substance is represented by the following formula (A):

[0033] AP1-L A -AP2(A)

[0034] Where,

[0035] AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain,

[0036] AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain,

[0037] L A Indicates a connector.

[0038] [8] A compound or a salt thereof according to any one of [1] to [7], wherein the affinity substance: (i) (i-1) contains only one specific reactive group, and (i-2) is linked to the antibody reactive group via the specific reactive group.

[0039] [9] The compound or salt thereof according to any one of [1] to [8], wherein the affinity substance is an affinity polypeptide comprising a first affinity peptide and a second affinity peptide having affinity for the constant region of the antibody heavy chain.

[0040]

[10] The compound or salt thereof according to any one of [1] to [6], wherein the affinity polypeptide is represented by the following formula (A'):

[0041] AP1-PL A -AP2(A')

[0042] Where,

[0043] AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and present at the N-terminal side of the affinity polypeptide.

[0044] AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain and present at the C-terminal side of the affinity polypeptide.

[0045] PL A represents a peptide linker.

[0046]

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

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

[0047]

[12] The compound or salt thereof of

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

[0048]

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

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

[0049]

[14] The compound or salt thereof of

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

[0050]

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

[14] , wherein one of the first affinity peptide and the second affinity peptide is an affinity peptide having affinity for the constant region of the antibody heavy chain and having one lysine residue, and

[0051] The other of the first affinity peptide and the second affinity peptide is an affinity peptide having affinity for the constant region of the antibody heavy chain and having no lysine residue.

[0052]

[16] The compound or salt thereof of

[15] , wherein the affinity peptide having affinity for the constant region of the antibody heavy chain and having one lysine residue is any one of the following (1) to (4):

[0053] (1) an affinity peptide comprising the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38);

[0054] (2) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​in the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38) are substituted with amino acid residues other than lysine and cysteine;

[0055] (3) an affinity peptide comprising the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and

[0056] (4) An affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​in the amino acid sequence of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) are substituted with amino acid residues other than lysine and cysteine;

[0057] Here, the two cysteine ​​residues contained in the amino acid sequence can be cross-linked via a disulfide bond; and / or

[0058] The affinity peptide having affinity for the constant region of the antibody heavy chain and having no lysine residue is any one of the following (5) to (10):

[0059] (5) an affinity peptide comprising the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40);

[0060] (6) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: 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 lysine residues and amino acid residues other than cysteine ​​residues;

[0061] (7) an affinity peptide comprising the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41);

[0062] (8) An affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: 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 amino acid residues other than lysine residues and cysteine ​​residues;

[0063] (9) an affinity peptide comprising the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and

[0064] (10) An affinity peptide having affinity for the constant region of an antibody heavy chain comprising the following amino acid sequence: 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 amino acid residues other than cysteine ​​residues;

[0065] Here, the two cysteine ​​residues contained in the amino acid sequence may be cross-linked via a disulfide bond.

[0066]

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

[16] , wherein the compound is represented by the following formula (I):

[0067] [Chemical Formula 1]

[0068] RLA (I)

[0069] Where,

[0070] R represents the reactive group,

[0071] L represents a connector,

[0072] A represents the affinity substance.

[0073]

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

[17] , further comprising (iii) a cleavable portion between (i) the affinity substance and (ii) the reactive group.

[0074]

[19]

[18] The compound or salt thereof, wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the side of the reactive group by cleavage.

[0075]

[20] The compound or a salt thereof of

[19] , wherein the compound is represented by the following formula (Ia):

[0076] [Chemical Formula 2]

[0077] R-L1-CLE(B)-L2-A (Ia)

[0078] Where,

[0079] R represents the reactive group,

[0080] L1 represents the first connector,

[0081] L2 represents the second connector,

[0082] CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the side of the reactive group by cleavage,

[0083] A represents the affinity substance.

[0084]

[21] The compound of

[19] or a salt thereof, wherein the compound is represented by the following formula (Ia-1):

[0085] [Chemical Formula 3]

[0086]

[0087] Where,

[0088] X represents a leaving group,

[0089] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[0090] L3 represents the third connector,

[0091] L4 represents the 4th connector,

[0092] S represents a sulfur atom,

[0093] A represents the affinity substance.

[0094]

[22]

[21] or a salt thereof, wherein the leaving group is selected from the following groups:

[0095] (a)R A -S, here, 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;

[0096] (b)R A -O, here, 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;

[0097] (c)R A -(R B -)N, here, 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

[0098] (d) Halogen atoms.

[0099]

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

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

[0100]

[24] The compound of

[23] or a salt thereof, wherein the compound is represented by the following formula (Ib):

[0101] [Chemical Formula 4]

[0102]

[0103] Where,

[0104] R represents the reactive group,

[0105] L5 represents the fifth connector,

[0106] L6 represents the 6th connector,

[0107] B represents a group containing a bioorthogonal functional group,

[0108] CLE stands for cutting element.

[0109] A represents the affinity substance.

[0110]

[25] The compound of

[23] or a salt thereof, wherein the compound is represented by the following formula (Ib-1):

[0111] [Chemical Formula 5]

[0112]

[0113] Where,

[0114] X represents a leaving group,

[0115] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[0116] L7 represents the 7th connector,

[0117] L8 represents the 8th connector,

[0118] B represents a group containing a bioorthogonal functional group,

[0119] V represents an oxygen atom or a sulfur atom,

[0120] A represents the affinity substance.

[0121]

[26] The compound or 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 olefin residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue or a halogenated carbonyl residue.

[0122]

[27] A reagent for derivatizing an antibody, comprising the compound according to any one of [1] to

[26] or a salt thereof.

[0123]

[28] An antibody modified with an affinity substance or a salt thereof, wherein the affinity substance is contained in the constant region of the antibody heavy chain, and the affinity substance comprises a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain.

[0124]

[29]

[28] An antibody or a salt thereof modified with an affinity substance, comprising: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, and (b) the affinity substance, wherein (c) the affinity substance is introduced only into the constant region of one heavy chain in the immunoglobulin unit.

[0125]

[30]

[29] An antibody or a salt thereof modified with an affinity substance, wherein the affinity substance is introduced only into the constant region of one heavy chain via modification of an amino group in the side chain of a lysine residue present at one or more sites in the constant region of one heavy chain.

[0126]

[31]

[30] An antibody or a salt thereof modified with an affinity substance, wherein the one or more sites are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

[0127]

[32] The antibody modified with an affinity substance or a salt thereof according to any one of

[28] to

[31] , wherein the antibody modified with an affinity substance comprises a structural unit represented by the following formula (II):

[0128] [Chemical Formula 6]

[0129]

[0130] Where,

[0131] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[0132] L represents a connector,

[0133] A represents the affinity substance,

[0134] The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

[0135]

[33] The antibody or salt thereof modified with an affinity substance according to any one of

[28] to

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

[0136]

[34]

[33] An antibody or a salt thereof modified with an affinity substance, wherein the cleavable portion is a cleavable portion that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[0137]

[35]

[34] An antibody or a salt thereof modified with an affinity substance, wherein the antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIa):

[0138] [Chemical Formula 7]

[0139]

[0140] Where,

[0141] Ig represents the immunoglobulin unit,

[0142] L1 represents the first connector,

[0143] L2 represents the second connector,

[0144] CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage,

[0145] A represents the affinity substance,

[0146] The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

[0147]

[36]

[34] An antibody or a salt thereof modified with an affinity substance, wherein the antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIa-1):

[0148] [Chemical Formula 8]

[0149]

[0150] Where,

[0151] Ig represents the immunoglobulin unit,

[0152] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[0153] L3 represents the third connector,

[0154] L4 represents the 4th connector,

[0155] S represents a sulfur atom,

[0156] A represents the affinity substance,

[0157] The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

[0158]

[37]

[33] An antibody or a salt thereof modified with an affinity substance, wherein the antibody or the salt thereof further comprises (iv') a bioorthogonal functional group between (ii') the antibody and (iii') the cleavage moiety.

[0159]

[38]

[37] An antibody or a salt thereof modified with an affinity substance, wherein the antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIb):

[0160] [Chemical Formula 9]

[0161]

[0162] Where,

[0163] Ig represents the immunoglobulin unit,

[0164] L5 represents the fifth connector,

[0165] L6 represents the 6th connector,

[0166] B represents a group containing a bioorthogonal functional group,

[0167] CLE stands for cutting element.

[0168] A represents the affinity substance,

[0169] The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

[0170]

[39]

[37] An antibody or a salt thereof modified with an affinity substance, wherein the antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIb-1):

[0171] [Chemical Formula 10]

[0172]

[0173] Where,

[0174] Ig represents the immunoglobulin unit,

[0175] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[0176] L7 represents the 7th connector,

[0177] L8 represents the 8th connector,

[0178] B represents a group containing a bioorthogonal functional group,

[0179] V represents an oxygen atom or a sulfur atom,

[0180] A represents the affinity substance,

[0181] The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

[0182]

[40] The antibody modified with an affinity substance or a salt thereof according to any one of

[28] to

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

[0183]

[41]

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

[0184]

[42]

[41] An antibody or a salt thereof modified with an affinity substance, wherein the additional affinity substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

[0185]

[43]

[42] An antibody or a salt thereof modified with an affinity substance, wherein one or more sites in the constant regions of the two heavy chains are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

[0186]

[44] A method for producing an antibody or a salt thereof modified with an affinity substance, which comprises reacting a compound or a salt thereof with an antibody comprising an immunoglobulin unit to generate an antibody or a salt thereof modified with an affinity substance containing the affinity substance in the constant region of the heavy chain of the immunoglobulin unit, wherein the compound or the salt thereof comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain, and (B) an antibody reactive group, wherein the immunoglobulin unit comprises two heavy chains and, as required, two light chains.

[0187]

[45] The method of

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

[0188]

[46]

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

[0189]

[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.

[0190]

[48] ​​The method of

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

[29] to

[43] .

[0191]

[49] An antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, 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 of the immunoglobulin unit.

[0192]

[50]

[49] An antibody derivative or a salt thereof, wherein the bioorthogonal functional group is introduced only into the constant region of one heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the one heavy chain.

[0193]

[51]

[49] or

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

[0194]

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

[49] to

[51] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group comprises a structural unit represented by the following formula (IIIa):

[0195] [Chemical Formula 11]

[0196]

[0197] Where,

[0198] Ig represents the immunoglobulin unit,

[0199] L1 represents the first connector,

[0200] B represents a group containing a bioorthogonal functional group,

[0201] The average modification percentage r of the immunoglobulin unit by the bioorthogonal functional groups is 65-135%.

[0202]

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

[49] to

[51] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group comprises a structural unit represented by the following formula (IIIa-1):

[0203] [Chemical Formula 12]

[0204]

[0205] Where,

[0206] Ig represents the immunoglobulin unit,

[0207] W1 represents an oxygen atom or a sulfur atom,

[0208] L3 represents the third connector,

[0209] SH represents a thiol group,

[0210] The average modification percentage r of the immunoglobulin unit by the bioorthogonal functional groups is 65-135%.

[0211]

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

[49] to

[51] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group comprises a structural unit represented by the following formula (IIIb):

[0212] [Chemical Formula 13]

[0213]

[0214] Where,

[0215] Ig represents the immunoglobulin unit,

[0216] L5 represents the fifth connector,

[0217] B represents a group containing a bioorthogonal functional group,

[0218] T1 represents a monovalent group,

[0219] The average modification percentage r of the immunoglobulin unit by the bioorthogonal functional groups is 65-135%.

[0220]

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

[49] to

[51] , wherein the antibody derivative or salt thereof containing a bioorthogonal functional group comprises a structural unit represented by the following formula (IIIb-1):

[0221] [Chemical Formula 14]

[0222]

[0223] Where,

[0224] Ig represents the immunoglobulin unit,

[0225] W1 and W2 each independently represent an oxygen atom or a sulfur atom,

[0226] L7 represents the 7th connector,

[0227] B represents a group containing a bioorthogonal functional group,

[0228] T2 represents a monovalent group,

[0229] The modification percentage r of the immunoglobulin unit by the bioorthogonal functional group is 65-135%.

[0230]

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

[49] to

[55] , wherein the antibody derivative further comprises an additional modified portion.

[0231]

[57]

[56] An antibody derivative or a salt thereof, wherein the additional modification portion is an additional modification portion comprising a bioorthogonal functional group, and the additional affinity substance comprising the bioorthogonal functional group is contained in the constant region of the antibody heavy chain.

[0232]

[58]

[57] An antibody derivative or a salt thereof, wherein the additional modification portion comprising a bioorthogonal functional group is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

[0233]

[59]

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

[0234]

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

[0235]

[61]

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

[0236]

[62] The conjugate of

[60] or

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

[0237]

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

[60] to

[62] , wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (IVa):

[0238] [Chemical Formula 15]

[0239]

[0240] Where,

[0241] Ig represents the immunoglobulin unit,

[0242] L1 represents the first connector,

[0243] Z represents a functional substance,

[0244] The average modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

[0245]

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

[60] to

[62] , wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (IVa-1):

[0246] [Chemical Formula 16]

[0247]

[0248] Where,

[0249] Ig represents the immunoglobulin unit,

[0250] W1 represents an oxygen atom or a sulfur atom,

[0251] L3 represents the third connector,

[0252] Z represents a functional substance,

[0253] The average modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

[0254]

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

[60] to

[62] , wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (IVb):

[0255] [Chemical Formula 17]

[0256]

[0257] Where,

[0258] Ig represents the immunoglobulin unit,

[0259] L5 represents the fifth connector,

[0260] Z represents a functional substance,

[0261] T1 represents a monovalent group,

[0262] The average modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

[0263]

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

[60] to

[62] , wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (IVb-1):

[0264] [Chemical Formula 18]

[0265]

[0266] Where,

[0267] Ig represents the immunoglobulin unit,

[0268] W1 and W2 each independently represent an oxygen atom or a sulfur atom,

[0269] L7 represents the 7th connector,

[0270] Z represents a functional substance,

[0271] T2 represents a monovalent group,

[0272] The modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

[0273]

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

[60] to

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

[0274]

[68]

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

[0275]

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

[60] to

[68] , wherein the conjugate further comprises an additional modification portion.

[0276]

[70]

[69] The conjugate or a salt thereof, wherein the additional modification portion is an additional modification portion containing a functional substance, and the additional modification portion containing a functional substance is contained in the constant region of the antibody heavy chain.

[0277]

[71]

[70] A conjugate or a salt thereof, wherein the additional modification portion containing the functional substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

[0278]

[72]

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

[0279]

[73] A method for producing an antibody or a salt thereof that does not contain an affinity substance, which comprises: subjecting an antibody or a salt thereof modified with an affinity substance to a cleavage treatment at a cleavage portion to generate an antibody or a salt thereof that does not contain an affinity substance, wherein the antibody or the salt thereof modified with the affinity substance comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain, and (B) an antibody, and (C) further comprising a cleavage portion between (A) the affinity substance and (B) the antibody.

[0280]

[74]

[73] , wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody side by cleavage,

[0281] The antibody or salt thereof not containing an affinity substance is an antibody derivative or salt thereof containing a bioorthogonal functional group.

[0282]

[75]

[74] The method, wherein the antibody derivative or salt thereof comprising a bioorthogonal functional group is any one of the above-mentioned antibody derivatives or salts thereof.

[0283]

[76] The method of

[73] , wherein the antibody or a salt thereof not containing an affinity substance further comprises a bioorthogonal functional group between the antibody and the cleavage moiety,

[0284] The antibody or salt thereof not containing an affinity substance is an antibody derivative or salt thereof containing a bioorthogonal functional group.

[0285]

[77]

[76] The method of claim 7, wherein the antibody derivative or salt thereof comprising a bioorthogonal functional group is any one of the above-mentioned antibody derivatives or salts thereof.

[0286]

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

[0287] (1) producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group by the method of

[74] : and

[0288] (2) An antibody derivative containing a bioorthogonal functional group or a salt thereof is reacted with a functional substance to produce a conjugate containing the antibody and the functional substance or a salt thereof.

[0289]

[79] The method of

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

[0290]

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

[0291] (1) producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group by the method of

[76] : and

[0292] (2) An antibody derivative containing a bioorthogonal functional group or a salt thereof is reacted with a functional substance to produce a conjugate containing the antibody and the functional substance or a salt thereof.

[0293]

[81] The method of

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

[0294]

[82] A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, the method comprising: reacting an antibody derivative comprising a bioorthogonal functional group or a salt thereof with a functional substance to produce a conjugate comprising the antibody and the functional substance or a salt thereof;

[0295] The antibody derivative or salt thereof comprising a bioorthogonal functional group comprises: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, and (b) a bioorthogonal functional group, wherein (c) the bioorthogonal functional group is introduced only into the constant region of one heavy chain of the immunoglobulin unit.

[0296] The conjugate comprising an antibody and a functional substance or a salt thereof comprises: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, and (b) a functional substance, wherein (c) the functional substance is introduced only into the constant region of one heavy chain of the immunoglobulin unit.

[0297]

[83]

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

[0298]

[84] The method of

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

[0299]

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

[0300]

[86]

[85] or a salt thereof, wherein the affinity substance is represented by the following formula (A):

[0301] AP1-L A -AP2(A)

[0302] Where,

[0303] AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain,

[0304] AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain,

[0305] L A Indicates a connector.

[0306]

[87]

[85] or

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

[0307]

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

[85] to

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

[0308]

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

[85] to

[88] , wherein the affinity polypeptide is represented by the following formula (A'):

[0309] AP1-PL A -AP2(A')

[0310] Where,

[0311] AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and present at the N-terminal side of the affinity polypeptide.

[0312] AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain and present at the C-terminal side of the affinity polypeptide.

[0313] PL A represents a peptide linker.

[0314]

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

[85] to

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

[0315]

[91]

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

[0316]

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

[85] to

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

[0317]

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

[89] to

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

[0318]

[94]

[85] to

[93] , or a salt thereof, wherein one of the first affinity peptide and the second affinity peptide is an affinity peptide having affinity for the constant region of the antibody heavy chain and having one lysine residue, and

[0319] The other of the first affinity peptide and the second affinity peptide is an affinity peptide having affinity for the constant region of the antibody heavy chain and having no lysine residue.

[0320]

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

[0321]

[96] An expression vector comprising the polynucleotide of

[95] and a promoter operably linked thereto.

[0322]

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

[95] and a promoter operably linked thereto.

[0323]

[98] An antibody or a salt thereof modified with an affinity substance comprising a first modification portion and a second modification portion, wherein the first modification portion and the second modification portion are contained in the constant region of the antibody heavy chain, the first modification portion comprises: a first affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion, and the second modification portion comprises: a second affinity substance comprising a third affinity portion having affinity for the constant region of the antibody heavy chain and a fourth affinity portion.

[0324]

[99]

[98] An antibody or a salt thereof modified with an affinity substance, comprising: (a) an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains, and (b) the first modified portion and the second modified portion,

[0325] (c) the first modified portion is introduced into the constant region of the first heavy chain in the immunoglobulin unit, and

[0326] (d) The second modified portion is introduced into the constant region of the second heavy chain in the immunoglobulin unit.

[0327]

[100]

[98] or

[99] , an antibody or a salt thereof modified with an affinity substance, wherein the first modification portion is introduced into the constant region of the first heavy chain via modification of an amino group in the side chain of a lysine residue present at one or more sites in the constant region of the first heavy chain, and the second modification portion is introduced into the constant region of the second heavy chain via modification of an amino group in the side chain of a lysine residue present at one or more sites in the constant region of the second heavy chain.

[0328]

[101]

[100] An antibody or a salt thereof modified with an affinity substance, wherein one or more sites in the constant region of the first heavy chain and one or more sites in the constant region of the second heavy chain are selected from the following sites: positions 246 / 248, 288 / 290 and 317 of the human IgG heavy chain according to EU numbering, and combinations thereof.

[0329]

[102] The antibody modified with an affinity substance or a salt thereof according to any one of

[98] to

[101] , wherein the antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (V):

[0330] [Chemical Formula 19]

[0331]

[0332] Where,

[0333] Ig represents an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains.

[0334] L L and L R Each independently represents a joint,

[0335] A L represents the first affinity substance,

[0336] A R represents the second affinity substance,

[0337] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[0338]

[103] An antibody or a salt thereof modified with an affinity substance according to any one of

[98] to

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

[0339]

[104]

[103] An antibody or a salt thereof modified with an affinity substance, wherein the first cleavage moiety and the second cleavage moiety are cleavage moieties that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[0340]

[105] The antibody modified with an affinity substance or a salt thereof according to any one of

[98] to

[104] , wherein the antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Va):

[0341] [Chemical Formula 20]

[0342]

[0343] Where,

[0344] Ig represents the immunoglobulin unit,

[0345] L L1 and L R1 Each independently represents the first joint,

[0346] L L2 and L R2 Each independently represents the second joint,

[0347] CLE(B) L and CLE(B) R Each independently represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage,

[0348] A L represents the first affinity substance,

[0349] A R represents the second affinity substance,

[0350] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[0351]

[106] The affinity substance-modified antibody or a salt thereof according to any one of

[98] to

[104] , wherein the affinity substance-modified antibody comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Va-1):

[0352] [Chemical Formula 21]

[0353]

[0354] Where,

[0355] Ig represents the immunoglobulin unit,

[0356] 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,

[0357] L L3 and L R3 Each independently represents a third joint,

[0358] L L4 and L R4 Each independently represents the fourth joint,

[0359] S represents a sulfur atom,

[0360] A L represents the first affinity substance,

[0361] A R represents the second affinity substance,

[0362] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[0363]

[107]

[98] to

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

[0364]

[108] The antibody modified with an affinity substance or a salt thereof according to any one of

[98] to

[107] , wherein the antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vb):

[0365] [Chemical Formula 22]

[0366]

[0367] Where,

[0368] Ig represents the immunoglobulin unit,

[0369] L L5 and L R5 Each independently represents the fifth joint,

[0370] L L6 and L R6 Each independently represents the sixth joint,

[0371] B L represents a first group comprising a first bioorthogonal functional group,

[0372] B R represents a second group comprising a second bioorthogonal functional group,

[0373] CLE L and CLE R Each independently represents the cutting part,

[0374] A L represents the first affinity substance,

[0375] A R represents the second affinity substance,

[0376] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[0377]

[109] The antibody modified with an affinity substance or a salt thereof according to any one of

[98] to

[108] , wherein the antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vb-1):

[0378] [Chemical Formula 23]

[0379]

[0380] Where,

[0381] Ig represents the immunoglobulin unit,

[0382] 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,

[0383] L L7 and L R7 Each independently represents the seventh joint,

[0384] L L8 and L R8 Each independently represents the 8th joint,

[0385] B L represents a first group comprising a first bioorthogonal functional group,

[0386] B R represents a second group comprising a second bioorthogonal functional group,

[0387] V L and V R each independently represents an oxygen atom or a sulfur atom,

[0388] A L represents the first affinity substance,

[0389] A R represents the second affinity substance,

[0390] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[0391]

[110] The antibody or salt thereof modified with an affinity substance according to any one of

[98] to

[109] , wherein the antibody or salt thereof further comprises (iii') a first cleavage moiety between (i') the first affinity substance and (ii') the immunoglobulin unit, and further comprises (iv") a first bioorthogonal functional group between (ii") the immunoglobulin unit and (iii") the second cleavage moiety,

[0392] The first cleavable moiety is a cleavable moiety that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[0393]

[111] The antibody modified with an affinity substance or a salt thereof according to any one of

[98] to

[110] , wherein the antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vc):

[0394] [Chemical Formula 24]

[0395]

[0396] Where,

[0397] Ig represents the immunoglobulin unit,

[0398] L R1 Indicates the first connector,

[0399] L R2 Indicates the second connector,

[0400] L L5 Indicates the 5th connector,

[0401] L L6 Indicates the 6th connector,

[0402] B L represents a group comprising a first bioorthogonal functional group,

[0403] CLE L Indicates the first cutting part,

[0404] CLE(B) R represents a second cleavable moiety that can generate a second bioorthogonal functional group by cleavage on the immunoglobulin unit side,

[0405] A L represents the first affinity substance,

[0406] A R represents the second affinity substance,

[0407] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[0408]

[112] The affinity-substance-modified antibody or a salt thereof according to any one of

[98] to

[111] , wherein the affinity-substance-modified antibody comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vc-1):

[0409] [Chemical Formula 25]

[0410]

[0411] Where,

[0412] Ig represents the immunoglobulin unit,

[0413] 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,

[0414] L R3 Each independently represents a third joint,

[0415] L R4 Each independently represents the fourth joint,

[0416] L L7 Each independently represents the seventh joint,

[0417] L L8 Each independently represents the 8th joint,

[0418] B L represents a group comprising a first bioorthogonal functional group,

[0419] V L represents an oxygen atom or a sulfur atom,

[0420] A L represents the first affinity substance,

[0421] A R represents the second affinity substance,

[0422] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion RThey are 65 to 135% respectively.

[0423]

[113] The antibody modified with an affinity substance or a salt thereof according to any one of

[98] to

[112] , wherein the antibody modified with an affinity substance further comprises an additional modification portion.

[0424]

[114]

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

[0425]

[115]

[114] An antibody or a salt thereof modified with an affinity substance, wherein the additional affinity substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

[0426]

[116]

[115] An antibody or a salt thereof modified with an affinity substance, wherein one or more sites in the constant regions of the two heavy chains are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

[0427]

[117] A method for producing an antibody or a salt thereof modified with an affinity substance comprising a first modification portion and a second modification portion, comprising the following (1) and (2):

[0428] (1) reacting a compound or a salt thereof with an antibody comprising an immunoglobulin unit, wherein the compound or salt thereof comprises: (A) a first affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion, and (B) a first reactive group for the antibody, to produce an antibody or a salt thereof modified with an affinity substance comprising a first modification portion in the constant region of the heavy chain of the immunoglobulin unit; the immunoglobulin unit comprises two heavy chains and, if necessary, two light chains, and the first modification portion comprises the first affinity substance; and

[0429] (2) reacting an antibody or its salt modified with an affinity substance containing the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with a compound or its salt to generate an antibody or its salt modified with an affinity substance containing the first modification portion and the second modification portion in the constant region of the heavy chain of the immunoglobulin unit, wherein the compound or its salt comprises: a second affinity substance comprising a third affinity portion and a fourth affinity portion having affinity for the constant region of the antibody heavy chain, and (B) a second reactive group for the antibody.

[0430]

[118]

[117] The method of claim 1, wherein the antibody or salt thereof modified with an affinity substance comprising a first modification portion and a second modification portion is any one of the above-mentioned antibodies or salts thereof modified with an affinity substance.

[0431]

[119] An antibody derivative or a salt thereof comprising a first modified portion and a second modified portion, wherein the first modified portion comprises: (a) an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains, and (b) a first bioorthogonal functional group, and the second modified portion comprises a second bioorthogonal functional group,

[0432] (c) the first modified portion is introduced into the constant region of the first heavy chain,

[0433] (d) a second modified portion is introduced into the constant region of the second heavy chain, and

[0434] (e) The first modified portion and the second modified portion are different from each other.

[0435]

[120]

[119] An antibody derivative or a salt thereof, wherein the first modified portion is introduced into the constant region of the first heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the first heavy chain, and the second modified portion is introduced into the constant region of the second heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the second heavy chain.

[0436]

[121]

[119] or

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

[0437]

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

[119] to

[121] , wherein the antibody derivative or salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIa):

[0438] [Chemical Formula 26]

[0439]

[0440] Where,

[0441] Ig represents the immunoglobulin unit,

[0442] L L1 and L R1Each independently represents the first joint,

[0443] B L represents a first group comprising a first bioorthogonal functional group,

[0444] B R represents a second group comprising a second bioorthogonal functional group,

[0445] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0446]

[123]

[119] to

[122] , wherein the antibody derivative or salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIa-1):

[0447] [Chemical Formula 27]

[0448]

[0449] Where,

[0450] Ig represents the immunoglobulin unit,

[0451] W L1 and W R1 each independently represents an oxygen atom or a sulfur atom,

[0452] L L3 and L R3 Each independently represents a third joint,

[0453] SH represents a thiol group which is a bioorthogonal functional group,

[0454] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0455]

[124]

[119] to

[123] , wherein the antibody derivative or salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIb):

[0456] [Chemical Formula 28]

[0457]

[0458] Where,

[0459] Ig represents the immunoglobulin unit,

[0460] L L5 and L R5 Each independently represents the fifth joint,

[0461] B L represents a first group comprising a first bioorthogonal functional group,

[0462] B R represents a second group comprising a second bioorthogonal functional group,

[0463] T L1 and T R1 Each independently represents a monovalent group,

[0464] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0465]

[125]

[119] to

[124] , wherein the antibody derivative or salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIb-1):

[0466] [Chemical Formula 29]

[0467]

[0468] Where,

[0469] Ig represents the immunoglobulin unit,

[0470] W L1 and W L2 , and W R1 and W R2 each independently represents an oxygen atom or a sulfur atom,

[0471] L L7 and L R7 Each independently represents the seventh joint,

[0472] B L represents a first group comprising a first bioorthogonal functional group,

[0473] B R represents a second group comprising a second bioorthogonal functional group,

[0474] T L2 and T R2 Each independently represents a monovalent group,

[0475] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0476]

[126]

[119] to

[125] , wherein the antibody derivative or salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIc):

[0477] [Chemical formula 30]

[0478]

[0479] Where,

[0480] Ig represents the immunoglobulin unit,

[0481] L R1 Indicates the first connector,

[0482] L L5 Indicates the 5th connector,

[0483] B L represents a first group comprising a first bioorthogonal functional group,

[0484] B R represents a second group comprising a second bioorthogonal functional group,

[0485] T L1 represents a monovalent group,

[0486] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0487]

[127]

[119] to

[126] , wherein the antibody derivative or salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIc-1):

[0488] [Chemical Formula 31]

[0489]

[0490] Where,

[0491] Ig represents the immunoglobulin unit,

[0492] W L1 and W L2 , and W R1 each independently represents an oxygen atom or a sulfur atom,

[0493] L R3 Indicates the third connector,

[0494] L L7 Indicates the 7th connector,

[0495] B L represents a first group comprising a first bioorthogonal functional group,

[0496] SH represents a thiol group as the second bioorthogonal functional group,

[0497] T L2 represents a monovalent group,

[0498] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0499]

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

[119] to

[127] , wherein the antibody derivative further comprises an additional modified portion.

[0500]

[129]

[128] An antibody derivative or a salt thereof, wherein the additional modification portion is an additional modification portion comprising a bioorthogonal functional group, and the additional modification portion comprising a bioorthogonal functional group is contained in the constant region of the antibody heavy chain.

[0501]

[130]

[129] An antibody derivative or a salt thereof, wherein the additional modification portion comprising a bioorthogonal functional group is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

[0502]

[131]

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

[0503]

[132] An antibody and a conjugate of a first modified portion and a second modified portion, or a salt thereof, comprising the first modified portion and the second modified portion, wherein the first modified portion comprises: (a) an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains, and (b) a first functional substance, and the second modified portion comprises a second functional substance.

[0504] (c) the first modified portion is introduced into the constant region of the first heavy chain,

[0505] (d) a second modified portion is introduced into the constant region of the second heavy chain, and

[0506] (e) The first modified portion and the second modified portion are different from each other.

[0507]

[133]

[132] or a salt thereof, wherein the first modification portion is introduced into the constant region of the first heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the first heavy chain, and the second modification portion is introduced into the constant region of the second heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the second heavy chain.

[0508]

[134]

[132] or

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

[0509]

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

[132] to

[134] , wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (VIIa):

[0510] [Chemical Formula 32]

[0511]

[0512] Where,

[0513] Ig represents the immunoglobulin unit,

[0514] L L1 and L R1 Each independently represents the first joint,

[0515] Z L Indicates the first functional substance,

[0516] Z R Indicates the second functional substance,

[0517] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0518]

[136]

[132] to

[135] or a salt thereof, wherein the conjugate or the salt thereof comprises a structural unit represented by the following formula (VIIa-1):

[0519] [Chemical Formula 33]

[0520]

[0521] Where,

[0522] Ig represents the immunoglobulin unit,

[0523] W L1 and W R1 each independently represents an oxygen atom or a sulfur atom,

[0524] L L3 and L R3 Each independently represents a third joint,

[0525] Z L Indicates the first functional substance,

[0526] Z R Indicates the second functional substance,

[0527] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0528]

[137]

[132] to

[136] or a salt thereof, wherein the conjugate or the salt thereof comprises a structural unit represented by the following formula (VIIb):

[0529] [Chemical Formula 34]

[0530]

[0531] Where,

[0532] Ig represents the immunoglobulin unit,

[0533] L L5 and L R5 Each independently represents the fifth joint,

[0534] Z L Indicates the first functional substance,

[0535] Z R Indicates the second functional substance,

[0536] T L1 and T R1Each independently represents a monovalent group,

[0537] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0538]

[138]

[132] to

[137] or a salt thereof, wherein the conjugate or the salt thereof comprises a structural unit represented by the following formula (VIIb-1):

[0539] [Chemical Formula 35]

[0540]

[0541] Where,

[0542] Ig represents the immunoglobulin unit,

[0543] W L1 and W L2 , and W R1 and W R2 each independently represents an oxygen atom or a sulfur atom,

[0544] L L7 and L R7 Each independently represents the seventh joint,

[0545] Z L Indicates the first functional substance,

[0546] Z R Indicates the second functional substance,

[0547] T L2 and T R2 Each independently represents a monovalent group,

[0548] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0549]

[139]

[132] to

[138] or a salt thereof, wherein the conjugate or the salt thereof comprises a structural unit represented by the following formula (VIIc):

[0550] [Chemical Formula 36]

[0551]

[0552] Where,

[0553] Ig represents the immunoglobulin unit,

[0554] L R1 Indicates the first connector,

[0555] L L5 Indicates the 5th connector,

[0556] Z L Indicates the first functional substance,

[0557] Z R Indicates the second functional substance,

[0558] T L1 represents a monovalent group,

[0559] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0560]

[140]

[132] to

[139] or a salt thereof, wherein the conjugate or the salt thereof comprises a structural unit represented by the following formula (VIIc-1):

[0561] [Chemical Formula 37]

[0562]

[0563] Where,

[0564] Ig represents the immunoglobulin unit,

[0565] W L1 and W L2 , and W R1 each independently represents an oxygen atom or a sulfur atom,

[0566] L R3 Indicates the third connector,

[0567] L L7 Indicates the 7th connector,

[0568] Z L Indicates the first functional substance,

[0569] Z R Indicates the second functional substance,

[0570] T L2 represents a monovalent group,

[0571] The average modification percentage r of the immunoglobulin unit by the first modification part Land the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

[0572]

[141]

[132] to

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

[0573]

[142]

[132] to

[141] or a salt thereof, wherein one of the first functional substance and the second functional substance is a full-length antibody or a fragment thereof, or both the first functional substance and the second functional substance are independently a full-length antibody or a fragment thereof.

[0574]

[143] The conjugate of

[132] to

[142] or a salt thereof, wherein the conjugate further comprises an additional modification portion.

[0575]

[144]

[143] The conjugate or a salt thereof, wherein the additional modification portion is an additional modification portion containing a functional substance, and the additional modification portion containing a functional substance is contained in the constant region of the antibody heavy chain.

[0576]

[145]

[144] A conjugate or a salt thereof, wherein the additional modification portion containing the functional substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

[0577]

[146]

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

[0578] Effects of the Invention

[0579] According to the present invention, it is possible to easily modify only one heavy chain among the constituent units of an antibody (an immunoglobulin unit comprising two heavy chains).

[0580] Furthermore, according to the present invention, it is possible to provide an antibody that can be easily modified by modifying only one heavy chain in the constituent units of the antibody while performing site-selective modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0581] [ Figure 1 ] Figure 1 This is a schematic diagram showing modification of an antibody structural unit by the compound of the present invention represented by formula (I) or a salt thereof.

[0582] [ Figure 2 ] Figure 2It is a diagram showing an outline of one embodiment of the present invention.

[0583] [ Figure 3 ] Figure 3 It is a diagram showing an outline of another embodiment of the present invention.

[0584] [ Figure 4 ] Figure 4 This is a diagram showing an outline of still another embodiment of the present invention.

[0585] [ Figure 5 ] Figure 5 This is a diagram showing an outline of still another embodiment of the present invention.

[0586] [ Figure 6 ] Figure 6 This is a diagram showing an outline of still another embodiment of the present invention.

[0587] [ Figure 7 ] Figure 7 It is a diagram showing an outline of one embodiment of the present invention.

[0588] [ Figure 8 ] Figure 8 It is a diagram showing an outline of another embodiment of the present invention.

[0589] [ Figure 9 ] Figure 9 This is a diagram showing an outline of still another embodiment of the present invention.

[0590] [ Figure 10 ] Figure 10 This is a diagram showing an outline of still another embodiment of the present invention.

[0591] [ Figure 11 ] Figure 11 This is a diagram showing an outline of still another embodiment of the present invention.

[0592] [ Figure 12 ] Figure 12 This is a diagram showing an outline of still another embodiment of the present invention.

[0593] [ Figure 13 ] Figure 13 This is a diagram showing an outline of still another embodiment of the present invention.

[0594] [ Figure 14 ] Figure 14 This is a diagram showing an outline of still another embodiment of the present invention.

[0595] [ Figure 15 ] Figure 15 This is a diagram showing an outline of still another embodiment of the present invention.

[0596] [ Figure 16 ] Figure 16 It is a graph showing the expression of each polypeptide in each transformant.

[0597] [ Figure 17-1 ] Figure 17-1 This is a sensorgram showing the affinity of the polypeptide QET-Z34CM-PA32-Fc3K (SEQ ID NO: 1) to the Fc region of an IgG1 antibody.

[0598] [ Figure 17-2 ] Figure 17-2 This is a sensorgram showing the affinity of the polypeptide QET-Z34CM-PA48-Fc3K (SEQ ID NO: 2) to the Fc region of an IgG1 antibody.

[0599] [ Figure 17-3 ] Figure 17-3 This is a sensorgram showing the affinity of the polypeptide QET-Fc3-PA32-Z34CM (SEQ ID NO: 3) for the Fc region of an IgG1 antibody.

[0600] [ Figure 17-4 ] Figure 17-4 It is a sensorgram showing the affinity of the polypeptide QET-Fc3K-PA48-Z34CM (SEQ ID NO: 4) to the Fc region of an IgG1 antibody.

[0601] [ Figure 17-5 ] Figure 17-5 It is a sensorgram showing the affinity of the polypeptide QET-Fc3K-PA32-ProAR (SEQ ID NO: 5) to the Fc region of an IgG1 antibody.

[0602] [ Figure 17-6 ] Figure 17-6 It is a sensorgram showing the affinity of the polypeptide QET-Fc3K-PA48-ProAR (SEQ ID NO: 6) to the Fc region of an IgG1 antibody.

[0603] [ Figure 17-7 ] Figure 17-7 It is a sensorgram showing the affinity of the polypeptide QET-ProAR-PA32-Z34CK (SEQ ID NO: 7) to the Fc region of an IgG1 antibody.

[0604] [ Figure 17-8 ] Figure 17-8 It is a sensorgram showing the affinity of the polypeptide QET-ProAR-PA48-Z34CK (SEQ ID NO: 8) to the Fc region of an IgG1 antibody.

[0605] [ Figure 18 ] Figure 18 It is a graph showing the results of confirming the peptide / antibody binding ratio.

[0606] [ Figure 19-1 ] Figure 19-1 This is a diagram showing modification sites of lysine residues in the constant region of the antibody heavy chain based on LC-MS / MS.

[0607] [ Figure 19-2 ] Figure 19-2 This is a diagram showing modifications of the lysine residue at position 246 or 248 of the heavy chain according to EU numbering based on the CID spectrum.

[0608] [ Figure 19-3 ] Figure 19-3 This is a graph showing the modification selectivity of the lysine residue at position 248 based on BioPharma Finder.

[0609] [ Figure 20-1 ] Figure 20-1 This is a sensorgram showing the affinity between EGFR and cetuximab.

[0610] [ Figure 20-2 ] Figure 20-2 This is a sensorgram showing the affinity of neonatal Fc receptor (FcRn) for cetuximab.

[0611] [ Figure 20-3 ] Figure 20-3 This is a sensorgram showing the pH-dependent affinity between FcRn and cetuximab.

[0612] [ Figure 21-1 ] Figure 21-1 This is a sensorgram showing the affinity between EGFR and the bispecific antibody (cetuximab-trastuzumab Fab) (M-3).

[0613] [ Figure 21-2 ] Figure 21-2 This is a sensorgram showing the affinity between HER2 and the bispecific antibody (cetuximab-trastuzumab Fab) (M-3).

[0614] [ Figure 21-3 ] Figure 21-3 This is a sensorgram showing the affinity between FcRn and the bispecific antibody (cetuximab-trastuzumab Fab) (M-3).

[0615] [ Figure 21-4 ] Figure 21-4 This is a sensorgram showing the pH-dependent affinity between FcRn and the bispecific antibody (cetuximab-trastuzumab Fab) (M-3).

[0616] [ Figure 22-1] Figure 22-1 This is a sensorgram showing the affinity between EGFR and the bispecific antibody (trastuzumab-cetuximab Fab) (M-4).

[0617] [ Figure 22-2 ] Figure 22-2 This is a sensorgram showing the affinity between HER2 and the bispecific antibody (trastuzumab-cetuximab Fab) (M-4).

[0618] [ Figure 22-3 ] Figure 22-3 This is a sensorgram showing the affinity between FcRn and the bispecific antibody (trastuzumab-cetuximab Fab) (M-4).

[0619] [ Figure 22-4 ] Figure 22-4 This is a sensorgram showing the pH-dependent affinity between FcRn and the bispecific antibody (trastuzumab-cetuximab Fab) (M-4).

[0620] [ Figure 23-1 ] Figure 23-1 This is a sensorgram showing the affinity between EGFR and the trispecific antibody (M-2).

[0621] [ Figure 23-2 ] Figure 23-2 This is a sensorgram showing the affinity between HER2 and the trispecific antibody (M-2).

[0622] [ Figure 23-3 ] Figure 23-3 This is a sensorgram showing the affinity between PD-1 and the trispecific antibody (M-2).

[0623] [ Figure 23-4 ] Figure 23-4 This is a sensorgram showing the affinity between FcRn and the trispecific antibody (M-2).

[0624] [ Figure 23-5 ] Figure 23-5 This is a sensorgram showing the pH-dependent affinity between FcRn and the trispecific antibody (M-2).

[0625] [ Figure 24-1 ] Figure 24-1 This graph shows the positive rates of binding of cetuximab (Cmab), trastuzumab (Tmab), bispecific antibody (cetuximab-trastuzumab Fab) (M-3), (trastuzumab-cetuximab Fab) (M-4), and trispecific antibody (pembrolizumab Fab-cetuximab-trastuzumab Fab) (M-2) to SKBR-3 cells (HER2-positive) and A-431 cells (EGFR-positive), calculated by flow cytometry.

[0626] [ Figure 24-2 ] Figure 24-2 The graph shows the evaluation of the binding of cetuximab (Cmab) to SKBR-3 cells (HER2 positive), A-431 cells (EGFR positive), and T cells (PD-1 positive) by flow cytometry.

[0627] [ Figure 24-3 ] Figure 24-3 The graph shows the evaluation of trastuzumab (Tmab) binding to SKBR-3 cells (HER2 positive) and A-431 cells (EGFR positive) by flow cytometry.

[0628] [ Figure 24-4 ] Figure 24-4 The graph shows the evaluation of the binding of the bispecific antibody (cetuximab-trastuzumab Fab) (M-3) to SKBR-3 cells (HER2-positive) and A-431 cells (EGFR-positive) by flow cytometry.

[0629] [ Figure 24-5 ] Figure 24-5 The graph shows the evaluation of the binding of the bispecific antibody (trastuzumab-cetuximab Fab) (M-4) to SKBR-3 cells (HER2 positive) and A-431 cells (EGFR positive) by flow cytometry.

[0630] [ Figure 24-6 ] Figure 24-6 This graph shows the evaluation of the binding of pembrolizumab (Pbl) to T cells (PD-1 positive) by flow cytometry.

[0631] [ Figure 24-7 ] Figure 24-7 The graph shows the evaluation of the binding of the trispecific 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.

[0632] [ Figure 24-8 ] Figure 24-8 It is a graph showing the positive rates of T cells (PD-1 positive) bound to cetuximab (Cmab), pembrolizumab (Pbl), and a trispecific antibody (pembrolizumab Fab-cetuximab-trastuzumab Fab) (M-2), calculated by flow cytometry.

[0633] [ Figure 25 ] Figure 25 This is a picture showing SDS-PAGE of CD3-VHH-PA24H6-AzF (V-1) expression.

[0634] [ Figure 26 ] Figure 26 This is a graph showing the results of affinity evaluation of CD3-VHH (V-1).

[0635] [ Figure 27 ] Figure 27 This is a graph showing the results of affinity evaluation of CD3-VHH (V-1).

[0636] [ Figure 28 ] Figure 28 It is a figure showing SDS-PAGE of 6 kinds of affinity peptides. DETAILED DESCRIPTION

[0637] 1. Definition of general terms

[0638] In this specification, the terms and expressions used to describe a specific invention or matter may also be cited in the description of another invention or matter. Therefore, the definitions, examples, and preferred examples of the terms and expressions used to describe a specific invention or matter may also be the same as those of the other invention or matter described in such terms and expressions.

[0639] In the present invention, the term "antibody" is as follows. In addition, the term "immunoglobulin unit" corresponds to the bivalent monomer unit that is the constituent unit of such an antibody, and is an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains. Therefore, with respect to the immunoglobulin unit, the definitions, examples, and preferred examples of terms and expressions such as its source, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, amino acid residue (e.g., lysine residue) site, and site selectivity are the same as those for the antibody described below and can be used interchangeably with the expression "antibody".

[0640] The source of the antibody is not particularly limited, and for example, it can be an antibody derived from mammals, birds (e.g., chickens), or other animals. 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., cattle, pigs, goats), and working animals (e.g., horses, sheep). Primates or rodents are preferred, and humans are more preferred.

[0641] The type of antibody can be a polyclonal antibody or a monoclonal antibody. The antibody can also be a bivalent antibody (e.g., IgG, IgD, IgE) or an antibody with more than four valences (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. As monoclonal antibodies, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies with added specified sugar chains (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond-reduced antibodies can be mentioned. As the isotype of the monoclonal antibody, for example, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY can be mentioned. In the present invention, as a monoclonal antibody, a full-length antibody or an antibody fragment comprising a variable region and a CH1 domain and a CH2 domain can be utilized, but a full-length antibody is preferred. The antibody is preferably a human IgG monoclonal antibody, more preferably a human IgG full-length monoclonal antibody.

[0642] Any antigen can be used as an antigen for the antibody. For example, such antigens include proteins [including oligopeptides and polypeptides. Proteins modified with biomolecules such as sugars (e.g., glycoproteins)], sugar chains, nucleic acids, and small molecule compounds. Preferably, the antibody is one 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.

[0643] More specifically, the protein serving as an antigen for the antibody may be a disease target protein. Examples of disease target proteins include the following.

[0644] (1) Cancer field

[0645] PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, CD123, gp A33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, tissue factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor-associated macrophages), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GI TR, fucosyl GM1, IGF-1, angiopoietin 2, CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2 , TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, immunosuppressive protein, NaPi2b, Lewis blood group B antigen, A34, lysyl oxidase, DLK-1, TROP-2, α9 integrin, TAG-72 (CA72-4), CD70.

[0646] (2) Autoimmune diseases / inflammatory diseases

[0647] 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 (α4β7 integrin), IL-23, GM-CSFR, TSLP, CD28, CD 40, 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 ligand, IL-21, cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT.

[0648] (3) Brain and nerve diseases

[0649] CGRP, CD20, beta-amyloid, beta-amyloid fibrils, calcitonin gene-related peptide receptor, LINGO (1 including Ig domains), alpha-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus.

[0650] (4) Infectious diseases

[0651] Clostridium Difficile toxin B, cytomegalovirus, RS virus, LPS, S. Aureus alpha-toxin, M2e protein, Psl, PcrV, S. aureus toxin, influenza A, alginate, S. aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Pneumococcus.

[0652] (5) Genetic diseases / rare diseases

[0653] Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin protein.

[0654] (6) Eye diseases

[0655] Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (endoglin), IGF-1R, beta-amyloid.

[0656] (7) Orthopedics / Plastic Surgery

[0657] Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15.

[0658] (8) Blood diseases

[0659] vWF, factor IXa, factor X, IFNγ, C5, BMP-6, ferroportin, TFPI.

[0660] (9) Other diseases

[0661] BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP.

[0662] Specific examples of monoclonal antibodies include specific chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, datuximab, and ortatuximab), specific humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), moglizumab, pertuzumab, obtuzumab, vedolizumab, pembrolizumab (IgG4), mepolizumab, and elotuzumab). The present invention also provides the following antibodies: monoclonal antibody, daratumumab, ixekizumab (IgG4), retuzumab (IgG4), atezolizumab), specific human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, ranixicumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necituzumab, berdalumab (IgG2), olaratumumab) (where IgG isotype is not mentioned, it is indicated as IgG1).

[0663] 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 residues, tyrosine residues, serine residues, and threonine residues. For example, in human IgGs such as human IgG1, the following amino acid residues present in the heavy chain constant region are exposed on the antibody surface, and therefore these amino acid residues can be used to introduce specific cleavage sites (amino acid residue sites are numbered according to the EU; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0664] (1) Exposed lysine residues

[0665] CH2 domain (e.g., positions 246, 248, 274, 288, 290, 317, 320, 322)

[0666] CH3 domain (e.g., positions 360, 414, 439)

[0667] (2) Exposed tyrosine residues

[0668] CH2 domain (e.g., positions 278, 296, 300)

[0669] CH3 domain (e.g., position 436)

[0670] (3) Exposed serine residues

[0671] CH2 domain (e.g., positions 254, 267, 298)

[0672] CH3 domain (e.g., positions 400, 415, 440)

[0673] (4) Exposed threonine residues

[0674] CH2 domain (e.g., positions 256, 289)

[0675] CH3 domain (e.g., positions 335, 359)

[0676] Regarding the positions of amino acid residues in antibodies and positions in the constant region of the heavy chain (e.g., CH2 domain), EU numbering is used (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, in the case of human IgG, the lysine residue at position 246 corresponds to the 16th amino acid residue in the CH2 region of human IgG, the lysine residue at position 248 corresponds to the 18th amino acid residue in the CH2 region of human IgG, the lysine residue at position 288 corresponds to the 58th amino acid residue in the CH2 region of human IgG, the lysine residue at position 290 corresponds to the 60th amino acid residue in the CH2 region of human IgG, and the lysine residue at position 317 corresponds to the 87th amino acid residue in the CH2 region of human IgG. The expression 246 / 248 indicates that either the lysine residue at position 246 or 248 is the target. The expression 288 / 290 indicates that either the lysine residue at position 288 or 290 is the target.

[0677] Preferably, with respect to a specific amino acid residue in the heavy chain constant region that is site-selectively modified, a lysine residue (e.g., a lysine residue at positions 246 / 248 or 288 / 290) can be site-selectively modified. In this specification, "site-selective" or "site selectivity" means that, although a specific amino acid residue in an antibody is not biased towards a specific region, a prescribed structural unit that can be bound to a specific amino acid residue in an antibody is biased towards a specific region in the antibody. Therefore, expressions related to site selectivity such as "site-selectively having", "site-selective binding", and "site-selective binding" refer to that the holding rate or binding rate of a prescribed structural unit in a target region comprising more than one specific amino acid residue is significantly higher than the holding rate or binding rate of the structural unit in a non-target region comprising multiple amino acid residues of the same species as the specific amino acid residue in the target region. Such site selectivity can be 50% or greater, preferably 60% or greater, more preferably 70% or greater, even more preferably 80% or greater, and particularly preferably 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, or 100%. According to the present invention, specific lysine residues in the heavy chain of an antibody can be site-selectively modified without using a peptide-containing linker. Peptide moieties are potentially immunogenic and easily hydrolyzed in blood. Therefore, avoiding the use of peptide-containing linkers is desirable in clinical applications.

[0678] In the present invention, specific amino acid residues at other sites may also be further site-selectively modified, as long as the specific amino acid residues in the heavy chain constant region (e.g., lysine residues at specific sites) are site-selectively modified. For example, methods for site-selectively modifying specific amino acid residues at specified sites in antibodies are described in International Publication No. 2018 / 199337, International Publication No. 2019 / 240288, International Publication No. 2019 / 240287, and International Publication No. 2020 / 090979. As such specific amino acid residues, amino acid residues having easily modifiable side chains (e.g., amino group, carboxyl group, amide group, hydroxyl group, thiol group) (e.g., lysine residue, aspartic acid residue, glutamic acid residue, asparagine residue, glutamine residue, threonine residue, serine residue, tyrosine residue, cysteine ​​residue) can be used, but preferably, a lysine residue having a side chain containing an amino group, a tyrosine residue, a serine residue, and a threonine residue having a side chain containing a hydroxyl group, or a cysteine ​​residue having a side chain containing a thiol group, and more preferably, a lysine residue (i.e., two lysine residues among the lysine residues at positions 246 / 248, the lysine residues at positions 288 / 290, and the lysine residue at position 317 can be site-selectively doubly modified, and three lysine residues can be site-selectively triply modified).

[0679] (Halogen atom)

[0680] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0681] (monovalent group)

[0682] Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

[0683] The monovalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, further preferably 1 to 5, particularly preferably 1 to 3) substituents described below.

[0684] (monovalent hydrocarbon group and related terms)

[0685] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0686] A monovalent chain hydrocarbon group refers to a hydrocarbon group consisting solely of a chain structure, with the main chain not containing a cyclic structure. However, the chain structure may be either straight-chain or branched. Examples of monovalent chain hydrocarbon groups include alkyl groups, alkenyl groups, and alkynyl groups. Alkyl groups, alkenyl groups, and alkynyl groups may be straight-chain or branched.

[0687] The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 6 carbon atoms, and even more preferably an alkyl group having 1 to 4 carbon atoms. When the alkyl group has a substituent, the carbon number of the substituent is not included in the above carbon number. Examples of the alkyl group having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0688] The alkenyl group is preferably an alkenyl group having 2 to 12 carbon atoms, more preferably an alkenyl group having 2 to 6 carbon atoms, and even more preferably an alkenyl group having 2 to 4 carbon atoms. When the alkenyl group has a substituent, the carbon atom number of the substituent is not included in the above carbon atom number. Examples of the alkenyl group having 2 to 12 carbon atoms include ethenyl, propenyl, and n-butenyl.

[0689] The alkynyl group is preferably an alkynyl group having 2 to 12 carbon atoms, more preferably an alkynyl group having 2 to 6 carbon atoms, and even more preferably an alkynyl group having 2 to 4 carbon atoms. When the alkynyl group has a substituent, the carbon atom number of the substituent is not included in the above carbon atom number. Examples of the alkynyl group having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0690] As the monovalent chain hydrocarbon group, an alkyl group is preferred.

[0691] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group containing only an alicyclic hydrocarbon as a ring structure and not an aromatic ring. The alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not need to be composed solely of alicyclic hydrocarbons and may partially contain a chain structure. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl groups, cycloalkenyl groups, and cycloalkynyl groups, which may be either monocyclic or polycyclic.

[0692] The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 carbon atoms, more preferably a cycloalkyl group having 3 to 6 carbon atoms, and even more preferably a cycloalkyl group having 5 to 6 carbon atoms. When the cycloalkyl group has a substituent, the carbon number of the substituent is not included in the above carbon number. Examples of the cycloalkyl group having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0693] The cycloalkenyl group is preferably a cycloalkenyl group having 3 to 12 carbon atoms, more preferably a cycloalkenyl group having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl group having 5 to 6 carbon atoms. When the cycloalkenyl group has a substituent, the carbon atom number of the substituent is not included in the above carbon atom number. Examples of the cycloalkenyl group having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0694] The cycloalkynyl group is preferably a cycloalkynyl group having 3 to 12 carbon atoms, more preferably a cycloalkynyl group having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl group having 5 to 6 carbon atoms. When the cycloalkynyl group has a substituent, the carbon atom number of the substituent is not included in the above carbon atom number. Examples of the cycloalkynyl group having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0695] As the monovalent alicyclic hydrocarbon group, a cycloalkyl group is preferred.

[0696] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to be composed solely of an aromatic ring; a portion thereof may contain a chain structure or an alicyclic hydrocarbon, and the aromatic ring may be either a monocyclic or a polycyclic ring. As a 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 further preferred. When the monovalent aromatic hydrocarbon group has a substituent, the carbon atom number of the substituent is not included in the above-mentioned carbon atom number. Examples of the aryl group having 6 to 12 carbon atoms include phenyl and naphthyl.

[0697] As the monovalent aromatic hydrocarbon group, a phenyl group is preferred.

[0698] Among these, as the monovalent hydrocarbon group, an alkyl group, a cycloalkyl group, and an aryl group are preferred.

[0699] (monovalent heterocyclic group and related terms)

[0700] A monovalent heterocyclic group is a group formed by removing one hydrogen atom from the heterocyclic ring of a heterocyclic compound. A monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic group preferably contain one or more selected from oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contain one or more selected from oxygen, sulfur, and nitrogen atoms.

[0701] As the monovalent aromatic heterocyclic group, an aromatic heterocyclic group having 1 to 15 carbon atoms is preferred, an aromatic heterocyclic group having 1 to 9 carbon atoms is more preferred, and an aromatic heterocyclic group having 1 to 6 carbon atoms is further preferred. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above-mentioned number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furyl, thienyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, Azolyl, iso oxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinoneyl, carbazolyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl and phthalazinyl.

[0702] As the monovalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferred, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferred, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is further preferred. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above-mentioned number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridine, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothienyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydro Azinyl, tetrahydro oxazinyl, dihydropyrimidinyl and tetrahydropyrimidinyl.

[0703] Among these, as the monovalent heterocyclic group, a 5-membered or 6-membered heterocyclic group is preferred.

[0704] (divalent group)

[0705] The divalent group is selected from a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, -C(=O)-, -C(=S)-, -NR1-, -C(=O)-NR1-, -NR1-C(=O)-, -C(=S)-NR1-, -NR1-C(=S)-, -O-, -S-, -(O-R2) n -and-(S-R2) m -, or a group having a main chain structure containing two or more (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, further preferably 2 to 5, and particularly preferably 2 or 3) of these groups. R1 represents a hydrogen atom or a substituent described below. R2 represents a divalent straight-chain hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. n and m are each an integer from 1 to 10, preferably an integer from 1 to 8, more preferably an integer from 1 to 6, further preferably an integer from 1 to 5, and particularly preferably an integer from 1 to 3.

[0706] The divalent straight-chain hydrocarbon group is a straight-chain alkylene group, a straight-chain alkenylene group, or a straight-chain alkynylene group.

[0707] The linear alkylene group is a linear alkylene group having 1 to 6 carbon atoms, preferably a linear alkylene group having 1 to 4 carbon atoms. Examples of the linear alkylene group include a methylene group, an ethylene group, an n-propylene group, an n-butylene group, an n-pentylene group, and an n-hexylene group.

[0708] The straight-chain alkenylene group is a straight-chain alkenylene group having 2 to 6 carbon atoms, preferably a straight-chain alkenylene group having 2 to 4 carbon atoms. Examples of the straight-chain alkenylene group include ethenylene, n-propenylene, n-butenylene, n-pentenylene, and n-hexenylene.

[0709] The straight-chain alkynylene group is a straight-chain alkynylene group having 2 to 6 carbon atoms, preferably a straight-chain alkynylene group having 2 to 4 carbon atoms. Examples of the straight-chain alkynylene group include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene.

[0710] As the divalent straight-chain hydrocarbon group, a straight-chain alkylene group is preferred.

[0711] The divalent cyclic hydrocarbon group is an arylene group or a divalent non-aromatic cyclic hydrocarbon group.

[0712] The arylene group is preferably an arylene group having 6 to 14 carbon atoms, more preferably an arylene group having 6 to 10 carbon atoms, and particularly preferably an arylene group having 6 carbon atoms. Examples of the arylene group include phenylene, naphthylene, and anthracene.

[0713] 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 the divalent non-aromatic cyclic hydrocarbon group include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene.

[0714] As the divalent cyclic hydrocarbon group, an arylene group is preferred.

[0715] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocyclic ring preferably contain one or more selected from oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably contain one or more selected from oxygen atoms, sulfur atoms, and nitrogen atoms.

[0716] 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 the divalent aromatic heterocyclic group include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, Azoles, isoxadiazoles oxadiyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl and phthalazinediyl.

[0717] The divalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a non-aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of the divalent non-aromatic heterocyclic group include a pyrroledionediyl group, a pyrrolidinedionediyl group, an oxiranediyl group, an aziridinediyl group, an azetidinediyl group, an oxetanediyl group, a thietanediyl group, a pyrrolidinediyl group, a dihydrofurandiyl group, a tetrahydrofurandiyl group, a dioxolanediyl group, a tetrahydrothiophenediyl group, a pyrrolidinediyl group, an imidazolinediyl group, Oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholindiyl, thiomorpholindiyl, piperazinediyl, dihydro Oxadiazole, tetrahydro oxazinediyl, dihydropyrimidinediyl and tetrahydropyrimidinediyl.

[0718] As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

[0719] Preferably, the divalent group is a group having a radical selected from the group consisting of an alkylene group, an arylene group, -C(=O)-, -NR1-, -C(=O)-NR1-, -NR1-C(=O)-, -O- and -(O-R2) n - a divalent group in the main chain structure of one group, or

[0720] is a group containing a group selected from alkylene, arylene, -C(=O)-, -NR1-, -C(=O)-NR1-, -NR1-C(=O)-, -O- and -(O-R2) n - a divalent group of a main chain structure of two or more groups,

[0721] R1 is a hydrogen atom or an alkyl group,

[0722] R2 is an alkylene group or an arylene group,

[0723] n may be an integer from 1 to 5 (ie, 1, 2, 3, 4, or 5).

[0724] The alkylene group, arylene group and alkyl group are the same as those described above.

[0725] 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, further preferably 1 to 5, particularly preferably 1 to 3) substituents described below.

[0726] (Substituent)

[0727] As the substituent, the following can be mentioned:

[0728] (i) halogen atoms;

[0729] (ii) a monovalent hydrocarbon group;

[0730] (iii) a monovalent heterocyclic group;

[0731] (iv) aralkyl;

[0732] (v)R a -O-、R a -C(=O)-、R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or a monovalent hydrocarbon group); or

[0733] (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 (a) represents, identically or differently, a hydrogen atom or a monovalent hydrocarbon group;

[0734] (vii) nitro, sulfate, sulfonate, cyano and carboxyl groups.

[0735] The definitions, examples and preferred examples of the halogen atom, monovalent hydrocarbon group and monovalent heterocyclic group in the above-mentioned substituents are the same as those described above.

[0736] Aralkyl refers to an arylalkyl group. The definitions, examples, and preferred examples of the aryl and alkyl groups in the arylalkyl group are as described above. Aralkyl groups preferably have 3 to 15 carbon atoms. Examples of such aralkyl groups include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0737] Preferably, the substituents may be as follows:

[0738] (i) halogen atoms;

[0739] (ii) an alkyl group having 1 to 12 carbon atoms, a phenyl group or a naphthyl group;

[0740] (iii) an aralkyl group having 3 to 15 carbon atoms;

[0741] (iv) a 5-membered or 6-membered heterocyclic ring;

[0742] (v)R a -O-、R a -C(=O)-、R a -OC(=O)- or R a -C(=O)-O-(R arepresents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms);

[0743] (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 represent, identically or differently, a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or

[0744] (vii) The same groups as exemplified in the above (vii).

[0745] More preferably, the substituents may be as follows:

[0746] (i) halogen atoms;

[0747] (ii) an alkyl group having 1 to 12 carbon atoms;

[0748] (iii)R a -O-、R a -C(=O)-、R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms);

[0749] (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 represent, identically or differently, a hydrogen atom or an alkyl group having 1 to 12 carbon atoms); or

[0750] (v) The same groups as exemplified in the above (vii).

[0751] More preferably, the substituents may be as follows:

[0752] (i) halogen atoms;

[0753] (ii) an alkyl group having 1 to 6 carbon atoms;

[0754] (iii)Ra -O-、R a -C(=O)-、R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms);

[0755] (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 the same or different hydrogen atoms or alkyl groups having 1 to 6 carbon atoms); or

[0756] (v) The same groups as exemplified in the above (vii).

[0757] Particularly preferably, the substituents may be as follows:

[0758] (i) halogen atoms;

[0759] (ii) an alkyl group having 1 to 4 carbon atoms;

[0760] (iii)R a -O-、R a -C(=O)-、R a -OC(=O)- or R a -C(=O)-O-(R a represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms);

[0761] (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 (a) the same or different hydrogen atoms or alkyl groups having 1 to 4 carbon atoms; or

[0762] (v) The same groups as exemplified in the above (vii).

[0763] (Bioorthogonal functional groups)

[0764] Bioorthogonal functional groups are groups that do not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates) or react slowly with biological components, but selectively react with components other than biological components. Bioorthogonal functional groups are known in the art (e.g., see Sharpless KB et al., Angew. Chem. Int. 40th ed., 2004 (2015); Bertozzi CR et al., Science 291, 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).

[0765] In the present invention, as bioorthogonal functional groups, bioorthogonal functional groups for proteins are used. This is because the antibodies suitable for being derived from the reagents of the present invention are proteins. Bioorthogonal functional groups for proteins are groups that do not react with the side chains of the 20 natural amino acid residues that constitute proteins or react slowly with the side chains, but react with the target functional groups. The 20 natural amino acids that constitute proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H) and lysine (K). Among these 20 natural amino acids, glycine, which has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., the side chains do not contain heteroatoms selected from sulfur, nitrogen, and oxygen atoms), are inert to typical reactions. Therefore, bioorthogonal functional groups for proteins are groups that do not react with the side chains of these amino acids with side chains inert to typical reactions, as well as groups that do not react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, or react slowly but react with the desired functional group.

[0766] Examples of such bioorthogonal functional groups include: azide residues, aldehyde residues, thiol residues, olefin residues (in other words, any residue having an ethenylene moiety as the minimum unit having a carbon-carbon double bond, the same below), alkyne residues (in other words, any residue having an ethynylene moiety as the minimum unit having a carbon-carbon triple bond, the same 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 (for example, a carbonyl residue having a fluorine atom, a chlorine atom, a bromine atom or an iodine atom at the α position, the same below), isonitrile residues, sydney ketone residues, and selenium residues.

[0767] The bioorthogonal functional group may be protected or unprotected. The bioorthogonal functional group refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. The unprotected bioorthogonal functional group is equivalent to the above-mentioned bioorthogonal functional group. The protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of a protecting group. The cleavage of the protecting group can be carried out by a specific treatment under conditions (mild conditions) that do not cause denaturation / degradation of the protein (for example, cleavage of an amide bond). As such specific treatments, for example, there can be mentioned: (a) treatment with one or more substances selected from acidic substances, alkaline substances, reducing agents, oxidizing agents, and enzymes; (b) treatment with a physicochemical stimulus selected from light; or (c) placement in the case of using a cleavable linker containing a self-degradable cleavable portion. Such protecting groups and their cleavage conditions are common technical knowledge in the field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry, 20, 571 (2012); Feng P. et al., Journal of American Chemical Society, 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society, 132, 179-28 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)). Mild reaction conditions (e.g., reaction temperature, reaction time, reaction solution) are described below.

[0768] Examples of the bioorthogonal functional group to be protected include a disulfide residue, an ester residue, an acetal residue, a ketal residue, an imine residue, and a vicinal diol residue.

[0769] Preferably, the bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0770] More preferably, the bioorthogonal functional group can be a specific bioorthogonal functional group that has excellent reactivity (e.g., reaction level and / or reaction specificity) with other bioorthogonal functional groups. Examples of such bioorthogonal functional groups include: azide residues, alkyne residues (preferably cyclic groups having carbon-carbon triple bonds that can be substituted with the above-mentioned substituents), tetrazine residues, olefin residues, thiol residues, maleimide residues, thiol residues, furan residues, and halogenated carbonyl residues. Regarding the combination of two bioorthogonal functional groups that can react with each other, examples include: a combination of an azide residue and an alkyne residue, a combination of a tetrazine residue and an olefin residue, a combination of a tetrazine residue and an alkyne residue, a combination of a thiol residue and a maleimide residue, a combination of a furan residue and a maleimide residue, a combination of a thiol residue and a halogenated carbonyl residue (halogen is replaced by thiol through a substitution reaction), and a combination of a thiol residue and another thiol residue (forming a disulfide bond).

[0771] (Functional substances)

[0772] The functional substance is not particularly limited, as long as it imparts any function to the antibody. Examples thereof include drugs, labeling substances, affinity substances, transfer substances, and stabilizers. Preferably, it is a drug, labeling substance, affinity substance, or transfer substance. The functional substance may be a single functional substance or a combination of two or more functional substances.

[0773] As a medicine, it can be a medicine for any disease. As such diseases, for example, can be mentioned: cancer (for example, lung cancer, gastric cancer, colorectal 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 disease / inflammatory disease (for example, allergic disease, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve disease (for example, cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infection (for example, bacterial infection, viral infection), genetic disease / rare disease (for example, hereditary spherocytosis, non-dystrophic myotonia), eye disease (for example, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone / orthopedic field disease (for example, osteoarthritis), blood disease (for example, leukemia, purpura), other diseases (for example, metabolic disorders such as diabetes, hyperlipidemia, liver disease, kidney disease, lung disease, circulatory system disease, digestive system disease). The medicine can be a drug for preventing or treating a disease, or a drug for alleviating side effects.

[0774] More specifically, the drug may be an anticancer drug. Examples of anticancer drugs include: chemotherapeutics, toxins, radioactive isotopes, or substances containing the same. Examples of chemotherapeutics include: DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, microtubule binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include: bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin). Examples of radioactive isotopes 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., 153 Sm), radioactive isotopes of rhenium (e.g. 186 Re), radioactive isotopes of astatine (e.g. 211 At), radioactive isotopes of bismuth (e.g. 212 More specifically, the drugs include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine ), IGN, camptothecin analogs, Calichemicin, Duocarmycin, Eribulin, anthracycline antibiotics, dmDNA31, Tubulysin.

[0775] A labeling substance is a substance that can detect 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'-bipyridine)ruthenium, luminol), radioactive isotopes (e.g., the aforementioned isotopes), or substances containing the same.

[0776] Affinity substances are substances that have affinity for the target. Examples of affinity substances include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary strands of target nucleic acids. Affinity substances are preferably affinity proteins or affinity peptides, and more preferably antibodies. The animal species from which the antibodies used as functional substances are derived are the same as those described above.

[0777] The type of antibody used as a functional substance can be a polyclonal antibody or a monoclonal antibody. The antibody can 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. As monoclonal antibodies, for example, chimeric antibodies, humanized antibodies, human antibodies, antibodies with added specified sugar chains (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond-reduced antibodies can be mentioned. As isotypes of monoclonal antibodies, for example, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY can be mentioned. As antibodies used as functional substances, for example, full-length antibodies and fragments thereof (fragment antibodies) can be mentioned. Fragment antibodies may be any antibody as long as they maintain binding properties to the desired antigen, and examples thereof include Fab, Fab', F(ab')2, scFv, and VHH antibodies.

[0778] The antigenicity of the antibody used as a functional substance may be the same as or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, preferably different. In addition, the source of the antibody used as a functional substance may be the same as or different from the source of the immunoglobulin unit, preferably different. Therefore, the antibody used as a 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 a functional substance may also be IgG1, IgG2, IgG3, or IgG4 mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom.

[0779] The substance for transport is a substance that has the ability to transport a compound. Preferred substances for transport are substances that can encapsulate a compound in a protein shell (e.g., a polymer) (e.g., ferritin, virus particles, virus-like particles).

[0780] Stabilizers are substances that stabilize antibodies. Examples of stabilizers include glycols, glycerol, nonionic surfactants, anionic surfactants, natural surfactants, sugars, and polyols.

[0781] In addition, the functional substance can be a peptide, protein, nucleic acid, organic compound, inorganic compound, sugar chain, lipid, high molecular polymer, metal (e.g., gold), chelating agent. Examples of peptides include: cell penetrating peptides, blood-brain barrier penetrating peptides, peptide drugs. Examples of proteins include: enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, antibodies, ferritin. Examples of nucleic acids include: DNA, RNA, artificial nucleic acids. In addition, examples of nucleic acids include: RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, antisense nucleic acids. Examples of organic compounds include: small molecule organic compounds such as chimeric molecules that induce protein degradation, pigments, and photodegradable compounds. Examples of inorganic compounds include: silica, talc, and alumina.

[0782] (Salt)

[0783] In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium and potassium), alkaline earth metals (e.g., calcium and magnesium), other metals such as zinc and aluminum, and ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0784] 2. Affinity substances or their salts, and related inventions

[0785] 2-1. Affinity substance or its salt

[0786] The present invention provides an affinity substance or a salt thereof comprising a first affinity portion and a second affinity portion having affinity for the constant region of an antibody heavy chain.

[0787] The affinity substance used in the present invention comprises a first affinity portion and a second affinity portion having affinity for the constant region of the heavy chain in the constituent unit of the antibody. The constituent unit of the antibody is an immunoglobulin unit comprising two heavy chains and two light chains as needed. Therefore, the constituent unit of the antibody is an immunoglobulin unit comprising two heavy chains and two light chains, or an immunoglobulin unit comprising two heavy chains and not containing two light chains. As antibodies comprising an immunoglobulin unit comprising two heavy chains and two light chains, for example, bivalent antibodies (for example, IgG, IgD, IgE) or antibodies with four or more valences (for example, IgA antibodies, IgM antibodies), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies and disulfide bond reduction antibodies. As antibodies comprising an immunoglobulin unit comprising two heavy chains and not containing two light chains, for example, Fc region proteins, Fc fusion proteins and disulfide bond reduction antibodies can be mentioned. This is because the Fc region contains the CH2 domain and the CH3 domain as heavy chains, but does not contain light chains.

[0788] The first affinity portion and the second affinity portion may be the same or different. When the first affinity portion and the second affinity portion are different, the first affinity portion and the second affinity portion may be affinity substances having affinities for different regions in the constant region of the antibody heavy chain (for example, one of the first affinity portion and the second affinity portion has affinity for the CH2 domain, and the other has affinity for the CH3 domain). Alternatively, they may be different affinity portions having affinities for the same region in the constant region of the antibody heavy chain (for example, both the first affinity portion and the second affinity portion have affinity for the CH2 domain or the CH3 domain). Preferably, the different affinity portions have affinities for the same region in the constant region of the antibody heavy chain. Furthermore, the first affinity portion and the second affinity portion preferably sterically interfere with the first binding site of the heavy chain constant region and the second binding site of the heavy chain constant region, and are in a relationship such that the association of one inhibits the association of the other, thereby inhibiting the association of the first affinity portion and the second affinity portion with a single heavy chain constant region. If such a relationship exists, in an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains), it is possible to inhibit the competitive association of two molecules (the first molecule and the second molecule) of a compound comprising an affinity substance and an antibody-reactive group within a single antibody constituent unit (the aforementioned immunoglobulin unit), thereby promoting specific modification of only one heavy chain constant region. Examples of the first affinity portion and the second affinity portion include polymer substances of predetermined structural units [e.g., peptides (including oligopeptides, polypeptides, proteins), nucleic acids (including oligonucleic acids and polysaccharides), sugars (including oligosaccharides and polysaccharides)] and non-polymer substances (e.g., small molecule compounds). As substances that can be used as the first affinity portion and the second affinity portion having affinity for the constant region of the antibody heavy chain, many substances such as peptides, nucleic acids, sugars, and small molecule compounds have been reported (for example, see International Publication Nos. 2007 / 004748, 2008 / 054030, 2013 / 027796, 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979; Nomura Y et al., Nucleic Acids Res., 2010 Nov; 38(21): 7822-9; Miyakawa et al., 2010 Nov; 38(21): 7822-9). S et al., RNA., 2008 Jun; 14(6): 1154-63 and the scientific paper mentioned below). The first affinity part and the second affinity part are preferably the same.

[0789] In addition, the first affinity portion and the second affinity portion that can be used as a substance having affinity for the constant region of the antibody heavy chain can be obtained by any method. For example, such a substance can be obtained by screening a substance having affinity for the constant region of the antibody heavy chain from a library of any substance (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) (e.g., a high throughput screening method, a phage display method, a SELEX method, an mRNA display method, a ribosome display method, a cDNA display method, a yeast display method). In addition, when screening for a substance having affinity for a specific region (e.g., CH2 domain, CH3 domain) in the Fc region or constant region of an antibody, by using a partial peptide present in a specific region (e.g., CH1, CH2, CH3) of the Fc region of various antibodies (e.g., IgG, IgA, IgM, IgD, IgE), a substance that can selectively bind to such a region can be effectively obtained.

[0790] The first affinity portion and the second affinity portion may have affinity for the constant region of the antibody heavy chain. For example, the first affinity portion and the second affinity portion may also have affinity for the Fc region of the antibody heavy chain. Alternatively, the first affinity portion and the second affinity portion may have affinity for the CH1 domain, CH2 domain, or CH3 domain, or a region spanning these domains (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 affinity portion and the second affinity portion may also have affinity for the same or different CHX domains (X is 1, 2, or 3), preferably having affinity for the same domain. Here, the phrase "having affinity for a CHX domain" is not particularly limited; it suffices to have affinity for at least a portion of the CHX domain. It may have affinity for a portion of the CHX domain, or it may have affinity for a region spanning the CHX domain and another CHX domain (e.g., an adjacent region). Thus, the first affinity portion and the second affinity portion having affinity for the CH2 domain may have affinity for only a portion of the CH2 domain, or may have affinity for a region spanning the CH2 domain and the CH1 domain or the CH3 domain (e.g., an adjacent region between the CH1 domain and the CH2 domain, or an adjacent region between the CH2 domain and the CH3 domain). The first affinity portion and the second affinity portion having affinity for the CH2 domain may preferably have affinity for only a portion of the CH2 domain, or may have affinity for a region spanning the CH2 domain and the CH3 domain (e.g., the adjacent region between the CH2 domain and the CH3 domain). More preferably, they may have affinity for only a portion of the CH2 domain.

[0791] The affinity substance or salt thereof of the present invention can directly link the first affinity moiety and the second affinity moiety, and a linker can be inserted between the first affinity moiety and the second affinity moiety. If the first and second affinity moieties are close to each other in the binding site of the constant region of the antibody heavy chain and the first affinity substance and the second affinity moiety are sufficiently large, and there is no need to adjust the distance between the first and second affinity moieties, the first and second affinity moieties can be directly linked. On the other hand, if the first and second affinity moieties are not close to each other in the binding site of the constant region of the antibody heavy chain and the first affinity substance and the second affinity moiety are not sufficiently large, and there is a need to adjust the distance between the first and second affinity moieties, a linker can be inserted between the first affinity substance and the second affinity moiety. As the linker, a bivalent group can be used. The bivalent group may be substituted or unsubstituted. As the bivalent group, the above-mentioned groups can be mentioned. As the substituent when the bivalent group is substituted, the above-mentioned substituents can be mentioned. In addition, as a linker, for example, peptides, nucleic acids, sugars, other polymeric substances (for example, polyethylene glycol), bivalent hydrocarbon groups (for example, alkyl chains) and other substances can be used. Those skilled in the art can appropriately determine the presence or absence of a linker and the type of linker based on the type of the first affinity part and the second affinity part used. Preferably, a linker can be inserted from the viewpoint of optimizing the binding of the first affinity part and the second affinity part to the constant region of the antibody heavy chain.

[0792] The constant region of the antibody heavy chain to which the first affinity portion and the second affinity portion have affinity can be derived from the above-mentioned animals (e.g., mammals, birds). The constant region of the antibody heavy chain can preferably be a mammalian constant region, more preferably a primate constant region or a rodent constant region, and even more preferably a human constant region.

[0793] The constant region of the antibody heavy chain to which the first affinity portion and the second affinity portion have affinity may be a 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 a constant region of a bivalent antibody (e.g., IgG, IgD, IgE), more preferably a constant region of IgG.

[0794] Preferably, the first affinity portion and the second affinity portion can be affinity peptides having affinity for the constant region of the antibody heavy chain. Many such affinity peptides have been reported. For example, examples of such affinity peptides are shown below:

[0795] (1) Various IgG-binding peptides having affinity for a specific region (CH2 domain) of all human IgGs (i.e., human IgG1, IgG2, IgG3, and IgG4, hereinafter the same) (e.g., see International Publication Nos. 2008 / 054030, 2013 / 027796, and 2016 / 186206);

[0796] (2) Protein A mimetic peptides (PAM) that have affinity for the specific region (CH2 domain) of all human IgG (for example, see Fassina G et al., JOURNAL OF MOLECULAR RECOGNITION, 1996, Vol. 6, 564-569);

[0797] (3) EPIHRSTLTALL (SEQ ID NO: 27) having affinity for a specific region (CH2 domain) of all human IgG (for example, see Ehrlich GK et al., J. Biochem. Biophys. Methods, 2001, Vol. 49, 443-454);

[0798] (4) (NH2-Cys1-X1-X2-X3-X4)2-Lys-Gly-OH, which has affinity for the specific region (Fc region) of all human IgG (for example, see Ruvo M et al., ChemBioChem, 2005, Vol. 6, 1242-1253);

[0799] (5) FARLVSSIRY (SEQ ID NO: 28), FGRLVSSIRY (SEQ ID NO: 29), and TWKTSRISIF (SEQ ID NO: 30) that have affinity for the specific region (Fc region) of all human IgG (for example, see Krook M et al., Journal of Immunological Methods, 1998, Vol. 221, 151-157);

[0800] (6) QSYP (SEQ ID NO: 31) having affinity for a specific region of all human IgG (e.g., see Jacobs JM et al., Bio. Techniques, 2003, Vol. 34, 132-141);

[0801] (7) HWRGWV (SEQ ID NO: 32), HYFKFD (SEQ ID NO: 33), and HFRRHL (SEQ ID NO: 34), which have affinity for the specific region (Fc region) of all human IgG (for example, see Carbonell RG et al., Journal of Chromatography A, 2009, Vol. 1216, 910-918);

[0802] (8) DAAG (SEQ ID NO: 35) having affinity for the specific region (Fc region) of all human IgG (for example, see Lund LN et al., Journal of Chromatography A, 2012, Vol. 1225, 158-167);

[0803] (9) Fc-I, Fc-II, and Fc-III that have affinity for all human IgG specific regions (Fc regions) (e.g., see Warren L. Delano et al., Science, 2000, Vol. 287, 1279-1283; International Publication No. 2001 / 045746); and

[0804] (10) NARKFYKG (SEQ ID NO: 36) and NKFRGKYK (SEQ ID NO: 37) that have affinity for the specific region (Fc region) of all human IgG (for example, see Biochemical Engineering Journal, 2013, Vol. 79, 33-40);

[0805] (11) Protein A, protein G, protein L or protein Z, or fragments thereof, that have affinity for the specific region (Fc region) of all human IgG (e.g., Moks T et al., Eur J Biochem. 1986 May 2; 156(3): 637-43; Sjobring UJ et al., Biol Chem. 1991 Jan 5; 266(1): 399-405; Graille M et al., Structure. 2001 Aug; 9(8): 679-87; Nilsson B et al., Protein Eng. 1987 Feb-Mar; 1(2): 107-13);

[0806] (12) A variety of IgG-binding peptides that have affinity for specific regions (Fc region or CH2 domain) of all human IgG (for example, see International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, and International Publication No. 2020 / 090979).

[0807] Alternatively, affinity peptides can be obtained by the aforementioned screening methods (eg, methods using the aforementioned libraries or the aforementioned display methods).

[0808] As the amino acid residues constituting the affinity peptide, residues of the 20 common natural amino acids or non-natural amino acids constituting proteins can be used. Examples of the 20 common natural amino acids constituting proteins include L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (hereinafter, the symbol L is omitted).

[0809] In a specific embodiment, one of the first affinity peptide and the second affinity peptide may be an affinity peptide having one lysine residue, and the other may be an affinity peptide having no lysine residue.

[0810] As affinity peptides having affinity for the constant region of the antibody heavy chain and having one lysine residue, many peptides have been reported (for example, see International Publication No. 2016 / 186206, International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, and International Publication No. 2020 / 090979). Therefore, in the present invention, such peptides can be used as one of the first affinity peptide and the second affinity peptide.

[0811] More specifically, as an affinity peptide having affinity for the constant region of the antibody heavy chain and having one lysine residue, the following affinity peptides can be used:

[0812] (1) an affinity peptide comprising the amino acid sequence of SEQ ID NOs: 39 to 72 of International Publication No. 2018 / 199337;

[0813] (2) an affinity peptide comprising the amino acid sequence of SEQ ID NOs: 5, 6, 37 to 100 of International Publication No. 2019 / 240288;

[0814] (3) an affinity peptide comprising the amino acid sequence of SEQ ID NOs: 5, 8 to 57, 68 to 92 of International Publication No. 2019 / 240287;

[0815] (4) an affinity peptide having a QET at the N-terminus (SEQ ID NOs: 7 to 10, 22 to 25, 52, 53 of International Publication No. 2020 / 090979); and

[0816] (5) An affinity peptide having one lysine residue among the affinity peptides listed above (1) to (12) as examples of the affinity peptide.

[0817] In a specific embodiment, examples of affinity peptides having affinity for the constant region of the antibody heavy chain and having one lysine residue include the following affinity peptides (1) to (4):

[0818] (1) an affinity peptide comprising the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38);

[0819] (2) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​in the amino acid sequence of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38) are substituted with amino acid residues other than lysine and cysteine;

[0820] (3) an affinity peptide comprising the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and

[0821] (4) An affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: an amino acid sequence in which one or two amino acid residues other than lysine and cysteine ​​in the amino acid sequence of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) are substituted with amino acid residues other than lysine and cysteine.

[0822] Here, the two cysteine ​​residues contained in the above amino acid sequence may be cross-linked via a disulfide bond.

[0823] As the constant region of antibody heavy chain, there is affinity and the affinity peptide without lysine residues, many peptides have been reported. In addition, the constant region of antibody heavy chain has affinity and the lysine residue in the above-mentioned affinity peptide with 1 lysine residue, in many cases it is not for maintaining the affinity of the constant region of antibody heavy chain, but for covalent bonding with other parts (for example, partial compounds comprising reactive groups) to derive affinity material introduced (for example, with reference to 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), therefore even if such lysine residue is substituted into other amino acid residues, the affinity of the constant region of antibody heavy chain can also be maintained. Therefore, as an affinity peptide that has affinity for the constant region of the antibody heavy chain and does not have a lysine residue, an affinity peptide that has affinity for the constant region of the antibody heavy chain can be used, which is an affinity peptide that has affinity for the constant region of the antibody heavy chain and has one lysine residue, in which the lysine residue is substituted with another amino acid residue (preferably a normal natural amino acid residue constituting a protein other than a lysine residue and a cysteine ​​residue).

[0824] More specifically, as affinity peptides having affinity for the constant region of the antibody heavy chain and having no lysine residues, the following affinity peptides can be used:

[0825] (1) SEQ ID NOs: 20 to 38, 73 to 75 (when Xaa1 is other than a lysine residue) and SEQ ID NO: 92 of International Publication No. 2018 / 199337;

[0826] (2) SEQ ID NOs: 7, 11-14, 108 of International Publication No. 2019 / 240288;

[0827] (3) Affinity peptides in which the lysine residue in the above (1) to (4) listed as examples of affinity peptides having affinity for the constant region of the antibody heavy chain and having one lysine residue is substituted with another amino acid residue (preferably an amino acid residue other than a cysteine ​​residue); and

[0828] (4) An affinity peptide that does not have a lysine residue among the affinity peptides listed above (1) to (12) as examples of the affinity peptide.

[0829] In a specific embodiment, examples of affinity peptides having no lysine residue include the following affinity peptides (5) to (10):

[0830] (5) an affinity peptide comprising the amino acid sequence (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40);

[0831] (6) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: 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 lysine residues and amino acid residues other than cysteine ​​residues;

[0832] (7) an affinity peptide comprising the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41);

[0833] (8) An affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: 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 amino acid residues other than lysine residues and cysteine ​​residues;

[0834] (9) an affinity peptide comprising the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and

[0835] (10) An affinity peptide having affinity for the constant region of an antibody heavy chain comprising the following amino acid sequence: 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 amino acid residues other than cysteine ​​residues.

[0836] Here, the two cysteine ​​residues contained in the above amino acid sequence may be cross-linked via a disulfide bond.

[0837] The substitution of an amino acid residue may be a conservative substitution. The term "conservative substitution" refers to replacing a specified amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. For example, such a family includes 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 non-polar 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-containing (e.g., alcoholic, phenolic) 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 amino acids with non-polar side chains are sometimes collectively referred to as neutral amino acids. Preferably, conservative substitutions of amino acids may be substitutions between aspartic acid and glutamic acid, substitutions between arginine, lysine and histidine, substitutions between tryptophan and phenylalanine, substitutions between phenylalanine and valine, substitutions between leucine, isoleucine and alanine, and substitutions between glycine and alanine.

[0838] When the first affinity portion and the second affinity portion are affinity peptides, the affinity substance of the present invention or a salt thereof may be directly linked to the first affinity peptide and the second affinity peptide, or a linker may be inserted between the first affinity peptide and the second affinity peptide. However, from the perspective of optimizing the binding of the first affinity peptide and the second affinity peptide to the constant region of the antibody heavy chain, insertion of a linker is preferred. When the first affinity portion and the second affinity portion are affinity peptides, the linker is preferably a peptide linker.

[0839] The number of amino acid residues constituting the peptide linker can be appropriately set according to conditions such as the type of amino acid residue (e.g., α-amino acid, β-amino acid, γ-amino acid. Preferably, α-amino acid). For example, the peptide linker can be a peptide linker consisting of 20 or more amino acid residues. The peptide linker can be a peptide linker consisting 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 can also be a peptide linker consisting of less than 50, less than 49, less than 48, less than 47, less than 46, or less than 45 amino acid residues.

[0840] As the amino acid residue constituting the peptide linker, the residue of the above-mentioned natural amino acid or non-natural amino acid can be used. Preferably, the amino acid residue constituting the peptide linker can also only include the above-mentioned natural amino acid residue. As the amino acid residue suitable for the peptide linker, for example, alanine, proline, serine and glycine can be enumerated, but are not limited to these. As the peptide linker, the peptide linker disclosed in International Publication No. 2021 / 112249 and International Publication No. 2011 / 144756 can also be used.

[0841] Preferably, the affinity substance or salt thereof of the present invention may be an affinity substance or salt thereof wherein the first affinity portion and the second affinity portion are affinity peptides, and a linker is included between the first affinity peptide and the second affinity peptide. Such an affinity substance may be represented by the following formula (A):

[0842] AP1-L A -AP2(A)

[0843] Where,

[0844] AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain,

[0845] AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain,

[0846] L A Indicates a connector.

[0847] In formula (A) and other formulas related to the present invention, a hyphen (-) indicates that two units present on either side thereof 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.

[0848] The definitions, examples, and preferred examples of the first affinity peptide and the second affinity peptide represented by AP1 and AP2, respectively, are as described above.

[0849] The linker represented by LA is a divalent group. The divalent group may be substituted or unsubstituted. Examples of the divalent group include the divalent groups described above. When the divalent group is substituted, examples of the substituent include the substituents described above. Preferred linkers include, for example, peptides, nucleic acids, sugars, other polymeric substances (e.g., polyethylene glycol), and divalent hydrocarbon groups (e.g., alkyl chains).

[0850] More preferably, the affinity substance or salt thereof of the present invention may be an affinity polypeptide or salt thereof, wherein the first affinity portion and the second affinity portion are affinity peptides, and a peptide linker is included between the first affinity peptide and the second affinity peptide. Such an affinity polypeptide may be represented by the following formula (A'):

[0851] AP1-PL A -AP2(A')

[0852] Where,

[0853] AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and present at the N-terminal side of the affinity polypeptide.

[0854] AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain and present at the C-terminal side of the affinity polypeptide.

[0855] PL A represents a peptide linker.

[0856] Regarding the affinity substance or its salt of affinity polypeptide etc., as the amino acid residue constituting it, the residue of the above-mentioned natural amino acid or non-natural amino acid can be included. In the case where the affinity substance only comprises the above-mentioned natural amino acid residue, the affinity substance can be manufactured by, for example, an expression system of the polypeptide using a host cell, a cell-free synthesis system or an organic synthesis system (for example, solid phase synthesis). In the case where the affinity substance comprises a non-natural amino acid residue, the affinity substance can be manufactured by, for example, an organic synthesis system (for example, solid phase synthesis). Preferably, the affinity substance also can comprise only natural amino acid residues to allow a large amount of affinity substances to be manufactured by an expression system of the polypeptide using a host cell or a cell-free synthesis system.

[0857] 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 terminals of the affinity substance may be appropriately protected. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl (acyl) groups (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkoxycarbonyl 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 carboxyl group include groups capable of forming esters or amides. Examples of groups capable of forming esters or amides include alkoxy groups (e.g., methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy), aryloxy groups (e.g., phenoxy and naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups.

[0858] In addition, when the N-terminal amino acid of the affinity substance is glutamic acid (E) or glutamine (Q), the side chains thereof can be used to protect the N-terminus. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid can have a cyclic structure similar to pyroglutamic acid. In addition, when the N-terminal amino acid is glutamine, the protected N-terminal glutamine can have a cyclic structure similar to pyroglutamic acid by reacting the N-terminal amino group (NH2) with the amide group present in its side chain (pyroglutamylation). Therefore, the N-terminal amino acid can preferably be glutamic acid or glutamine.

[0859] When the affinity substance is an affinity polypeptide, the affinity polypeptide may further include a tripeptide consisting of Gln-Glu-Thr (QET) at the N-terminus. In this case, by using a polypeptide expression system of a host cell, the N-terminal amino group can be protected by pyroglutamylation of Q, and the affinity polypeptide can be secreted and produced in large quantities and simply (see Examples, International Publication No. 2013 / 062029, International Publication No. 2020 / 090979). In this case, a signal peptide (CspBss) such as a signal peptide consisting of the amino acid sequence of MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 42) can be added to the N-terminal side of QET (see Examples, International Publication No. 2013 / 062029, International Publication No. 2020 / 090979).

[0860] The affinity substance of the present invention or its salt can be used as a synthetic intermediate of the compound of the present invention or its salt comprising an affinity substance and an antibody reactive group. Therefore, in order to simply realize the uniform synthesis of the compound of the present invention or its salt comprising an antibody reactive group, the affinity substance of the present invention or its salt, in addition to the affinity substance, can also be derived to comprise only one specific reactive group, and the specific reactive group can react specifically with a portion of the compound comprising the antibody reactive group. In the case where the affinity substance only comprises one specific reactive group, since both the affinity substance and the antibody reactive group can react specifically via the specific reactive group in the affinity substance, the compound of the present invention or its salt comprising an affinity substance and an antibody reactive group can be easily manufactured into a uniform compound.

[0861] Examples of the specific reactive group include the following.

[0862] (1) amino group (NH2, NHR3, NR3R4. R3 and R4 are each independently a monovalent group as described above, preferably a monovalent hydrocarbon group, more preferably an alkyl group, and even more preferably an alkyl group having 1 to 6 carbon atoms);

[0863] (2) Residues that can react with amino groups. For example, activated ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethyl residues, and diazonium terephthalate residues;

[0864] (3) carboxyl group (COOH);

[0865] (4) Residues that can react with carboxyl groups, for example, the amino groups mentioned above;

[0866] (5) hydroxyl group (OH) (including alcoholic hydroxyl group and phenolic hydroxyl group); and

[0867] (6) Residues that can react with hydroxyl groups, for example, diazo residues, azodicarboxylate residues, and 2,3-dihydro-1H-pyrazin-6-one residues.

[0868] The specific reactive group may preferably be (1) to (4). More preferably, the specific reactive group may be (1) or (2), or (3) or (4). Alternatively, more preferably, the specific reactive group may be (1) or (3). The specific reactive group may further preferably be (1), and may particularly preferably be an amino group (NH2).

[0869] The affinity substance or its salt may contain only one amino acid residue containing a specific reactive group. In this case, the affinity polypeptide may contain only one amino acid residue (e.g., a lysine residue, an aspartic acid residue, a glutamic acid residue, a tyrosine residue, a threonine residue, or a serine residue) having (a) a specific reactive group (e.g., an amino group, a carboxyl group, or a hydroxyl group) on its side chain. When the affinity polypeptide contains only one lysine residue having an amino group on 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 having a carboxyl group on its side chain (e.g., an aspartic acid residue or a glutamic acid residue), the C-terminus of the affinity polypeptide is preferably protected (the N-terminus may also be protected).

[0870] Alternatively, the affinity polypeptide may be a polypeptide that does not contain an amino acid residue having an amino group in its side chain (e.g., a lysine residue) and, by having an amino group at its N-terminus, contains only one amino group as a specific reactive group. The affinity polypeptide may also be a polypeptide that does not contain an amino acid residue having a carboxyl group in its side chain (e.g., an aspartic acid residue, a glutamic acid residue) and, by having a carboxyl group at its C-terminus, contains only one carboxyl group as a specific reactive group.

[0871] Preferably, the affinity polypeptide containing only one specific reactive group may be a polypeptide containing an amino acid residue with an amino group in its side chain. When the affinity polypeptide is produced by an organic synthesis system (e.g., solid-phase synthesis), not only lysine residues, which are natural amino acids constituting proteins, but also other amino acid residues with amino groups in their side chains (e.g., ornithine) can be used. More preferably, to facilitate production not only by organic synthesis systems but also by polypeptide expression systems using host cells and cell-free synthesis systems, the affinity polypeptide containing only one specific reactive group may be a polypeptide containing only one lysine residue with an amino group (NH2) 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 at a position proximal to the first or second affinity peptide (e.g., a position 10 or fewer, 5 or fewer, or 1 to 3 amino acid residues from the first or second affinity peptide). Preferably, the lysine residue is contained in either the first affinity peptide or the second affinity peptide.

[0872] Preferably, the affinity substance represented by the above formula (A) or a salt thereof comprises the first affinity peptide and the second affinity peptide represented by AP1 and AP2, respectively, and L A The linker may further have the following properties (1) or (2), preferably the property (1).

[0873] (1) Affinity substances containing only one amino group as a specific reactive group or salts thereof:

[0874] (1-1) An affinity substance or a salt thereof, wherein: (i) the first affinity peptide is an affinity peptide comprising only one amino acid residue (preferably a lysine residue) having an amino group in its side chain and having a protected N-terminus; and (ii) the second affinity peptide is an affinity peptide not containing an amino acid residue having an amino group in its side chain, and the linker is a linker not containing an amino group;

[0875] (1-2) 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 an amino group in its side chain and has an unprotected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker that does not contain an amino group;

[0876] (1-3) 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 an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue (preferably a lysine residue) having an amino group in its side chain, and the linker is a linker that does not contain an amino group;

[0877] (1-4) 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 an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker is a linker containing only one amino group.

[0878] In order to suppress unwanted reactions (e.g., intramolecular reactions or intermolecular reactions), the 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 reactive with the amino group (e.g., a carboxyl group). Therefore, in (1-1) to (1-4) above, the first affinity peptide may be an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, the second affinity peptide may be an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and / or has a protected C-terminus, and the linker may be a linker that does not contain a carboxyl group.

[0879] (2) Affinity substances containing only one carboxyl group as a specific reactive group or salts thereof:

[0880] (2-1) An affinity substance or a salt thereof, wherein: (i) the first affinity peptide is an affinity peptide comprising only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid), (ii) the second affinity peptide is an affinity peptide not containing an amino acid residue having a carboxyl group in its side chain, and the linker is a linker not containing a carboxyl group;

[0881] (2-2) 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 carboxyl group in its side chain, (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) and has a protected C-terminus, and the linker is a linker that does not contain a carboxyl group;

[0882] (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 carboxyl group in its side chain, (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain and has an unprotected C-terminus, and the linker is a linker that does not contain a carboxyl group;

[0883] (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 carboxyl group in its side chain, (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain and has a protected C-terminus, and the linker is a linker containing only one carboxyl group.

[0884] In order to suppress unwanted reactions (e.g., intramolecular reactions or intermolecular reactions), the affinity substance or its salt containing only one carboxyl group as a specific reactive group may be an affinity substance or its salt that does not contain a group reactive with the carboxyl group (e.g., an amino group). Therefore, in (2-1) to (2-4) above, the first affinity peptide may be an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and / or has a protected N-terminus, the second affinity peptide may be an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the linker may be a linker that does not contain an amino group.

[0885] Preferably, the affinity substance represented by the above formula (A') or a salt thereof comprises the first affinity peptide and the second affinity peptide represented by AP1 and AP2, respectively, and PL A The peptide linker shown may further have the following property (1') or (2'), preferably the property (1').

[0886] (1') Affinity substances containing only one amino group as a specific reactive group or salts thereof:

[0887] (1-1′) an affinity substance or a salt thereof, wherein (i) the first affinity peptide is an affinity peptide comprising only one amino acid residue (preferably a lysine residue) having an amino group in its side chain and having a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide not containing an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker not containing an amino acid residue having an amino group in its side chain;

[0888] (1-2′) 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 an amino group in its side chain and has an unprotected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker that does not contain an amino acid residue having an amino group in its side chain;

[0889] (1-3′) 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 an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue (preferably a lysine residue) having an amino group in its side chain, and the peptide linker is a peptide linker that does not contain an amino acid residue having an amino group in its side chain;

[0890] (1-4') 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 an amino group in its side chain and has a protected N-terminus, and (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having an amino group in its side chain, and the peptide linker is a peptide linker that contains only one amino acid residue having an amino group in its side chain.

[0891] In order to suppress unwanted reactions (e.g., intramolecular reactions or intermolecular reactions), the 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 reactive with the amino group (e.g., a carboxyl group). Therefore, in (1-1') to (1-4') above, the first affinity peptide may be an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, the second affinity peptide may be an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain, and / or has a protected C-terminus, and the peptide linker may be a peptide linker that does not contain an amino acid residue having a carboxyl group in its side chain.

[0892] (2') Affinity substances containing only one carboxyl group as a specific reactive group or salts thereof:

[0893] (2-1′) An affinity substance or a salt thereof, wherein (i) the first affinity peptide is an affinity peptide comprising only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid), (ii) the second affinity peptide is an affinity peptide not containing an amino acid residue having a carboxyl group in its side chain, and the peptide linker is a peptide linker not containing an amino acid residue having a carboxyl group in its side chain;

[0894] (2-2′) 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 carboxyl group in its side chain, (ii) the second affinity peptide is an affinity peptide that contains only one amino acid residue having a carboxyl group in its side chain (preferably an acidic amino acid residue such as aspartic acid or glutamic acid) and has a protected C-terminus, and the peptide linker is a peptide linker that does not contain an amino acid residue having a carboxyl group in its side chain;

[0895] (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 carboxyl group in its side chain, (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group in its side chain and has an unprotected C-terminus, and the peptide linker is a peptide linker that does not contain an amino acid residue having a carboxyl group in its side chain;

[0896] (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 carboxyl group on its side chain, (ii) the second affinity peptide is an affinity peptide that does not contain an amino acid residue having a carboxyl group on its side chain and has a protected C-terminus, and the peptide linker is a linker containing only one amino acid residue having a carboxyl group on its side chain.

[0897] In order to suppress unwanted reactions (e.g., intramolecular reactions or intermolecular reactions), the affinity substance or its salt containing only one carboxyl group as a specific reactive group may be an affinity substance or its salt that does not contain a group reactive with the carboxyl group (e.g., an amino group). Therefore, in (2-1') to (2-4') above, the first affinity peptide may be an affinity peptide that does not contain an amino acid residue having an amino group in its side chain and / or has a protected N-terminus, the second affinity peptide may be an affinity peptide 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.

[0898] 2-2. Inventions Related to Affinity Substances or Salts thereof

[0899] When the affinity substance or salt thereof of the present invention is an affinity polypeptide comprising a first affinity peptide and a second affinity peptide having affinity for the constant region of the antibody heavy chain, such an affinity polypeptide can be produced 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. The present invention also provides such a polynucleotide and host cell, as well as an expression vector that can be used to produce the host cell.

[0900] The polynucleotide of the present invention is a polynucleotide encoding the affinity polypeptide of the present invention. The polynucleotide of the present invention can be DNA or RNA, but is preferably DNA.

[0901] The host cell of the present invention can be made, for example, by using a method (for example, competent cell method, electroporation) or genome modification technology of an expression vector comprising the polynucleotide of the present invention. In the case where the expression vector is an integrative (integrative) vector that produces 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, in the case where the expression vector is a non-integrative vector that does not produce 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, but can be present in the host cell independently of the genomic DNA with the state of the expression vector. Alternatively, according to genome editing technology (for example, CRISPR / Cas system, transcription activator-like effector nuclease (Transcription Activator-LikeEffector Nucleases, TALEN)), the expression unit can be integrated into the genomic DNA of the host cell, and the host cell-specific expression unit can be modified.

[0902] 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 such as a terminator, a ribosome binding site, and a drug-resistance gene that function in the host cell. Examples of drug-resistance genes include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0903] The expression vector may further comprise a region capable of homologous recombination with the host cell's genome for homologous recombination with the host cell's genomic DNA. For example, the expression vector may be designed so that the expression unit contained therein is located between a pair of homology regions (e.g., homology arms or loxP or FRT homologous to specific sequences in the host cell's genome). The host cell genomic region into which the expression unit is to be introduced (the target of the homology region) is not particularly limited and may be the locus of a gene that is highly expressed in the host cell.

[0904] The expression vector can be a plasmid, a viral vector, a phage or an artificial chromosome. The expression vector can also be an integrative vector or a non-integrative vector. The integrative vector can be a vector of a type that is integrated into the genome of the host cell. Alternatively, the integrative vector can be a vector of a type that is only integrated into the genome of the host cell by a portion thereof (e.g., an expression unit). Moreover, the expression vector can be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector can also be a universal 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.

[0905] As host cells for expressing the affinity polypeptides of the present invention, for example, various prokaryotic cells represented by Escherichia coli, Corynebacterium bacteria [e.g., Corynebacterium glutamicum], and Bacillus bacteria [e.g., Bacillus subtilis], and various eukaryotic cells represented by Saccharomyces bacteria [e.g., Saccharomyces cerevisiae], Pichia bacteria [e.g., Pichia stipitis], and Aspergillus bacteria [e.g., Aspergillus oryzae] can be used. Alternatively, insect cells, plant cells, and animal cells (e.g., mammalian cells such as Chinese hamster ovary (CHO) cells) can be used as hosts. Strains lacking a predetermined gene can also be used as hosts. Examples of host cells include those harboring an expression vector in the cytoplasm and those into which the gene of interest has been introduced into the genome.

[0906] When the affinity polypeptide of the present invention comprises a tripeptide composed of Gln-Glu-Thr (QET) at the end, it is preferred to use an affinity polypeptide that can be modulated by a secretory production method for a polypeptide using coryneform bacteria as a host (International Publication No. 2013 / 062029). This method is suitable for modulating affinity polypeptides because the three N-terminal residues Gln-Glu-Thr (QET) of the Csp mature protein can be added to the N-terminus of the target polypeptide, and polypeptides containing a glutamine residue (Q) at the N-terminus can be easily and in large quantities. In this case, various signal peptides such as a signal peptide (CspBss) composed of the amino acid sequence of MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 42) can be added to the N-terminal side of QET (see Examples, International Publication No. 2013 / 062029, International Publication No. 2020 / 090979). Examples of coryneform bacteria that can be used in this method include bacteria belonging to the genus Corynebacterium (eg, Corynebacterium glutamicum and Corynebacterium stationis) and bacteria belonging to the genus Brevibacterium.

[0907] The host cells of the present invention can be cultured using a prescribed culture device (e.g., a test tube, a flask, a small fermenter), for example, in a culture medium having the composition described below. The culture conditions can be set appropriately. Specifically, the culture temperature can be 10°C to 37°C, the pH can be 6.5 to 7.5, and the culture time can be 1 hour to 100 hours. In addition, the culture can be carried out while managing the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture solution is sometimes used as a control index. The aeration / agitation conditions can be controlled so that the relative dissolved oxygen concentration DO value when the oxygen concentration in the atmosphere is 21% is, for example, not less than 1 to 10%, preferably not less than 3% to 8%. In addition, the culture can be a batch culture or a fed-batch culture. In the case of fed-batch culture, a solution as a sugar source or a solution containing phosphoric acid can be added to the culture solution in sequence, either continuously or discontinuously, to continue the culture.

[0908] As promoters for expressing the polynucleotide of the present invention, promoters commonly used for heterologous protein production in Escherichia coli can be used. Examples include strong promoters such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, and T5 promoter, with PhoA, PhoC, and lac being preferred. Furthermore, as vectors, for example, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and derivatives thereof can be used.

[0909] Alternatively, a transcription termination sequence, i.e., a terminator, may be linked downstream of the polynucleotide of the present invention. Examples of such terminators include the T7 terminator, the fd phage terminator, the T4 terminator, the tetracycline resistance gene terminator, and the Escherichia coli trpA gene terminator.

[0910] As culture medium, M9-Casamino Acids culture medium, LB culture medium etc. can be used to be generally used for cultivating the culture medium of Escherichia coli. Culture medium can contain the carbon source, nitrogen source, coenzyme (for example, pyridoxine hydrochloride) of regulation. Specifically, peptone, yeast extract, NaCl, glucose, MgSO , ammonium sulfate, potassium dihydrogen phosphate, ferrous sulfate, manganese sulfate etc. can be used. In addition, culture condition, production induction condition are suitably selected according to the kind of marker, promoter, host bacteria etc. of used carrier.

[0911] The following methods are available for recovering the affinity polypeptides of the present invention. After recovering the transformed cells of the present invention, the affinity polypeptides of the present invention can be obtained as a fragment or lysate by disrupting the cells (e.g., by sonication or homogenization) or lysing them (e.g., by lysozyme treatment). If the affinity polypeptide is secreted or leaked out of the cells, a sterilized solution containing the affinity polypeptide can be obtained from the culture medium by centrifugation or membrane filtration. The affinity polypeptides of the present invention can be obtained by subjecting these fragments, lysates, and sterilized solutions to extraction, precipitation, filtration, column chromatography, and other methods.

[0912] 3. Compound or its salt

[0913] The compound of the present invention or a salt thereof comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of an antibody heavy chain, and (B) an antibody-reactive group. Definitions, examples, and preferred examples of the affinity substance and its constituent elements (e.g., an affinity portion such as an affinity peptide and a linker between the affinity portions such as a peptide linker) are as described above.

[0914] As antibody reactive groups, reactive groups for amino acid residues with easily reactive side chains in the amino acid residues constituting the antibody (protein) can be used. Among the 20 natural amino acids constituting the protein, glycine without side chains and alanine, isoleucine, leucine, phenylalanine and valine, whose side chains are hydrocarbon groups, are inert to common reactions. Therefore, antibody reactive groups are groups that can react with any one or more (e.g., two, three, 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, four) reactive groups can be included in the compound of the present invention or its salt, but one reactive group can preferably be included in the compound of the present invention or its salt.

[0915] Preferably, the antibody reactive group is a group that can react with the side chain of any one of the 14 amino acids constituting the protein. More preferably, the antibody reactive group is a reactive group specific to the side chain of one of lysine, tyrosine, tryptophan or cysteine, and further preferably, it is a reactive group specific to the side chain of any one of lysine, tyrosine or tryptophan. Particularly preferably, it is a reactive group specific to the side chain of lysine or tyrosine, preferably to the side chain of lysine. For details of such reactive groups, for example, reference is made to International Publication No. 2016 / 186206, International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, and International Publication No. 2020 / 090979.

[0916] The reactive group specific for the side chain of a lysine residue is a group that reacts specifically with the amino group (NH2) present in the side chain of the lysine residue. Examples thereof include an activated ester residue (e.g., 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, a diazonium terephthalate residue, an α-halogen-substituted acetamide, and an α-halogen-substituted methyl ketone. The reaction between the reactive group specific for the side chain of a lysine residue and the amino group (NH2) present in the side chain of the lysine residue can generate, for example, an amide residue, a urea residue, a pyridine residue, a carbamate residue, or a sulfoneamide residue as a linker.

[0917] In a specific embodiment, the compound of the present invention or a salt thereof can be represented by the following formula (I):

[0918] [Chemical Formula 38]

[0919] RLA (I)

[0920] Where,

[0921] R represents an antibody reactive group,

[0922] L represents a connector,

[0923] A represents an affinity substance comprising a first affinity portion and a second affinity portion 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.

[0924] The linker is a divalent group. The divalent group may be substituted or unsubstituted. Examples of the divalent group include the divalent groups described above. When the divalent group is substituted, examples of the substituent include the substituents described above.

[0925] In a specific embodiment, the compound of the present invention or its salt may further comprise a cleavable moiety between the affinity substance and the antibody reactive group. In this case, the compound represented by the above formula (I) or its salt may also comprise a linker comprising a cleavable moiety.

[0926] The cleavage moiety is a site that can be cleaved under conditions (mild conditions) that do not cause protein denaturation / degradation (e.g., cleavage of an amide bond). Therefore, it can be said that the cleavage moiety is a site (bonds other than amide bonds) that can be cleaved under mild conditions by a specific cleavage treatment. Examples of such specific treatments include: (a) treatment with one or more substances selected from acidic substances, alkaline substances, reducing agents, oxidizing agents, and enzymes; (b) treatment with physicochemical stimulation such as light; or (c) standing (incubation) when using a cleavage linker containing a self-degradable cleavage moiety. Such cleavable linkers and their cleavage conditions are common technical knowledge in the field (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry, 20, 571 (2012); Feng P. et al., Journal of American Chemical Society, 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, JM, Journal of American Chemical Society, 132, 179-28 (2010); Thompson, DH, Journal of Controlled Release, 91, 187 (2003); Schoenmarks, RG, Journal of Controlled Release, 95, 291 (2004)). Reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are described below. Examples of the cleavable moiety include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tert-alkoxycarbamate residues (e.g., tert-butoxycarbamate residues), silane residues, hydrazone residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, residues containing an aromatic ring having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, chain residues containing unsaturated bonds, and glycosyl residues.

[0927] The aromatic ring group having an electron-withdrawing group is preferably selected from an aryl group, an aralkyl group, an aromatic heterocyclic group, an alkyl group having an aromatic heterocyclic group, more preferably an aralkyl group, an alkyl group having an aromatic heterocyclic group. The electron-withdrawing group is preferably bound to the 2-position of the ring. Further preferably, the residue containing an aromatic ring having an electron-withdrawing group is, for example, an aralkyl group having an electron-withdrawing group at the 2-position (for example, a benzyl group). As the electron-withdrawing group, for example, a halogen atom, an alkyl group substituted with a halogen atom (for example, a trifluoromethyl group), a boronic acid residue, a mesyl group, a tosyl group, a triflate group, a nitro group, a cyano group, a phenyl group, a keto group (for example, an acyl group) can be mentioned.

[0928] The definitions, examples and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, and aralkyl that appear as prefixes, suffixes, etc. in association with the name of the residue as the cleavable portion are the same as those described above.

[0929] Examples of the ester residue include conventional ester residues composed of carbon atoms and oxygen atoms [e.g., alkyl esters (e.g., tert-alkoxycarbonyl groups such as tert-butoxycarbonyl), aryl esters (e.g., phenacyl ester, 2-(diphenylphosphino)benzoate), glycosyl ester residues, orthoester residues], ester residues containing sulfur and oxygen atoms (e.g., thioester residues such as α-thiophenyl ester residues and alkylthio ester residues), ester residues containing phosphorus and oxygen atoms (e.g., phosphodiester residues, phosphotriester residues), and activated ester residues (e.g., N-hydroxysuccinimide residues).

[0930] Examples of the sulfone-containing residue include a sulfone residue and a quinolylbenzenesulfonate residue.

[0931] The silane residue is preferably a silane residue having a group selected from an alkyl group, an aryl group, an aralkyl group, and an alkoxy group. Examples of such silane residues include dialkyldialkoxysilane residues (e.g., dimethyldialkoxysilane, diethyldialkoxysilane) and diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane).

[0932] The alkoxyalkyl (ie, alkyloxyalkyl) residue is a group obtained by combining the above-mentioned alkoxy group and alkyl group, and examples thereof include, but are not limited to, a methoxymethyl residue, an ethoxymethyl residue, a methoxyethyl residue, and an ethoxyethyl residue.

[0933] An unsaturated bond-containing chain residue is a residue containing an unsaturated bond portion composed solely of carbon atoms [e.g., vinyl (vinyl / ethenyl), which is the smallest unit having a carbon-carbon double bond, or acetylenyl (ethynyl), which is the smallest unit having a carbon-carbon triple bond], or a residue containing an unsaturated bond portion composed of carbon atoms and heteroatoms (e.g., nitrogen, sulfur, or oxygen atoms) (e.g., aldehyde or cyano). Examples of unsaturated bond-containing chain residues include vinyl ether residues, cyanoethyl residues, ethylene residues, and malondialdehyde residues.

[0934] Examples of acidic substances (also referred to as electrophilic reagents) include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid; and organic acidic substances such as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3-morpholinopropanesulfonic acid, sodium dihydrogen phosphate, citric acid, dodecylsulfuric acid, N-lauroylsarcosine, and trifluoroacetic acid. Examples of sites cleavable by acidic substances include alkoxyarylalkyl residues, tertiary alkoxycarbamate 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.

[0935] Examples of alkaline substances (also called nucleophiles) include inorganic alkaline substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, and ammonium acetate; and organic alkaline substances such as hydroxylamine, triethylamine, and N,N'-diisopropylamine. Examples of sites cleavable by alkaline 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.

[0936] Examples of the reducing agent include cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Examples of the site cleavable by the reducing agent include a disulfide residue, an alkoxyalkyl residue, and an azo residue.

[0937] Examples of the oxidizing agent include sodium periodate and oxidized glutathione, and examples of the site that can be cleaved by the oxidizing agent include a vicinal diol residue and a selenium residue.

[0938] Examples of the enzyme include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspases, proteases, matrix metalloproteinases, lipases, endoglycosidases, and peptide N-glycosidase F (PNGase F). Examples of sites that can be cleaved by the enzyme include ester residues, phosphodiester residues, and glycosyl residues.

[0939] Examples of the photocleavable site include a 2-nitrobenzyl residue, a phenacyl ester residue, an 8-quinolinolbenzenesulfonate residue, a coumarin residue, a phosphotriester residue, a bisarylhydrazone residue, and a bimane dithiopropionic acid residue.

[0940] Examples of the self-degradable cleavable moiety include activated ester residues (eg, N-hydroxysuccinimide residues).

[0941] When the compound of the present invention or a salt thereof contains a cleavable moiety, the cleavable moiety can generate a bioorthogonal functional group on the side of the reactive group by 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.

[0942] When the compound of the present invention or a salt thereof contains a cleavable portion that can generate a bioorthogonal functional group on the side of the reactive group, the compound can be represented by the following formula (Ia):

[0943] [Chemical Formula 39]

[0944] R-L1-CLE(B)-L2-A (Ia)

[0945] Where,

[0946] R represents an antibody reactive group,

[0947] L1 represents the first connector,

[0948] L2 represents the second connector,

[0949] CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the side of the reactive group by cleavage,

[0950] A represents an affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion. The definitions, examples, and preferred examples of the antibody reactive group represented by R, the affinity substance represented by A, and the cleavable portion represented by CLE(B), which can generate a bioorthogonal functional group on the side of the reactive group by cleavage, are as described above.

[0951] The first linker represented by L1 and the second linker represented by L2 may be the same or different divalent groups. The divalent group may be substituted or unsubstituted. Examples of the divalent group include the above-mentioned divalent groups. When the divalent group is substituted, examples of the substituent include the above-mentioned substituents.

[0952] In specific 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.

[0953] The number of atoms constituting the main chain of the first linker and the second linker may be 1 to 9, respectively. This number of atoms may be 2 or more, or 3 or more. This number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, this number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0954] The main chain in the 1st joint and the 2nd joint is made of chain structure or cyclic structure or the structure comprising their combination. In the case that the main chain is a chain structure without 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, in the case that the main chain is a structure comprising 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 the cyclic structure can be determined by counting the number of atoms of the shortest path connecting the two bonds in the cyclic structure (for example, with reference to the bold path of (a) to (d) below). In the case that the main chain is a structure comprising a combination of chain structure and cyclic structure, the number of atoms in the main chain can be determined by adding the number of atoms in the chain structure without cyclic structure and the number of atoms of the shortest path connecting the two bonds in the cyclic structure. For other joints, the counting method of the number of atoms in the main chain is also the same.

[0955] [Chemical Formula 40]

[0956]

[0957] ·It is a bonding key.

[0958] In the case of (a), since the shortest path is the bold path, the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 2.

[0959] In the case of (b), since the shortest path is the bold path, the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 3.

[0960] In the case of (c), since all the paths are the shortest paths (equal distances), the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4.

[0961] In the case of (d), since the path of the fused portion is the shortest path, the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4.

[0962] In a specific embodiment, the compound of the present invention or a salt thereof may be a compound represented by the following formula (Ia-1) or a salt thereof:

[0963] [Chemical Formula 41]

[0964]

[0965] Where,

[0966] X represents a leaving group,

[0967] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[0968] L3 represents the third connector,

[0969] L4 represents the 4th connector,

[0970] S represents a sulfur atom,

[0971] A represents an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain. The definition, examples, and preferred examples of the affinity substance represented by A are as described above.

[0972] The leaving group represented by X is a group that can leave by reacting with the carbon atom in C=W1 adjacent to X and the amino group. Those skilled in the art can appropriately set such a leaving group. Examples of such a leaving group include the following:

[0973] (a)R A -S (here, 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);

[0974] (b)R A -O (here, R Arepresents 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);

[0975] (c)R A -(R B -)N(Here, R A and R B (where N represents a nitrogen atom) 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; or

[0976] (d) Halogen atoms.

[0977] Preferably, the leaving group represented by X can be the following groups:

[0978] (a)R A -S (here, 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);

[0979] (b)R A -O (here, 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); or

[0980] (c)R A -(R B -)N(Here, R A and R B (N represents a nitrogen atom, and 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.)

[0981] More preferably, the leaving group represented by X can be the following groups:

[0982] (a)R A -S (here, 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); or

[0983] (b)R A -O (here, 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).

[0984] More preferably, the leaving group represented by X can be the following groups:

[0985] (a)R A -S (here, R Arepresents 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).

[0986] Particularly preferably, the leaving group represented by X can be the following groups:

[0987] (a')R A -S (here, R A represents a monovalent aromatic hydrocarbon group which may have a substituent (for example, a phenyl group), and S represents a sulfur atom).

[0988] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom. Preferably, W1, W2 and W3 are oxygen atoms.

[0989] The third linker represented by L3 and the fourth linker represented by L4 may be the same or different divalent groups. The divalent group may be substituted or unsubstituted. Examples of the divalent group include the above-mentioned divalent groups. When the divalent group is substituted, examples of the substituent include the above-mentioned substituents.

[0990] In specific 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.

[0991] The number of atoms constituting the main chain of the third linker and the fourth linker can each be 1 to 9. This number of atoms can be 2 or more, or 3 or more. This number of atoms can be 8 or less, 7 or less, or 6 or less. More specifically, this number of atoms can be 2 to 8, 3 to 7, or 3 to 6.

[0992] In addition, when the compound of the present invention or its salt 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 includes: an azide residue, an alkyne residue (preferably a cyclic group having a carbon-carbon triple bond that can be substituted with the above-mentioned substituents), a tetrazine residue, an olefin residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, and a halogenated carbonyl residue.

[0993] 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):

[0994] [Chemical Formula 42]

[0995]

[0996] Where,

[0997] R represents an antibody reactive group,

[0998] L5 represents the fifth connector,

[0999] L6 represents the 6th connector,

[1000] B represents a group containing a bioorthogonal functional group,

[1001] CLE stands for cutting element.

[1002] A represents an affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion. The definitions, examples, and preferred examples of the antibody reactive group represented by R, the affinity substance represented by A, and the cleavage portion represented by CLE are as described above.

[1003] The fifth linker represented by L5 and the sixth linker represented by L6 may be the same or different divalent groups. The divalent group may be substituted or unsubstituted. Examples of the divalent group include the above-mentioned divalent groups. When the divalent group is substituted, examples of the substituent include the above-mentioned substituents.

[1004] In specific embodiments, the total number of atoms constituting the backbone of the fifth linker and the sixth 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.

[1005] The number of atoms constituting the main chain of the fifth linker and the sixth linker may be 1 to 9, respectively. 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.

[1006] The group containing a bioorthogonal functional group represented by B may be a group consisting solely of the bioorthogonal functional group or a group containing the bioorthogonal functional group and another moiety. Examples of the other moiety include a linker between the bioorthogonal functional group and the linker. The linker is, for example, a divalent group. The divalent group may or may not be substituted. The definitions, examples, and preferred examples of the bioorthogonal functional group, the divalent group, and the substituents when the divalent group is substituted are as described above.

[1007] In a specific embodiment, the compound of the present invention or a salt thereof may be a compound or a salt thereof represented by the following formula (Ib-1):

[1008] [Chemical Formula 43]

[1009]

[1010] Where,

[1011] X represents a leaving group,

[1012] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[1013] L7 represents the 7th connector,

[1014] L8 represents the 8th connector,

[1015] B represents a group containing a bioorthogonal functional group,

[1016] V represents an oxygen atom or a sulfur atom,

[1017] A represents an affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion. Definitions, examples, and preferred examples of the leaving group represented by X, the group comprising a bioorthogonal functional group represented by B, and the affinity substance represented by A are as described above.

[1018] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom. Preferably, W1, W2 and W3 may be an oxygen atom.

[1019] The seventh linker represented by L7 and the eighth linker represented by L8 may be the same or different divalent groups. The divalent group may be substituted or unsubstituted. Examples of the divalent group include the above-mentioned divalent groups. When the divalent group is substituted, examples of the substituent include the above-mentioned substituents.

[1020] In a specific embodiment, the total number of atoms constituting the main chain of the seventh linker and the eighth linker may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[1021] The number of atoms constituting the main chain of the seventh linker and the eighth linker may be 1 to 9, respectively. 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.

[1022] V represents an oxygen atom or a sulfur atom. Preferably, V may be a sulfur atom.

[1023] The compound of the present invention or its salt can easily modify only one heavy chain in the constituent units of an antibody. The compound of the present invention or its salt can also provide an antibody that can easily modify only one heavy chain in the constituent units of an antibody, and the modification is site-selective.

[1024] The above series of compounds or salts thereof can be produced by reacting the affinity substance of the present invention with a portion of a compound containing an antibody-reactive group. For example, such a reaction can be carried out in a suitable organic solvent system (e.g., an organic solvent containing an alkyl halide (e.g., a methyl halide) such as CH2Cl2 and an amine such as triethylamine) at a suitable 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.

[1025] The formation of the above series of compounds or their salts is also confirmed by the specific molecular weight of the raw materials and products, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), NMR or mass spectrometry. Such compounds or their salts can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[1026] 4. Antibodies modified with affinity substances or their salts

[1027] 4-1. Affinity-substance-modified antibody or salt thereof containing at least one affinity substance (comprising a first affinity portion and a second affinity portion)

[1028] The present invention provides an antibody or a salt thereof modified with an affinity substance, wherein the affinity substance is contained in the constant region of the antibody heavy chain, and the affinity substance comprises a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain. Definitions, examples, and preferred examples of the affinity substance and antibody, and the elements constituting them (e.g., affinity portions such as affinity peptides, linkers between affinity portions such as peptide linkers, and constant regions) are as described above.

[1029] Preferably, an antibody or a salt thereof modified with an affinity substance may comprise (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains) and (b) an affinity substance, and (c) the affinity substance is introduced only into the constant region of one heavy chain of the immunoglobulin unit (i.e., an antibody modified with an affinity substance in the constant region of one heavy chain of the immunoglobulin unit, and not in the constant region of the other heavy chain). Definitions, examples, and preferred examples of antibodies, immunoglobulin units, affinity substances, and elements constituting them (e.g., constant regions) are as described above.

[1030] The antibody or salt thereof modified with an affinity substance may contain an affinity substance by modification of a functional group in the side chain of any one or more (e.g., 2, 3, or 4) 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 above-mentioned constant region (preferably an Fc region or a CH2 domain). The antibody or salt thereof modified with an affinity substance may contain an affinity substance by modification of a functional group in the side chain of any one of lysine, tyrosine, tryptophan, or cysteine ​​present in the above-mentioned constant region (preferably an Fc region or a CH2 domain), more preferably by modification of a functional group in the side chain of any one of lysine, tyrosine, or tryptophan, further preferably by modification of a functional group in the side chain of lysine or tyrosine, and particularly preferably by modification of an amino group in the side chain of lysine. The sites of these amino acid residues in the above-mentioned constant region are as described above. The modification sites of the antibody or its salt by the affinity substance can be confirmed by peptide mapping. As described above, the modification can be site-selective. Therefore, in the formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin unit can site-selectively have the corresponding modification unit via the functional groups in the side chains of the above-mentioned amino acid residues.

[1031] Preferably, the antibody modified with an affinity substance contains the above-mentioned affinity substance via modification of the amino group in the side chain of one or more (preferably 1 or 2, more preferably 1) lysine residues in the constant region (preferably the Fc region or CH2 domain) of one heavy chain in the antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains) (in other words, the affinity substance is contained via the amino group in the side chain of a lysine residue in the constant region of one heavy chain of the immunoglobulin unit, and the affinity substance is not contained via the amino group in the side chain of a lysine residue in the constant region of the other heavy chain). More specifically, the site of one or more (preferably 1 or 2, more preferably 1) lysine residues can be 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering (for example, with reference to International Publication No. 2016 / 186206, International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, International Publication No. 2020 / 009165, International Publication No. 2020 / 090979). As described above, the modification can be site-selective. Therefore, in formula (II), (IIa), (IIa-1), (IIb) and (IIb-1) described later, the immunoglobulin unit can selectively have a corresponding modified unit via the amino group in the side chain of the above-mentioned lysine residue.

[1032] The manufacture of an antibody or a salt thereof modified with an affinity substance can be carried out by reacting a compound or a salt thereof of the present invention with an antibody or a salt thereof containing an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains. In the reaction, the equivalent of the compound or a salt thereof of the present invention relative to the antibody (compound or a salt thereof of the present invention / antibody) varies depending on factors such as the type of the compound or a salt thereof of the present invention and the antibody, and is therefore not particularly limited, and is, for example, 1 to 100, preferably 2 to 80, more preferably 4 to 60, further preferably 5 to 40, and particularly preferably 6 to 20.

[1033] Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation / degradation of the protein (e.g., cleavage of amide bonds). For example, the reaction under such mild conditions can be carried out in a suitable reaction system, such as a buffer, at room temperature (e.g., about 15 to 30° C.). The pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, refer to, for example, GJL Bernardes et al., Chem. Rev., 115, 2174 (2015); GJL Bernardes et al., Chem. Asian. J., 4, 630 (2009); BG Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[1034] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (II):

[1035] [Chemical Formula 44]

[1036]

[1037] Where,

[1038] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1039] L represents a connector,

[1040] A represents an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain,

[1041] The average modification percentage r of the immunoglobulin unit by 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 respect to the linker represented by L, refer to the linker represented by L in the compound of formula (I)).

[1042] The average modification percentage r of the immunoglobulin units by the affinity substance is 65-135%. The average modification percentage r can be 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 82% or more, 84% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, or 96% or more. The average modification percentage r can 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 analysis (which can be combined with a DAR calculator (Agilent software). See Examples).

[1043] The average modification percentage r may also be preferably 65-100%, more preferably 70-100%, even more preferably 75-100%, and particularly preferably 80-100%, 85-100%, 90-100%, or 95-100%. Within these average modification percentages, the upper limit may be a value below the aforementioned percentages, such as 98% or 96%. Alternatively, the average modification percentage r may be 96-100%, 97-100%, 98-100%, 99-100%, or 100%.

[1044] The above-mentioned average modification percentage r also applies to other average modification percentages r. That is, the above-mentioned average modification percentage r applies not only to the average modification percentage r described later based on affinity substances, but also to the average modification percentage r described later based on arbitrary modifications (e.g., bioorthogonal functional groups, functional substances).

[1045] An antibody or a salt thereof containing the structural unit represented by formula (II) can be produced by reacting a compound represented by formula (I) 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.

[1046] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may further comprise a cleavage moiety between the affinity substance and the antibody (immunoglobulin unit). In this case, the antibody or salt thereof comprising the structural unit represented by formula (II) may also contain a linker comprising a cleavage moiety. The definition, examples, and preferred examples of the cleavage moiety are as described above.

[1047] When the antibody or salt thereof modified with an affinity substance contains a cleavable moiety, the cleavable moiety can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by 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.

[1048] When the affinity substance-modified antibody or salt thereof contains a cleavable portion that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage, the antibody or salt thereof may be an antibody or salt thereof containing a structural unit represented by the following formula (IIa):

[1049] [Chemical Formula 45]

[1050]

[1051] Where,

[1052] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1053] L1 represents the first connector,

[1054] L2 represents the second connector,

[1055] CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage,

[1056] A represents an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain,

[1057] The average modification percentage r of the immunoglobulin unit by the affinity substance is 65 to 135%. The immunoglobulin unit represented by Ig, the first linker represented by L1, the second linker represented by L2, the cleavage portion represented by CLE(B), the affinity substance represented by A, the average modification percentage represented by r, and the definitions, examples, and preferred examples of antibodies are as described above.

[1058] An antibody or a salt thereof containing the structural unit represented by formula (IIa) can be produced by 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.

[1059] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (IIa-1):

[1060] [Chemical Formula 46]

[1061]

[1062] Where,

[1063] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1064] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[1065] L3 represents the third connector,

[1066] L4 represents the 4th connector,

[1067] S represents a sulfur atom,

[1068] A represents an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain,

[1069] The average modification percentage r of the immunoglobulin unit by the affinity substance is 65 to 135%. The immunoglobulin unit represented by Ig, the atoms represented by W1, W2, and W3, the third linker represented by L3, the fourth linker represented by L4, the affinity substance represented by A, the average modification percentage represented by r, and the definitions, examples, and preferred examples of antibodies are as described above.

[1070] An antibody or a salt thereof containing the structural unit represented by formula (IIa-1) can be produced by 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.

[1071] In addition, when the antibody or salt thereof modified with an affinity substance contains a cleavable moiety, a bioorthogonal functional group may be further included between the antibody (immunoglobulin unit) and the cleavable moiety. The bioorthogonal functional groups are as described above. Preferably, the bioorthogonal functional groups include: azide residues, alkyne residues (preferably cyclic groups having a carbon-carbon triple bond that may be substituted with the above-mentioned substituents), tetrazine residues, olefin residues, thiol residues, maleimide residues, thiol residues, furan residues, and halogenated carbonyl residues.

[1072] When the affinity substance-modified antibody or its salt further comprises a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavage moiety, the antibody or its salt may be an antibody or its salt comprising a structural unit represented by the following formula (IIb):

[1073] [Chemical Formula 47]

[1074]

[1075] Where,

[1076] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1077] L5 represents the fifth connector,

[1078] L6 represents the 6th connector,

[1079] B represents a group containing a bioorthogonal functional group,

[1080] CLE stands for cutting element.

[1081] A represents an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain,

[1082] The average modification percentage r of the immunoglobulin unit by the affinity substance is 65-135%. The immunoglobulin unit represented by Ig, the fifth linker represented by L5, the sixth linker represented by L6, the group containing a bioorthogonal functional group represented by B, the cleavage site represented by CLE, the affinity substance represented by A, the average modification percentage represented by r, and the definitions, examples, and preferred examples of the antibody are as described above.

[1083] An antibody or a salt thereof containing the structural unit represented by formula (IIb) can be produced by 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.

[1084] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (IIb-1):

[1085] [Chemical Formula 48]

[1086]

[1087] Where,

[1088] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1089] W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom,

[1090] L7 represents the 7th connector,

[1091] L8 represents the 8th connector,

[1092] B represents a group containing a bioorthogonal functional group,

[1093] V represents an oxygen atom or a sulfur atom, A represents an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain,

[1094] The modification percentage r of the immunoglobulin unit by the affinity substance is 65 to 135%. The immunoglobulin unit represented by Ig, the atoms represented by W1, W2, and W3, the seventh linker represented by L7, the eighth linker represented by L8, the group containing a bioorthogonal functional group represented by B, the atom represented by V, the affinity substance represented by A, the average modification percentage represented by r, and the antibody are defined, exemplified, and preferably exemplified as described above.

[1095] An antibody or a salt thereof containing the structural unit represented by formula (IIb-1) can be produced by 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.

[1096] The antibody or salt thereof modified with an affinity substance may further comprise an additional modification portion. Various methods are known for modifying antibodies. Therefore, in the present invention, the antibody or salt thereof modified with an affinity substance may be modified to further comprise an additional modification portion. The additional modification portion 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).

[1097] In a specific embodiment, the additional modifying moiety can 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 "third affinity moiety" and the "affinity substance" in "additional affinity substance" are the same as described above. The third affinity moiety can be the same as or different from the first affinity moiety and / or the second affinity moiety described above, and is preferably different.

[1098] In a specific embodiment, an additional modification portion comprising a third affinity moiety having affinity for the constant region of the antibody heavy chain can be introduced into the constant region of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the two heavy chains. An antibody or a salt thereof modified with an affinity substance can include an additional modification portion 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 an Fc region or CH2 domain) of the two heavy chains in the constituent unit of the antibody (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains). More specifically, the site of one or more (preferably 1 or 2, more preferably 1) lysine residues can be 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering (for example, see International Publication No. 2016 / 186206, International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, International Publication No. 2020 / 009165, International Publication No. 2020 / 090979). Preferably, the site at which the additional modification portion comprising the third affinity portion having affinity for the constant region of the antibody heavy chain is introduced is different from the site at which the affinity substance comprising the first affinity portion and the second affinity portion having affinity for the constant region of the antibody heavy chain is introduced. For example, when the site for introducing the affinity substance is the lysine residues at positions 246 / 248, the site for introducing the additional modification moiety is preferably the lysine residues at positions 288 / 290 or 317, more preferably the lysine residues at positions 288 / 290. When the site for introducing the affinity substance is the lysine residues at positions 288 / 290, the site for introducing the additional modification moiety is preferably the lysine residues at positions 246 / 248 or 317, more preferably the lysine residues at positions 246 / 248. When the site for introducing the affinity substance is the lysine residue at position 317, the site for introducing the additional modification moiety is preferably the lysine residues at positions 246 / 248 or 288 / 290.

[1099] 4-2. Antibodies or salts thereof modified with affinity substances comprising at least two affinity substances (comprising a first affinity portion, a second affinity portion, a third affinity portion, and a fourth affinity portion)

[1100] The present invention also provides an antibody or salt thereof modified with an affinity substance comprising a first modification portion and a second modification portion, wherein the first modification portion and the second modification portion are contained in the constant region of the antibody heavy chain, the first modification portion comprising: a first affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion, and the second modification portion comprising: a second affinity substance comprising a third affinity portion having affinity for the constant region of the antibody heavy chain and a fourth affinity portion. The definitions, examples, and preferred examples of affinity substances and antibodies, and the elements constituting them (e.g., affinity portions such as affinity peptides, linkers between affinity portions such as peptide linkers, and constant regions) are as described above. The first modification portion and the second modification portion may be the same or different, but are preferably different.

[1101] Preferably, such an affinity substance-modified antibody or a salt thereof may comprise: (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains), and (b) the first and second modified moieties described above, (c) the first modified moiety being introduced into the constant region of the first heavy chain in the immunoglobulin unit, and (d) the second modified moiety being introduced into the constant region of the second heavy chain in the immunoglobulin unit. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, affinity substances, and their constituent elements (e.g., constant regions) are as described above.

[1102] The antibody or salt thereof modified with an affinity substance may contain a first modification portion comprising a first affinity substance and a second modification portion comprising a second affinity substance through modification of a functional group in the side chain of any one or two 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 above-mentioned constant region (preferably the Fc region or CH2 domain). The antibody or salt thereof modified with an affinity substance is preferably modified via a functional group in the side chain of any one of lysine, tyrosine, tryptophan or cysteine ​​present in the above-mentioned constant region (preferably the Fc region or CH2 domain), more preferably via a functional group in the side chain of any one of lysine, tyrosine or tryptophan, further preferably via a functional group in the side chain of lysine or tyrosine, and particularly preferably via a modification of the amino group in the side chain of lysine. It may contain a first modification portion comprising a first affinity substance and a second modification portion comprising a second affinity substance. The sites of these amino acid residues in the above-mentioned constant region are as described above. The modification site of the antibody or its salt by the affinity substance can be confirmed by peptide mapping analysis. As described above, the modification can be site-selective. Therefore, in the formula described below, the immunoglobulin unit can selectively have the corresponding modification unit via the functional group in the side chain of the above-mentioned amino acid residue.

[1103] Preferably, an antibody modified with an affinity substance may contain a first modification portion comprising the first affinity substance via modification of the amino group of one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of the first heavy chain within the antibody structural unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains) in the constant region (preferably the Fc region or CH2 domain) of the first heavy chain. Furthermore, a second modification portion comprising the second affinity substance may be contained via modification of the amino group of one or more (preferably one or two, more preferably one) lysine residues in the constant region (preferably the Fc region or CH2 domain) of the second heavy chain. More specifically, the one or more (preferably one or two, more preferably one) lysine residues may be located at positions 246 / 248, 288 / 290, or 317 of the human IgG heavy chain according to EU numbering. As described above, the modification may be site-selective. Therefore, in the formula described below, the immunoglobulin unit can have a corresponding modification unit site-selectively via the amino group in the side chain of the above-mentioned lysine residue.

[1104] Antibodies or salts thereof modified with affinity substances can be produced by the following (1) and (2):

[1105] (1) reacting the compound of the present invention or a salt thereof with an antibody comprising an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, to produce an antibody or a salt thereof modified with an affinity substance, wherein the constant region of the heavy chain of the immunoglobulin unit comprises a first modification portion comprising the first affinity substance; and

[1106] (2) reacting an antibody or a salt thereof modified with an affinity substance containing the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with the compound or a salt thereof of the present invention to generate an antibody or a salt thereof modified with an affinity substance containing the first modification portion and the second modification portion in the constant region of the heavy chain of the immunoglobulin unit.

[1107] 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, but are preferably different. In the reaction, the equivalent amount of the compound of the present invention or its salt relative to the antibody (compound of the present invention or its salt / antibody) varies depending on factors such as the types of the compound of the present invention or its salt and the antibody, and is therefore not particularly limited. For example, it is 1 to 100, preferably 2 to 80, more preferably 4 to 60, further preferably 5 to 40, and particularly preferably 6 to 20.

[1108] Such a reaction can be suitably carried out under conditions (mild conditions) that do not cause denaturation / degradation of the protein (eg, cleavage of amide bonds). For example, the reaction under such mild conditions is the same as described above.

[1109] In a specific embodiment, such an affinity substance-modified antibody or a salt thereof may be an antibody or a salt thereof comprising a structural unit represented by the following formula (V):

[1110] [Chemical Formula 49]

[1111]

[1112] Where,

[1113] Ig represents an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains.

[1114] L L and L R Each independently represents a joint,

[1115] A L represents the first affinity substance,

[1116] A R represents the second affinity substance,

[1117] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1118] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may further comprise (iii') a first cleavage moiety between (i') the first affinity substance and (ii') the immunoglobulin unit; and / or may further comprise (iii") a second cleavage moiety between (i") the second affinity substance and (ii") the immunoglobulin unit. The first cleavage moiety and the second cleavage moiety are the same as the cleavage moieties described above. The first cleavage moiety and the second cleavage moiety may be the same or different.

[1119] When the antibody or salt thereof modified with an affinity substance contains a cleavable moiety, the cleavable moiety can generate a bioorthogonal functional group on the immunoglobulin unit side by 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.

[1120] When the affinity substance-modified antibody or salt thereof comprises a cleavable portion that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage, the antibody or salt thereof may comprise a structural unit represented by the following formula (Va):

[1121] [Chemical Formula 50]

[1122]

[1123] Where,

[1124] Ig represents the immunoglobulin unit,

[1125] L L1 and L R1 Each independently represents the first joint,

[1126] L L2 and L R2 Each independently represents the second joint,

[1127] CLE(B) L and CLE(B) R Each independently represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage,

[1128] A Lrepresents the first affinity substance,

[1129] A R represents the second affinity substance,

[1130] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1131] An antibody or a salt thereof containing the structural unit represented by formula (Va) can be produced by 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.

[1132] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (Va-1):

[1133] [Chemical Formula 51]

[1134]

[1135] Where,

[1136] Ig represents the immunoglobulin unit,

[1137] 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,

[1138] L L3 and L R3 Each independently represents a third joint,

[1139] L L4 and L R4 Each independently represents the fourth joint,

[1140] S represents a sulfur atom,

[1141] A L represents the first affinity substance,

[1142] A R represents the second affinity substance,

[1143] The average modification percentage r of the immunoglobulin unit by the first modification part Land the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1144] An antibody or a salt thereof containing the structural unit represented by formula (Va-1) can be produced by 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.

[1145] In addition, the antibody or salt thereof modified with an affinity substance may further comprise (iv') a first bioorthogonal functional group between the (ii') immunoglobulin unit and the (iii') first cleavable moiety; and / or may further comprise (iv") a second bioorthogonal functional group between the (ii") immunoglobulin unit and the (iii") second cleavable moiety. The bioorthogonal functional groups are as described above. Preferably, the bioorthogonal functional groups include: an azide residue, an alkyne residue (preferably a cyclic group having a carbon-carbon triple bond which may be substituted with the above-mentioned substituents), a tetrazine residue, an olefin residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, and a halogenated carbonyl residue.

[1146] When the affinity substance-modified antibody or its salt further comprises a bioorthogonal functional group between the immunoglobulin unit and the cleavage moiety, it may be an antibody or its salt comprising a structural unit represented by the following formula (Vb):

[1147] [Chemical Formula 52]

[1148]

[1149] Where,

[1150] Ig represents the immunoglobulin unit,

[1151] L L5 and L R5 Each independently represents the fifth joint,

[1152] L L6 and L R6 Each independently represents the sixth joint,

[1153] B L represents a first group comprising a first bioorthogonal functional group,

[1154] B R represents a second group comprising a second bioorthogonal functional group,

[1155] CLE L and CLE R Each independently represents the cutting part,

[1156] A L represents the first affinity substance,

[1157] A R represents the second affinity substance,

[1158] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1159] An antibody or a salt thereof containing the structural unit represented by formula (Vb) can be produced by 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.

[1160] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (Vb-1):

[1161] [Chemical Formula 53]

[1162]

[1163] Where,

[1164] Ig represents the immunoglobulin unit,

[1165] 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,

[1166] L L7 and L R7 Each independently represents the seventh joint,

[1167] L L8 and L R8 Each independently represents the 8th joint,

[1168] B L represents a first group comprising a first bioorthogonal functional group,

[1169] B R represents a second group comprising a second bioorthogonal functional group,

[1170] V L and V R each independently represents an oxygen atom or a sulfur atom,

[1171] A L represents the first affinity substance,

[1172] A R represents the second affinity substance,

[1173] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1174] An antibody or a salt thereof containing the structural unit represented by formula (Vb-1) can be produced by 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.

[1175] In addition, the antibody or salt thereof modified with an affinity substance may further comprise (iii') a first cleavable moiety between (i') the first affinity substance and (ii') the immunoglobulin unit; and may further comprise (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 by cleavage on the immunoglobulin unit side.

[1176] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (Vc):

[1177] [Chemical Formula 54]

[1178]

[1179] Where,

[1180] Ig represents the immunoglobulin unit,

[1181] L R1 Indicates the first connector,

[1182] L R2 Indicates the second connector,

[1183] L L5 Indicates the 5th connector,

[1184] L L6 Indicates the 6th connector,

[1185] B L represents a group comprising a first bioorthogonal functional group,

[1186] CLE L Indicates the first cutting part,

[1187] CLE(B) R represents a second cleavable moiety that can generate a second bioorthogonal functional group by cleavage on the immunoglobulin unit side,

[1188] A L represents the first affinity substance,

[1189] A R represents the second affinity substance,

[1190] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1191] The antibody or its salt containing the structural unit represented by formula (Vc) can be produced by reacting a compound represented by formula (Ia) or its salt and a compound represented by formula (Ib) or its salt with an antibody or its salt containing an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains.

[1192] In a specific embodiment, the antibody or salt thereof modified with an affinity substance may be an antibody or salt thereof comprising a structural unit represented by the following formula (Vc-1):

[1193] [Chemical Formula 55]

[1194]

[1195] Where,

[1196] Ig represents the immunoglobulin unit,

[1197] 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,

[1198] L R3 Each independently represents a third joint,

[1199] L R4 Each independently represents the fourth joint,

[1200] L L7 Each independently represents the seventh joint,

[1201] LL8 Each independently represents the 8th joint,

[1202] B L represents a group comprising a first bioorthogonal functional group,

[1203] V L represents an oxygen atom or a sulfur atom,

[1204] A L represents the first affinity substance,

[1205] A R represents the second affinity substance,

[1206] The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification portion R They are 65 to 135% respectively.

[1207] The antibody or its salt containing the structural unit represented by formula (Vc-1) can be produced by reacting a compound represented by formula (Ia-1) or its salt and a compound represented by formula (Ib-1) or its salt with an antibody or its salt containing an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains.

[1208] In the above formulae (V), (Va), (Va-1), (Vb), (Vb-1), (Vc) and (Vc-1), the definitions, examples and preferred examples of the immunoglobulin unit and antibody represented by Ig are as described above.

[1209] In the present specification, the subscript character "L" of any symbol is conveniently used for the symbol located on the left side (Left) relative to the immunoglobulin unit (Ig). On the other hand, the subscript character "R" of any symbol is conveniently used for the symbol located on the right side (Rignt) relative to the immunoglobulin unit (Ig). The meanings of the symbols with the subscript characters "L" and "R" are the same as those of the symbols without the subscript characters "L" and "R".

[1210] Therefore, in the above formulas (V), (Va), (Va-1), (Vb), (Vb-1), (Vc) and (Vc-1), the symbols with subscript characters "L" and "R" are as follows.

[1211] W L1 and W R1 As with W1, an oxygen atom is preferred.

[1212] W L2 and W R2 As with W2, an oxygen atom is preferred.

[1213] W L3 and W R3 As with W3, an oxygen atom is preferred.

[1214] L L and L R The connector shown is the same as the connector shown in L. L and L R The linkers shown may be the same or different, preferably they are different.

[1215] L L1 and L R1 The first joint shown is the same as the first joint shown in L1. L1 and L R1 The first linkers shown may be the same or different, but are preferably different.

[1216] L L2 and L R2 The second joint shown is the same as the second joint shown in L2. L2 and L R2 The second linkers shown may be the same or different, but are preferably different.

[1217] L L3 and L R3 The third joint shown is the same as the third joint shown in L3. L3 and L R3 The third linkers shown may be the same or different, but are preferably different.

[1218] L L4 and L R4 The fourth joint shown is the same as the fourth joint shown in L4. L4 and L R4 The fourth linkers shown may be the same or different, but are preferably different.

[1219] L L5 and L R5 The fifth joint shown is the same as the fifth joint shown in L5. L5 and L R5 The first linkers shown may be the same or different, but are preferably different.

[1220] L L6 and L R6 The sixth connector shown is the same as the sixth connector shown in L6. L6 and L R6 The sixth linkers shown may be the same or different, but are preferably different.

[1221] L L7 and L R7 The seventh connector shown is the same as the seventh connector shown in L7.L7 and L R7 The seventh linkers shown may be the same or different, but are preferably different.

[1222] L L8 and L R8 The 8th connector shown is the same as the 8th connector shown in L8. L8 and L R8 The eighth connectors shown may be the same or different, but are preferably different.

[1223] CLE(B) L and CLE(B) R The cuttable portion shown is the same as the cuttable portion shown in CLE(B). CLE(B) L and CLE(B) R The cutting portions shown may be the same or different, preferably different.

[1224] Contains B L The first group of the first bioorthogonal functional group shown and the group containing B R The second group of the second bioorthogonal functional group shown in is the same as the group containing the bioorthogonal functional group shown in B. L The first group of the first bioorthogonal functional group shown and the group containing B R The second groups of the second bioorthogonal functional groups shown may be the same or different, but are preferably different.

[1225] CLE L and CLE R The cutability portion shown is the same as the cutability portion shown in CLE. L and CLE R The cutting portions shown may be the same or different, preferably different.

[1226] A L and A R The affinity substance shown is the same as the affinity substance shown in A. L and A R The affinity substances shown may be the same or different.

[1227] r L and r R The degree of average modification percentage shown and the method of determination are the same as those of average modification percentage shown in r. L and r R The average modification percentages shown may be the same or different, and are preferably different.

[1228] The confirmation of the generation of the antibody or its salt modified by the target affinity substance also depends on the molecular weight of its specific raw materials and products, for example, by electrophoresis, chromatography (for example, gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC) or mass spectrometry. Site selectivity can be confirmed by peptide mapping. Peptide mapping can be carried out, for example, by protease treatment and mass spectrometry. As protease, endoprotease is preferred. As such endoprotease, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N can be mentioned. The confirmation of the number of introductions of affinity substances can be carried out by mass spectrometry (which can be combined with a DAR calculator (Agilent company software)). The antibody or its salt modified by the affinity substance can be appropriately purified by any method such as chromatography (for example, the above-mentioned chromatography and affinity chromatography).

[1229] The antibody or salt thereof modified with an affinity substance may further comprise an additional modification portion. Various methods are known for modifying antibodies. Therefore, in the present invention, the antibody or salt thereof modified with an affinity substance may be modified to further comprise an additional modification portion. The additional modification portion 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).

[1230] In a specific embodiment, the additional modifying moiety can be an additional affinity substance comprising a fifth affinity portion having affinity for the constant region of the antibody heavy chain. The "affinity portion" in "fifth affinity portion" and the "affinity substance" in "additional affinity substance" are the same as described above. The fifth affinity portion may be the same as or different from the first affinity portion, second affinity portion, third affinity portion, and / or fourth affinity portion described above, but is preferably different.

[1231] In a specific embodiment, an additional modification portion comprising a fifth affinity moiety having affinity for the constant region of the antibody heavy chain can be introduced into the constant region of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the two heavy chains. An affinity-substance-modified antibody or a salt thereof can include an additional modification portion 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 an Fc region or CH2 domain) of the two heavy chains in the constituent unit of the antibody (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains). More specifically, the position of one or more (preferably 1 or 2, more preferably 1) lysine residues can be 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering (for example, see International Publication No. 2016 / 186206, International Publication No. 2018 / 199337, International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, International Publication No. 2020 / 009165, International Publication No. 2020 / 090979). The site at which the additional modifying moiety comprising the fifth affinity moiety having affinity for the constant region of the antibody heavy chain is introduced is preferably different from the site at which the first modifying moiety comprising the first affinity substance comprising the first affinity moiety and the second affinity moiety having affinity for the constant region of the antibody heavy chain and the second modifying moiety comprising the third affinity moiety and the fourth affinity moiety having affinity for the constant region of the antibody heavy chain are introduced. For example, when both the first and second modifying moieties are introduced at lysine residues at positions 246 / 248, the site at which the additional modifying moiety is introduced is preferably lysine residues at positions 288 / 290 or 317, and more preferably lysine residues at positions 288 / 290. When the sites for introducing both the first and second modification moieties are lysine residues at positions 288 / 290, the sites for introducing the additional modification moiety are preferably lysine residues at positions 246 / 248 or 317, more preferably lysine residues at positions 246 / 248. When the sites for introducing both the first and second modification moieties are lysine residues at position 317, the sites for introducing the additional modification moiety are preferably lysine residues at positions 246 / 248 or 288 / 290.

[1232] 5. Antibodies or salts thereof without affinity substances

[1233] 5-1. Summary

[1234] An antibody or a salt thereof that does not contain an affinity substance can be produced using an antibody or a salt thereof modified with an affinity substance, wherein the antibody or a salt thereof modified with an affinity substance comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the heavy chain of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and (C) further comprises a cleavage portion between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

[1235] 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 method 1-2.

[1236] (Method 1-1) A method for producing an antibody or a salt thereof that does not contain an affinity substance, comprising: subjecting an antibody or a salt thereof modified with an affinity substance to a cleavage treatment at a cleavage moiety to produce the antibody or a salt thereof that does not contain the affinity substance, wherein the antibody or the salt thereof modified with the affinity substance comprises: (A) an affinity substance comprising a first affinity moiety and a second affinity moiety having affinity for the constant region of the heavy chain of an antibody (immunoglobulin unit), and (B) the antibody (immunoglobulin unit), and (C) further comprising a cleavage moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

[1237] (Method 1-2) A method for producing an antibody or a salt thereof that does not contain an affinity substance, comprising the following (1) and (2):

[1238] (1) reacting a compound or a salt thereof with an antibody containing an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, to produce an antibody or a salt thereof modified with an affinity substance containing a cleavage moiety between the affinity substance and the antibody, wherein the compound or the salt thereof comprises: (A) an affinity substance comprising a first affinity moiety and a second affinity moiety having affinity for a constant region of a heavy chain of the antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit) reactive group, and (C) further comprising a cleavage moiety between (A) the affinity substance and (B) the reactive group; and

[1239] (2) An antibody or a salt thereof modified with an affinity substance containing a cleavable moiety between the affinity substance and the antibody is subjected to a cleavage treatment at the cleavable moiety to produce an antibody or a salt thereof not containing the affinity substance.

[1240] Examples of cleavage treatments include: (a) treatment with one or more substances selected from the group consisting of the acidic substances, alkaline substances, reducing agents, oxidizing agents, and enzymes; (b) treatment with physical and chemical stimulation such as light; or (c) placement (incubation) in the presence of a cleavage linker containing a self-degradable cleavage portion. For these cleavage treatments, reference may also be made to International Publication No. 2019 / 240287, International Publication No. 2019 / 240288, International Publication No. 2020 / 009165, and International Publication No. 2020 / 090979.

[1241] Such a cleavage reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation / degradation of the protein (e.g., cleavage of amide bonds). For example, such mild conditions are as described above. In addition, when the cleavage site is an ester (e.g., a common 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 4.0-8.0, 10 mM-10 M) for an appropriate time (e.g., 1 hour) (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160).

[1242] The confirmation of the generation of the antibody or its salt without affinity substance obtained by the cleavage reaction also depends on the molecular weight of its specific raw materials and products, for example, can be carried out by electrophoresis, chromatography (for example, gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry. The confirmation of site selectivity can be carried out by the above-mentioned peptide map analysis. The confirmation of the number of introductions of affinity substances can be carried out by mass spectrometry (which can be combined with a DAR calculator (Agilent company software)). The antibody or its salt modified with affinity substance can be appropriately purified by any method such as chromatography (for example, the above-mentioned chromatography and affinity chromatography).

[1243] However, for antibodies or salts thereof modified with an affinity substance containing a cleavage moiety between (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the heavy chain of an antibody (immunoglobulin unit) and (B) the antibody (immunoglobulin unit), (a) when a cleavage moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side is included as the cleavage moiety, or (b) when a bioorthogonal functional group is included between the antibody (immunoglobulin unit) and the cleavage moiety, an antibody derivative or salt thereof containing a bioorthogonal functional group can be produced as an antibody or salt thereof that does not contain an affinity substance.

[1244] Furthermore, by reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance, a conjugate of an antibody and a functional substance or a salt thereof can be produced as an antibody or a salt thereof that does not contain an affinity substance.

[1245] Hereinafter, as antibodies or salts thereof not containing an affinity substance, (1) antibody derivatives or salts thereof containing a bioorthogonal functional group, and (2) conjugates of an antibody and a functional substance or salts thereof will be described in detail.

[1246] 5-2. Antibody derivatives or salts thereof containing at least one bioorthogonal functional group

[1247] The present invention provides an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising: (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains) and (b) a bioorthogonal functional group, wherein (c) the bioorthogonal functional group is introduced only into the constant region of one heavy chain of the immunoglobulin unit (i.e., the bioorthogonal functional group is introduced into the constant region of one heavy chain of the immunoglobulin unit, and the bioorthogonal functional group is not introduced into the constant region of the other heavy chain). Definitions, examples, and preferred embodiments of antibodies, immunoglobulin units, bioorthogonal functional groups, and elements constituting these (e.g., constant regions) are as described above.

[1248] Antibody derivatives or their salts may include bioorthogonal functional groups by modifying the functional groups in the side chains of any one or more (e.g., 2, 3, or 4) 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 above-mentioned constant region (preferably an Fc region or a CH2 domain). Antibody derivatives or their salts may include bioorthogonal functional groups by modifying the functional groups in the side chains of any one of lysine, tyrosine, tryptophan, or cysteine ​​present in the above-mentioned constant region (preferably an Fc region or a CH2 domain), more preferably by modifying the functional groups in the side chains of any one of lysine, tyrosine, or tryptophan, further preferably by modifying the functional groups in the side chains of lysine or tyrosine, and particularly preferably by modifying the amino group in the side chain of lysine. The sites of these amino acid residues in the above-mentioned constant region are as described above. The modification sites of the antibody or its salt with bioorthogonal functional groups can be confirmed by peptide mapping. As described above, the modification can be site-selective. Therefore, in Formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit can site-selectively possess the corresponding modified unit via the functional groups in the side chains of the above-mentioned amino acid residues.

[1249] Preferably, the antibody derivative may 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 heavy chain in the antibody constituent unit (an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains) (in other words, the bioorthogonal functional group is contained via the amino group in the side chain of the lysine residue in the constant region of one heavy chain in the immunoglobulin unit, and no bioorthogonal functional group is contained via the amino group in the side chain of the lysine residue in the constant region of the other heavy chain). More specifically, the site of one or more (preferably 1 or 2, more preferably 1) lysine residues can be 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering (for example, with reference to 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). As described above, the modification can be site-selective. Therefore, in formula (IIIa), (IIIa-1), (IIIb) and (IIIb-1) described later, the immunoglobulin unit can selectively have a corresponding modified unit via the amino group in the side chain of the above-mentioned lysine residue.

[1250] In a specific embodiment, the antibody derivative or its salt may be an antibody or its salt comprising a structural unit represented by the following formula (IIIa):

[1251] [Chemical Formula 56]

[1252]

[1253] Where,

[1254] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1255] L1 represents the first connector,

[1256] B represents a group containing a bioorthogonal functional group,

[1257] The average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65-135%. The immunoglobulin unit represented by Ig, the first linker represented by L1, the group containing the bioorthogonal functional group represented by B, the average modification percentage represented by r, and the definitions, examples, and preferred examples of the antibody are as described above. A particularly preferred bioorthogonal functional group is a thiol group.

[1258] In formula (IIIa), the molecular weight of the partial structure represented by L1-B can be 700 or less. When the molecular weight of the partial structure represented by L1-B is 700 or less, the antibody derivative or salt thereof having a bioorthogonal functional group is relatively difficult to purify based on molecular weight differences because the molecular weight of the partial structure relative to the molecular weight of the entire antibody is very small. However, according to the present invention, which allows for a high degree of control of the DAR, purification based on molecular weight differences is not necessarily required, and an antibody derivative exhibiting a desired DAR can be obtained at high purity. The molecular weight of the partial structure represented by L1-B can preferably be 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.

[1259] In another specific embodiment, the antibody derivative or its salt may be an antibody or its salt comprising a structural unit represented by the following formula (IIIa-1):

[1260] [Chemical Formula 57]

[1261]

[1262] Where,

[1263] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1264] W1 represents an oxygen atom or a sulfur atom,

[1265] L3 represents the third connector,

[1266] SH represents a thiol group,

[1267] The average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%. The immunoglobulin unit represented by Ig, the atom represented by W1, the third linker represented by L3, the average modification percentage represented by r, and the definitions, examples, and preferred examples of antibodies are as described above.

[1268] In formula (IIIa-1), the molecular weight of the partial structure represented by C(=W1)-L3-SH may be 700 or less. The molecular weight of the partial structure represented by C(=W1)-L3-SH may be preferably 600 or less, more preferably 500 or less, further preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[1269] In formula (IIIa-1), the third linker represented by L3 may be (CH2) n1n1 is an integer of 1 to 10. Preferably, n1 can be an integer greater than 2. n1 can also be an integer less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2. Particularly preferably, n1 is 2.

[1270] In another specific embodiment, the antibody derivative or its salt may be an antibody or its salt comprising a structural unit represented by the following formula (IIIb):

[1271] [Chemical Formula 58]

[1272]

[1273] Where,

[1274] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1275] L5 represents the fifth connector,

[1276] B represents a group containing a bioorthogonal functional group,

[1277] T1 represents a monovalent group,

[1278] The average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65-135%. The immunoglobulin unit represented by Ig, the fifth linker represented by L5, the group containing the bioorthogonal functional group represented by B, the average modification percentage represented by r, and the definitions, examples, and preferred examples of the antibody are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[1279] T1 is a monovalent group that can be generated by cleavage of a cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of the monovalent group include the monovalent groups described above. Examples of substituents when the monovalent group is substituted include the substituents described above.

[1280] In a specific embodiment, the monovalent group represented by T1 may be an optionally substituted hydroxyamino group. The optionally substituted hydroxyamino group may be represented by the following formula (α).

[1281] NR i -OR ii (α)

[1282] Where,

[1283] R i 、R ii Each independently represents a hydrogen atom or a monovalent hydrocarbon group.

[1284] Here, the monovalent hydrocarbon group may be substituted or unsubstituted. The definitions, examples and preferred examples of the monovalent hydrocarbon group and the substituent when the monovalent hydrocarbon group is substituted are as described above. Preferably, the hydroxyamino group which may be substituted may be NH-OR ii (Here, R ii More preferably, the substituted hydroxyamino group may be NH-OR ii (Here, R ii represents an alkyl group having 1 to 6 carbon atoms).

[1285] In formula (IIIb), the molecular weight of the partial structure represented by L5(-B)-T1 may be 700 or less. The molecular weight of the partial structure represented by L5(-B)-T1 may be preferably 600 or less, more preferably 500 or less, further preferably 400 or less, particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[1286] In another specific embodiment, the antibody derivative or its salt may be an antibody or its salt comprising a structural unit represented by the following formula (IIIb-1):

[1287] [Chemical Formula 59]

[1288]

[1289] Where,

[1290] Ig represents an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains.

[1291] W1 and W2 each independently represent an oxygen atom or a sulfur atom,

[1292] L7 represents the 7th connector,

[1293] B represents a group containing a bioorthogonal functional group,

[1294] T2 represents a monovalent group,

[1295] The modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65-135%. The immunoglobulin unit represented by Ig, the atoms represented by W1 and W2, the seventh linker represented by L7, the group containing the bioorthogonal functional group represented by B, the average modification percentage represented by r, and the definitions, examples, and preferred examples of antibodies are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[1296] T2 is a monovalent group that can be generated by cleavage of a cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of the monovalent group include the monovalent groups described above. When the monovalent group is substituted, examples of the substituent include the substituents described above. The monovalent group represented by T2 may be a hydroxyamino group that may be substituted. The details of the hydroxyamino group that may be substituted are the same as those described for T1.

[1297] In formula (IIIb-1), the molecular weight of the partial structure represented by C(=W1)-L7(-B)-C(=W2)-T2 may be 700 or less. The molecular weight of the partial structure represented by C(=W1)-L7(-B)-C(=W2)-T2 may preferably be 600 or less, more preferably 500 or less, further preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[1298] In formula (IIIb-1), the seventh linker represented by L7 may be (CH2) n2 n2 is an integer of 1 to 10. Preferably, n2 may be an integer of 2 or more or 3 or more. 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.

[1299] In formula (IIIb-1), the group containing a bioorthogonal functional group may be NH-C(=O)-(CH2) n3 -N3. n3 is an integer from 1 to 10. Preferably, n3 can be an integer of 2 or more, 3 or more, or 4 or more. n3 can 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 can be 4.

[1300] Antibody derivatives or salts thereof containing bioorthogonal functional groups can be produced using antibodies or salts thereof modified with affinity substances, wherein the antibodies or salts thereof comprise: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the heavy chain of an antibody (immunoglobulin unit), and (B) the antibody (immunoglobulin unit), and (C) further comprising a cleavage portion between (A) the affinity substance and (B) the antibody (immunoglobulin unit).

[1301] In a specific embodiment, when the antibody or salt thereof modified with the affinity substance comprises (a) a cleavable portion that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage, an antibody derivative or salt thereof comprising a bioorthogonal functional group can be produced.

[1302] More specifically, as such a production method, for example, the following (2-1) to (2-6) ( Figures 2 to 6 ).

[1303] (Method 2-1) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising: subjecting an antibody or a salt thereof modified with an affinity substance to a cleavage treatment at a cleavage moiety to generate an antibody derivative or a salt thereof containing a bioorthogonal functional group, wherein the antibody or the salt thereof modified with the affinity substance comprises: (A) an affinity substance comprising a first affinity moiety and a second affinity moiety having affinity for the constant region of the heavy chain of the antibody (immunoglobulin unit), and (B) the antibody (immunoglobulin unit), and (C) further comprising a cleavage moiety between (A) the affinity substance and (B) the antibody (immunoglobulin unit) (herein, the cleavage moiety is a cleavage moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage).

[1304] (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):

[1305] (1) reacting a compound or a salt thereof with an antibody containing an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, to generate an antibody or a salt thereof modified with an affinity substance containing a cleavage moiety (herein, the cleavage moiety is a cleavage moiety that can generate a bioorthogonal functional group on the antibody side by cleavage) between the affinity substance and the antibody, wherein the compound or the salt thereof comprises: (A) an affinity substance comprising a first affinity moiety and a second affinity moiety that have affinity for the constant region of the heavy chain of the antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit) reactive group, and (C) further comprising a cleavage moiety (herein, the cleavage moiety is a cleavage moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) reactive group side) between (A) the affinity substance and (B) the reactive group; and

[1306] (2) An antibody or a salt thereof modified with an affinity substance containing a cleavable moiety between the affinity substance and the antibody is subjected to a cleavage treatment at the cleavable moiety to produce an antibody derivative or a salt thereof containing a bioorthogonal functional group.

[1307] (Method 2-3) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, which comprises: cleaving an antibody or a salt thereof containing a structural unit represented by the above-mentioned formula (IIa) at a cleavable portion to generate an antibody derivative or a salt thereof containing a structural unit represented by the above-mentioned formula (IIIa).

[1308] (Method 2-4) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2):

[1309] (1) reacting a compound represented by the above 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 the above formula (IIa); and

[1310] (2) An antibody or a salt thereof containing the structural unit represented by the above formula (IIa) is subjected to cleavage treatment at a cleavable moiety to produce an antibody derivative or a salt thereof containing the structural unit represented by the above formula (IIIa).

[1311] (Method 2-5) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, which comprises: subjecting an antibody or a salt thereof containing a structural unit represented by the above-mentioned formula (IIa-1) to a cleavage treatment at a cleavable portion to generate an antibody derivative or a salt thereof containing a structural unit represented by the above-mentioned formula (IIIa-1).

[1312] (Method 2-6) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2):

[1313] (1) reacting a compound represented by the above 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 chai...

Claims

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

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

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

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

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

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

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

8. The compound or salt thereof according to claim 7, wherein Affinity substance: (i) (i-1) contains only one specific reactive group, and (i-2) is linked to the antibody reactive group via the specific reactive group.

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

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

11. The compound or salt thereof according to claim 10, wherein Affinity polypeptides: (i) (i-1) contain only one amino acid residue having an amino group in the side chain, and (ii-2) are linked to the antibody reactive group via the amino group; or (ii) are linked to the antibody reactive group via the N-terminal amino group in the first affinity peptide.

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

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

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

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

16. The compound or salt thereof according to claim 15, wherein The affinity peptide having affinity for the constant region of the antibody heavy chain and having one lysine residue is any one of the following (1) to (4): (1) an affinity peptide comprising the amino acid sequence (Fc3K) of RGNCAYHKGQIIWCTYH (SEQ ID NO: 38); (2) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: 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 amino acid residues other than lysine residues and cysteine ​​residues; (3) an affinity peptide comprising the amino acid sequence (Z34CK) of FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39); and (4) An affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: 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 FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 39) are substituted with amino acid residues other than lysine residues and cysteine ​​residues, Here, the two cysteine ​​residues contained in the amino acid sequence can be cross-linked via a disulfide bond; and / or The affinity peptide having affinity for the constant region of the 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 (Z34CM) of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40); (6) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: an amino acid sequence in which one or two amino acid residues other than the cysteine ​​residue in the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIREEC (SEQ ID NO: 40) are substituted with amino acid residues other than lysine residues and cysteine ​​residues; (7) an affinity peptide comprising the amino acid sequence (ProAR) of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQ SANLLAEA (SEQ ID NO: 41); (8) an affinity peptide having affinity for the constant region of the antibody heavy chain comprising the following amino acid sequence: an amino acid sequence in which one or two amino acid residues other than the cysteine ​​residue in the amino acid sequence of FNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA (SEQ ID NO: 41) are substituted with amino acid residues other than lysine residues and cysteine ​​residues; (9) an affinity peptide comprising the amino acid sequence of RGNCAYHRGQIIWCTYH (SEQ ID NO: 78); and (10) An affinity peptide having affinity for the constant region of an antibody heavy chain comprising the following amino acid sequence: 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 amino acid residues other than lysine residues and cysteine ​​residues, Here, the two cysteine ​​residues contained in the amino acid sequence may be cross-linked via a disulfide bond.

17. The compound or salt thereof according to claim 1, wherein The compound is represented by the following formula (I): R——L—A(I) In the formula, R represents the reactive group, L represents a connector. A represents the affinity substance.

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

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

20. The compound or salt thereof according to claim 19, wherein The compound is represented by the following formula (Ia): R——L-CLE(B)—L2-A(la) In the formula, R represents the reactive group, L1 indicates the first connector. L2 represents the second joint, CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the side of the reactive group by cleavage, A represents the affinity substance.

21. The compound or salt thereof according to claim 19, wherein The compound is represented by the following formula (Ia-1): In the formula, X represents a leaving group, W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom, L3 represents the third connector, L4 represents the 4th connector, S represents a sulfur atom, A represents the affinity substance.

22. The compound or salt thereof according to claim 21, wherein The leaving group is selected from the following groups: (a)R A -S, here, 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, here, 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, here, 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) Halogen atoms.

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

24. The compound or salt thereof according to claim 23, wherein The compound is represented by the following formula (Ib): In the formula, R represents the reactive group, L5 means the 5th connector. L6 means the 6th connector, B represents a group containing a bioorthogonal functional group, CLE stands for cutting part. A represents the affinity substance.

25. The compound or salt thereof according to claim 23, wherein The compound is represented by the following formula (Ib-1): In the formula, X represents a leaving group, W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom, L7 represents the 7th connector, L8 means the 8th connector, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A represents the affinity substance.

26. The compound or salt thereof according to claim 23, wherein The bioorthogonal functional group is an azide residue, an alkyne residue, a tetrazine residue, an olefin residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue or a halogenated carbonyl residue.

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

28. An antibody modified with an affinity substance or a salt thereof, wherein: The affinity substance is contained in the constant region of the antibody heavy chain, and comprises a first affinity part and a second affinity part having affinity for the constant region of the antibody heavy chain.

29. An antibody or a salt thereof modified with an affinity substance according to claim 28, comprising: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, and (b) the affinity substance, and (c) the affinity substance is introduced only into the constant region of one heavy chain of the immunoglobulin unit.

30. The antibody modified with an affinity substance or a salt thereof according to claim 29, wherein The affinity substance is introduced only into the constant region of the one heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the one heavy chain.

31. The antibody modified with an affinity substance or a salt thereof according to claim 30, wherein The one or more positions in the constant region of the one heavy chain are position 246 / 248, position 288 / 290 or position 317 of the human IgG heavy chain according to EU numbering.

32. The antibody modified with an affinity substance or a salt thereof according to claim 28, wherein The antibody modified with an affinity substance comprises a structural unit represented by the following formula (II): In the formula, Ig refers to an immunoglobulin unit consisting of two heavy chains and, if necessary, two light chains. L represents a connector. A represents the affinity substance, The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

33. The antibody modified with an affinity substance or a salt thereof according to claim 28, wherein The antibody or a salt thereof further comprises (iii') a cleavable portion between (i') the affinity substance and (ii') the antibody.

34. The antibody modified with an affinity substance or a salt thereof according to claim 33, wherein The cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

35. The antibody modified with an affinity substance or a salt thereof according to claim 34, wherein The antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIa): In the formula, Ig represents the immunoglobulin unit, L1 indicates the first connector. L2 represents the second joint, CLE(B) represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage, A represents the affinity substance, The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

36. The antibody modified with an affinity substance or a salt thereof according to claim 34, wherein The antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIa-1): In the formula, Ig represents the immunoglobulin unit, W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom, L3 represents the third connector, L4 represents the 4th connector, S represents a sulfur atom, A represents the affinity substance, The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

37. The antibody modified with an affinity substance or a salt thereof according to claim 33, wherein The antibody or salt thereof further comprises (iv') a bioorthogonal functional group between (ii') the antibody and (iii') the cleavable moiety.

38. The antibody modified with an affinity substance or a salt thereof according to claim 37, wherein The antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIb): In the formula, Ig represents the immunoglobulin unit, L5 means the 5th connector. L6 means the 6th connector, B represents a group containing a bioorthogonal functional group, CLE stands for cutting part. A represents the affinity substance, The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

39. The antibody modified with an affinity substance or a salt thereof according to claim 37, wherein The antibody modified with an affinity substance comprises a structural unit represented by the following formula (IIb-1): In the formula, Ig represents the immunoglobulin unit, W1, W2 and W3 each independently represent an oxygen atom or a sulfur atom, L7 represents the 7th connector, L8 means the 8th connector, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A represents the affinity substance, The average modification percentage r of the affinity substance to the immunoglobulin unit is 65-135%.

40. The antibody modified with an affinity substance or a salt thereof according to claim 28, wherein The antibody modified with an affinity substance further includes an additional modified portion.

41. The antibody modified with an affinity substance or a salt thereof according to claim 40, wherein The additional modification portion is an additional affinity substance comprising a third affinity portion having affinity for the constant region of the antibody heavy chain, and the additional affinity substance is contained in the constant region of the antibody heavy chain.

42. The antibody modified with an affinity substance or a salt thereof according to claim 41, wherein The additional affinity substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

43. The antibody modified with an affinity substance or a salt thereof according to claim 42, wherein The one or more positions in the constant regions of the two heavy chains are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

44. A method for producing an antibody or a salt thereof modified with an affinity substance, comprising: A compound or a salt thereof is reacted with an antibody comprising an immunoglobulin unit to generate an antibody or a salt thereof modified with an affinity substance containing the affinity substance in the constant region of the heavy chain of the immunoglobulin unit, wherein the compound or the salt thereof comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for the constant region of the antibody heavy chain, and (B) an antibody reactive group, wherein the immunoglobulin unit comprises two heavy chains and, if necessary, two light chains.

45. The method of claim 44, wherein: The compound or a salt thereof further comprises (iii) a cleavable portion between (i) the affinity substance and (ii) the reactive group.

46. ​​The method of claim 45, wherein: The cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the side of the reactive group by cleavage.

47. The method of claim 44, wherein: The compound or a salt thereof further comprises (iv) a bioorthogonal functional group between (ii) the reactive group and (iii) the cleavable moiety.

48. The method of claim 44, wherein: The antibody modified with an affinity substance or a salt thereof is the antibody modified with an affinity substance or a salt thereof according to any one of claims 29 to 43.

49. An antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, 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 of the immunoglobulin unit.

50. The antibody derivative or salt thereof according to claim 49, wherein The bioorthogonal functional group is introduced only into the constant region of the one heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the one heavy chain.

51. The antibody derivative or salt thereof according to claim 50, wherein The one or more positions in the constant region of the one heavy chain are position 246 / 248, position 288 / 290 or position 317 of the human IgG heavy chain according to EU numbering.

52. The antibody derivative or salt thereof according to claim 49, wherein The antibody derivative or salt thereof containing a bioorthogonal functional group contains a structural unit represented by the following formula (IIIa): In the formula, Ig represents the immunoglobulin unit, L1 indicates the first connector. B represents a group containing a bioorthogonal functional group, The average modification percentage r of the immunoglobulin unit by the bioorthogonal functional groups is 65-135%.

53. The antibody derivative or salt thereof according to claim 49, wherein The antibody derivative or salt thereof containing a bioorthogonal functional group contains a structural unit represented by the following formula (IIIa-1): In the formula, Ig represents the immunoglobulin unit, W1 represents an oxygen atom or a sulfur atom, L3 represents the third connector, SH represents a thiol group, The average modification percentage r of the immunoglobulin unit by the bioorthogonal functional groups is 65-135%.

54. The antibody derivative or salt thereof according to claim 49, wherein The antibody derivative or salt thereof containing a bioorthogonal functional group contains a structural unit represented by the following formula (IIIb): In the formula, Ig represents the immunoglobulin unit, L5 means the 5th connector. B represents a group containing a bioorthogonal functional group, T1 represents a monovalent group, The average modification percentage r of the immunoglobulin unit by the bioorthogonal functional groups is 65-135%.

55. The antibody derivative or salt thereof according to claim 49, wherein The antibody derivative or salt thereof containing a bioorthogonal functional group contains a structural unit represented by the following formula (IIIb-1): In the formula, Ig represents the immunoglobulin unit, W1 and W2 each independently represent an oxygen atom or a sulfur atom, L7 represents the 7th connector, B represents a group containing a bioorthogonal functional group, T2 represents a monovalent group, The modification percentage r of the immunoglobulin unit by the bioorthogonal functional group is 65-135%.

56. The antibody derivative or salt thereof according to claim 49, wherein Antibody derivatives further comprise additional modified moieties.

57. The antibody derivative or salt thereof according to claim 56, wherein The additional modification portion is an additional modification portion comprising a bioorthogonal functional group, and the additional affinity substance comprising a bioorthogonal functional group is contained in the constant region of the antibody heavy chain.

58. The antibody derivative or salt thereof according to claim 57, wherein The additional modification moiety comprising a bioorthogonal functional group is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

59. The antibody derivative or salt thereof according to claim 58, wherein The one or more positions in the constant regions of the two heavy chains are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

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

61. The conjugate or a salt thereof according to claim 60, wherein The functional substance is introduced only into the constant region of the one heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the one heavy chain.

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

63. The conjugate or salt thereof according to claim 60, wherein The conjugate or a salt thereof comprises a structural unit represented by the following formula (IVa): In the formula, Ig represents the immunoglobulin unit, L1 indicates the first connector. Z represents a functional substance, The average modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

64. The conjugate or salt thereof according to claim 60, wherein The conjugate or a salt thereof comprises a structural unit represented by the following formula (IVa-1): In the formula, Ig represents the immunoglobulin unit, W1 represents an oxygen atom or a sulfur atom, L3 represents the third connector, Z represents a functional substance, The average modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

65. The conjugate or salt thereof according to claim 60, wherein The conjugate or a salt thereof comprises a structural unit represented by the following formula (IVb): In the formula, Ig represents the immunoglobulin unit, L5 means the 5th connector. Z represents a functional substance, T1 represents a monovalent group, The average modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

66. The conjugate or salt thereof according to claim 60, wherein The conjugate or a salt thereof comprises a structural unit represented by the following formula (IVb-1): In the formula, Ig represents the immunoglobulin unit, W1 and W2 each independently represent an oxygen atom or a sulfur atom, L7 represents the 7th connector, Z represents a functional substance, T2 represents a monovalent group, The modification percentage r of the functional substance to the immunoglobulin unit is 65-135%.

67. The conjugate or salt thereof according to claim 60, wherein Functional substances are drugs, labeling substances, affinity substances, transfer substances or stabilizers.

68. The conjugate or salt thereof according to claim 67, wherein The affinity substance is a full-length antibody or a fragment thereof.

69. The conjugate or salt thereof according to claim 60, wherein The conjugate further comprises an additional modifying moiety.

70. The conjugate or salt thereof according to claim 69, wherein The additional modified portion is an additional modified portion comprising a functional substance, and the additional modified portion comprising a functional substance is contained in the constant region of the antibody heavy chain.

71. The conjugate or salt thereof according to claim 70, wherein The additional modification portion containing the functional substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

72. The conjugate or salt thereof according to claim 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 the human IgG heavy chain according to EU numbering.

73. A method for producing an antibody or a salt thereof that does not contain an affinity substance, comprising: An antibody or a salt thereof modified with an affinity substance is subjected to cleavage treatment at a cleavage portion to generate an antibody or a salt thereof that does not contain an affinity substance, wherein the antibody or a salt thereof modified with an affinity substance comprises: (A) an affinity substance comprising a first affinity portion and a second affinity portion having affinity for a constant region of an antibody heavy chain, and (B) an antibody, and (C) further comprises a cleavage portion between (A) the affinity substance and (B) the antibody.

74. The method of claim 73, wherein The cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody side by cleavage, The antibody or salt thereof not containing an affinity substance is an antibody derivative or salt thereof containing a bioorthogonal functional group.

75. The method of claim 74, wherein: The antibody derivative or salt thereof containing a bioorthogonal functional group is the antibody derivative or salt thereof according to any one of claims 49 to 53 and 56 to 59.

76. The method of claim 73, wherein: The antibody or salt thereof without an affinity substance further comprises a bioorthogonal functional group between the antibody and the cleavage moiety, The antibody or salt thereof not containing an affinity substance is an antibody derivative or salt thereof containing a bioorthogonal functional group.

77. The method of claim 76, wherein The antibody derivative or salt thereof containing a bioorthogonal functional group is the antibody derivative or salt thereof according to any one of claims 49 to 51 and 54 to 59.

78. A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group by the method according to claim 74: and (2) Reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to generate a conjugate containing the antibody and the functional substance or a salt thereof.

79. The method of claim 78, wherein The conjugate or a salt thereof is the conjugate or a salt thereof according to any one of claims 60 to 64.

80. A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group by the method according to claim 76: and (2) Reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to generate a conjugate containing the antibody and the functional substance or a salt thereof.

81. The method of claim 78, wherein The conjugate or a salt thereof is the conjugate or a salt thereof according to any one of claims 60 to 62, 65 and 66.

82. A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, the method comprising: reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to generate a conjugate containing the antibody and the functional substance or a salt thereof, The antibody derivative or salt thereof comprising a bioorthogonal functional group comprises: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, 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 of the immunoglobulin unit, The conjugate comprising an antibody and a functional substance or a salt thereof comprises: (a) an immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, and (b) a functional substance, and (c) the functional substance is introduced only into the constant region of one heavy chain of the immunoglobulin unit.

83. The method of claim 80, wherein The antibody derivative or salt thereof containing a bioorthogonal functional group is the antibody derivative or salt thereof according to any one of claims 49 to 59.

84. The method of claim 80, wherein The conjugate or a salt thereof is the conjugate or a salt thereof according to any one of claims 60 to 72.

85. An affinity substance or a salt thereof, comprising a first affinity portion and a second affinity portion having affinity for a constant region of an antibody heavy chain.

86. The affinity substance or salt thereof according to claim 85, wherein The affinity substance is represented by the following formula (A): AP1-L A -AP2(A) In the formula, AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain, AP2 represents a second affinity peptide having affinity for the constant region of the antibody heavy chain, L A Indicates a connector.

87. The affinity substance or a salt thereof according to claim 85, wherein Affinity substances contain only one specific reactive group.

88. The affinity substance or salt thereof according to claim 85, wherein The affinity substance is an affinity polypeptide comprising a first affinity peptide and a second affinity peptide having affinity for the constant region of the antibody heavy chain.

89. The affinity substance or a salt thereof according to claim 88, wherein The affinity polypeptide is represented by the following formula (A'): AP1-PL A -AP2(A’) In the formula, AP1 represents a first affinity peptide having affinity for the constant region of the antibody heavy chain and present at 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 present at the C-terminal side of the affinity polypeptide. PL A represents a peptide linker.

90. The affinity substance or a salt thereof according to claim 88, wherein The affinity polypeptide: (i) contains only one amino acid residue having an amino group at the side chain, or (ii) contains an unprotected N-terminal amino group.

91. The affinity substance or a salt thereof according to claim 90, wherein The amino acid residue having an amino group in the side chain is a lysine residue.

92. The affinity substance or salt thereof according to claim 88, wherein The affinity polypeptide further comprises a tripeptide consisting of Gln-Glu-Thr (QET) at the N-terminus.

93. The affinity substance or salt thereof according to claim 89, wherein The peptide linker has a length of 20 or more amino acid residues.

94. The affinity substance or salt thereof according to claim 85, wherein One of the first affinity peptide and the second affinity peptide is an affinity peptide having affinity for the constant region of the antibody heavy chain and having one lysine residue, and The other of the first affinity peptide and the second affinity peptide is an affinity peptide having affinity for the constant region of the antibody heavy chain and having no lysine residue.

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

96. An expression vector comprising the polynucleotide of claim 95 and a promoter operably linked thereto.

97. A host cell comprising an expression unit comprising the polynucleotide of claim 95 and a promoter operably linked thereto.

98. An antibody or a salt thereof modified with an affinity substance comprising a first modification portion and a second modification portion, wherein: The first modification part and the second modification part are contained in the constant region of the antibody heavy chain, the first modification part comprises: a first affinity substance comprising a first affinity part having affinity for the constant region of the antibody heavy chain and a second affinity part, and the second modification part comprises: a second affinity substance comprising a third affinity part having affinity for the constant region of the antibody heavy chain and a fourth affinity part.

99. The antibody or salt thereof modified with an affinity substance according to claim 98, comprising: (a) an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains, and (b) the first modified portion and the second modified portion, (c) the first modified portion is introduced into the constant region of the first heavy chain in the immunoglobulin unit, and (d) The second modified portion is introduced into the constant region of the second heavy chain in the immunoglobulin unit.

100. The antibody or salt thereof modified with an affinity substance according to claim 99, wherein The first modification portion is introduced into the constant region of the first heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the first heavy chain, and the second modification portion is introduced into the constant region of the second heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the second heavy chain.

101. The antibody modified with an affinity substance or a salt thereof according to claim 100, wherein One or more sites in the constant region of the first heavy chain and one or more sites in the constant region of the second heavy chain are selected from the following sites: positions 246 / 248, 288 / 290 and 317 of the human IgG heavy chain according to EU numbering, and combinations thereof.

102. The antibody or salt thereof modified with an affinity substance according to claim 98, wherein The antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (V): In the formula, Ig represents an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains. L L and L R Each independently represents a joint, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

103. The antibody or salt thereof modified with an affinity substance according to claim 98, wherein The antibody or salt thereof further comprises (iii') a first cleavage portion between (i') the first affinity substance and (ii') the immunoglobulin unit; and / or further comprises (iii") a second cleavage portion between (i") the second affinity substance and (ii") the immunoglobulin unit.

104. The antibody or salt thereof modified with an affinity substance according to claim 103, wherein The first cleavable moiety and the second cleavable moiety are cleavable moieties that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

105. The antibody or salt thereof modified with an affinity substance according to claim 98, wherein The affinity substance-modified antibody comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Va): In the formula, Ig represents the immunoglobulin unit, L L1 and L R1 Each independently represents the first joint, L L2 and L R2 Each independently represents the second joint, CLE(B) L and CLE(B) R Each independently represents a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

106. The antibody or salt thereof modified with an affinity substance according to claim 98, wherein The antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Va-1): In the formula, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represents an oxygen atom or a sulfur atom, L L3 and L R3 Each independently represents a third joint, L L4 and L R4 Each independently represents a fourth joint, S represents a sulfur atom, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

107. The antibody or salt thereof modified with an affinity substance according to claim 103, 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 antibody or salt thereof modified with an affinity substance according to claim 107, wherein The antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vb): In the formula, Ig represents the immunoglobulin unit, L L5 and L R5 Each independently represents the fifth joint, L L6 and L R6 Each independently represents the sixth joint, B L represents a first group comprising a first bioorthogonal functional group, B R represents a second group comprising a second bioorthogonal functional group, CLE L and CLE R Each independently represents the cutting part, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

109. The antibody or salt thereof modified with an affinity substance according to claim 108, wherein The antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vb-1): In the formula, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represents an oxygen atom or a sulfur atom, L L7 and L R7 Each independently represents the seventh joint, L L8 and L R8 Each independently represents the 8th joint, 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, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

110. The antibody modified with an affinity substance or a salt thereof according to claim 103, wherein The antibody or salt thereof further comprises (iii') a first cleavable portion between (i') the first affinity substance and (ii') the immunoglobulin unit, and further comprises (iv") a first bioorthogonal functional group between (ii") the immunoglobulin unit and (iii") the second cleavable portion, The first cleavable moiety is a cleavable moiety that can generate a second bioorthogonal functional group on the immunoglobulin unit side by cleavage.

111. The antibody modified with an affinity substance or a salt thereof according to claim 100, wherein The antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vc): In the formula, Ig represents the immunoglobulin unit, L R1 Indicates the first connector. L R2 Indicates the second connector. L L5 Indicates the 5th connector, L L6 Indicates the 6th connector, B L represents a group comprising a first bioorthogonal functional group, CLE L Indicates the first cutting part, CLE(B) R represents a second cleavable moiety that can generate a second bioorthogonal functional group by cleavage on the immunoglobulin unit side, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

112. The antibody or salt thereof modified with an affinity substance according to claim 111, wherein The antibody modified with an affinity substance comprising the first modification portion and the second modification portion comprises a structural unit represented by the following formula (Vc-1): In the formula, Ig represents the immunoglobulin unit, W L1 , W L2 and W L3 , and W R1 , W R2 and W R3 each independently represents an oxygen atom or a sulfur atom, L R3 Each independently represents a third joint, L R4 Each independently represents a fourth joint, L L7 Each independently represents the seventh joint, L L8 Each independently represents the 8th joint, B L represents a group comprising a first bioorthogonal functional group, V L represents an oxygen atom or a sulfur atom, A L represents the first affinity substance, A R represents the second affinity substance, The average modification percentage r of the first modified part to the immunoglobulin unit L and the average modification percentage r of the second modified part to the immunoglobulin unit R They are 65 to 135% respectively.

113. The antibody or salt thereof modified with an affinity substance according to claim 98, wherein The antibody modified with an affinity substance further includes an additional modified portion.

114. The antibody or salt thereof modified with an affinity substance according to claim 113, wherein The additional modification portion is an additional affinity substance comprising a fifth affinity portion having affinity for the constant region of the antibody heavy chain, and the additional affinity substance is contained in the constant region of the antibody heavy chain.

115. The antibody modified with an affinity substance or a salt thereof according to claim 114, wherein The additional affinity substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

116. The antibody or salt thereof modified with an affinity substance according to claim 115, wherein The one or more positions in the constant regions of the two heavy chains are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

117. A method for producing an antibody or a salt thereof modified with an affinity substance comprising a first modification portion and a second modification portion, comprising the following (1) and (2): (1) reacting a compound or a salt thereof with an antibody comprising an immunoglobulin unit to generate an antibody or a salt thereof modified with an affinity substance comprising a first modification portion in the constant region of the heavy chain of the immunoglobulin unit, the compound or the salt thereof comprising: (A) a first affinity substance comprising a first affinity portion having affinity for the constant region of the antibody heavy chain and a second affinity portion, and (B) a first reactive group for the antibody, the immunoglobulin unit comprising two heavy chains and, if necessary, two light chains, the first modification portion comprising the first affinity substance; and (2) reacting an antibody or a salt thereof modified with an affinity substance containing the first affinity substance in the constant region of the heavy chain of the immunoglobulin unit with a compound or a salt thereof to generate an antibody or a salt thereof modified with an affinity substance containing the first modification portion and the second modification portion in the constant region of the heavy chain of the immunoglobulin unit, wherein the compound or the salt thereof comprises: a second affinity substance comprising a third affinity portion and a fourth affinity portion having affinity for the constant region of the antibody heavy chain, and (B) a second reactive group for the antibody.

118. The method of claim 117, wherein: The antibody modified with an affinity substance or a salt thereof comprising the first modification portion and the second modification portion is the antibody modified with an affinity substance or a salt thereof according to any one of claims 86 to 100.

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

120. The antibody derivative or salt thereof according to claim 119, wherein The first modification portion is introduced into the constant region of the first heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the first heavy chain, and the second modification portion is introduced into the constant region of the second heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the second heavy chain.

121. The antibody derivative or salt thereof according to claim 120, wherein One or more sites in the constant region of the first heavy chain and one or more sites in the constant region of the second heavy chain are selected from the following sites: positions 246 / 248, 288 / 290 and 317 of the human IgG heavy chain according to EU numbering, and combinations thereof.

122. The antibody derivative or salt thereof according to claim 119, wherein The antibody derivative or a salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIa): In the formula, Ig represents the immunoglobulin unit, L L1 and L R1 Each independently represents the first joint, B L represents a first group comprising a first bioorthogonal functional group, B R represents a second group comprising a second bioorthogonal functional group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

123. The antibody derivative or salt thereof according to claim 122, wherein The antibody derivative or a salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIa-1): In the formula, 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 joint, SH represents a thiol group which is a bioorthogonal functional group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

124. The antibody derivative or salt thereof according to claim 119, wherein The antibody derivative or a salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIb): In the formula, Ig represents the immunoglobulin unit, L L5 and L R5 Each independently represents the fifth joint, 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, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

125. The antibody derivative or salt thereof according to claim 124, wherein The antibody derivative or a salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIb-1): In the formula, 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 the seventh joint, 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, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

126. The antibody derivative or salt thereof according to claim 119, wherein The antibody derivative or a salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIc): In the formula, Ig represents the immunoglobulin unit, L R1 Indicates the first connector. L L5 Indicates the 5th connector, 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, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

127. The antibody derivative or salt thereof according to claim 126, wherein The antibody derivative or a salt thereof comprising the first modified portion and the second modified portion comprises a structural unit represented by the following formula (VIc-1): In the formula, 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 Indicates the third connector. L L7 Indicates the 7th joint, B L represents a first group comprising a first bioorthogonal functional group, SH represents a thiol group as the second bioorthogonal functional group, T L2 represents a monovalent group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

128. The antibody derivative or salt thereof according to claim 119, wherein Antibody derivatives further comprise additional modified moieties.

129. The antibody derivative or salt thereof according to claim 128, wherein The additional modification portion is an additional modification portion comprising a bioorthogonal functional group, and the additional modification portion comprising a bioorthogonal functional group is contained in the constant region of the antibody heavy chain.

130. The antibody derivative or salt thereof according to claim 129, wherein The additional modification moiety comprising a bioorthogonal functional group is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

131. The antibody derivative or salt thereof according to claim 130, wherein The one or more positions in the constant regions of the two heavy chains are positions 246 / 248, 288 / 290 or 317 of the human IgG heavy chain according to EU numbering.

132. An antibody and a conjugate of a first modified portion and a second modified portion, or a salt thereof, comprising the first modified portion and the second modified portion, wherein the first modified portion comprises: (a) an immunoglobulin unit comprising two heavy chains consisting of a first heavy chain and a second heavy chain and, if necessary, two light chains, and (b) a first functional substance, and the second modified portion comprises a second functional substance, (c) the first modified portion is introduced into the constant region of the first heavy chain, (d) the second modified portion is introduced into the constant region of the second heavy chain, and (e) The first modified portion and the second modified portion are different from each other.

133. The conjugate or salt thereof according to claim 132, wherein The first modification portion is introduced into the constant region of the first heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the first heavy chain, and the second modification portion is introduced into the constant region of the second heavy chain via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant region of the second heavy chain.

134. The conjugate or salt thereof according to claim 133, wherein One or more sites in the constant region of the first heavy chain and one or more sites in the constant region of the second heavy chain are selected from the following sites: positions 246 / 248, 288 / 290 and 317 of the human IgG heavy chain according to EU numbering, and combinations thereof.

135. The conjugate or salt thereof according to claim 132, wherein The conjugate or its salt comprises a structural unit represented by the following formula (VIIa): In the formula, Ig represents the immunoglobulin unit, L L1 and L R1 Each independently represents the first joint, Z L Indicates the first functional substance, Z R Indicates the second functional substance, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

136. The conjugate or salt thereof according to claim 135, wherein The conjugate or its salt comprises a structural unit represented by the following formula (VIIa-1): In the formula, 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 joint, Z L Indicates the first functional substance, Z R Indicates the second functional substance, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

137. The conjugate or salt thereof according to claim 132, wherein The conjugate or its salt comprises a structural unit represented by the following formula (VIIb): In the formula, Ig represents the immunoglobulin unit, L L5 and L R5 Each independently represents the fifth joint, Z L Indicates the first functional substance, Z R Indicates the second functional substance, T L1 and T R1 Each independently represents a monovalent group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

138. The conjugate or salt thereof according to claim 137, wherein The conjugate or its salt comprises a structural unit represented by the following formula (VIIb-1): In the formula, 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 the seventh joint, Z L Indicates the first functional substance, Z R Indicates the second functional substance, T L2 and T R2 Each independently represents a monovalent group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

139. The conjugate or salt thereof according to claim 132, wherein The conjugate or its salt comprises a structural unit represented by the following formula (VIIc): In the formula, Ig represents the immunoglobulin unit, L R1 Indicates the first connector. L L5 Indicates the 5th connector, Z L Indicates the first functional substance, Z R Indicates the second functional substance, T L1 represents a monovalent group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

140. The conjugate or salt thereof according to claim 139, wherein The conjugate or its salt comprises a structural unit represented by the following formula (VIIc-1): In the formula, 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 Indicates the third connector, L L7 Indicates the 7th joint, Z L Indicates the first functional substance, Z R Indicates the second functional substance, T L2 represents a monovalent group, The average modification percentage r of the immunoglobulin unit by the first modification part L and the average modification percentage r of the immunoglobulin unit by the second modification part R They are 65 to 135% respectively.

141. The conjugate or salt thereof according to claim 132, wherein The first functional substance and the second functional substance are independently a drug, a labeling substance, an affinity substance, a transfer substance or a stabilizer.

142. The conjugate or salt thereof according to claim 141, wherein One of the first functional substance and the second functional substance is a full-length antibody or a fragment thereof, or both of the first functional substance and the second functional substance are independently a full-length antibody or a fragment thereof.

143. The conjugate or salt thereof according to claim 132, wherein The conjugate further comprises an additional modifying moiety.

144. The conjugate or salt thereof according to claim 143, wherein The additional modified portion is an additional modified portion comprising a functional substance, and the additional modified portion comprising a functional substance is contained in the constant region of the antibody heavy chain.

145. The conjugate or salt thereof according to claim 144, wherein The additional modification portion containing the functional substance is introduced into the constant regions of the two heavy chains via modification of the amino group in the side chain of a lysine residue present at one or more sites in the constant regions of the two heavy chains.

146. The conjugate or salt thereof according to claim 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 the human IgG heavy chain according to EU numbering.

Images