Compound containing antibody affinity substance, modified antibody and salts thereof, and method for producing antibody derivative or antibody-functional substance conjugate

Abstract

A compound or a salt thereof comprising: (A) an affinity polypeptide including an affinity moiety having affinity for a heavy chain CH2-CH3 interdomain region of an antibody; (B) a first antibody reactive group; and (C) a second antibody reactive group. The affinity polypeptide includes first and second amino acid residues each having a side chain amino group, and the first antibody reactive group and the second antibody reactive group are linked to the affinity polypeptide via linkers that are introduced into the side chain amino groups of the first and second amino acid residues each having the side chain amino group, respectively.

Description

Methods for producing compounds containing antibody affinity substances, modified antibodies and salts thereof, and antibody derivatives or antibody-functional substance conjugates.

[0001] The present invention relates to compounds containing antibody affinity substances, modified antibodies, salts thereof, and methods for producing antibody derivatives or antibody-functional substance conjugates.

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

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

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

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

[0006] As an improvement to the above C-CAP method, a technique has been reported that allows for the preparation of antibodies that regioselectively contain a functional substance (e.g., a drug) without including the peptide portion as a linker, by a chemical synthesis method using a predetermined compound containing affinity polypeptides (Patent Documents 2-5). Avoiding the use of linkers containing the peptide portion is desirable in clinical applications. These techniques propose multiple positions in the antibody that can be regioselectively modified by drugs, corresponding to various amino acid residues in the CH2 and CH3 domains (e.g., lysine residues, tyrosine residues, serine residues, and threonine residues).

[0007] 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

[0008] The objective of the present invention is to develop a technique that enables the simultaneous, regioselective chemical modification of two amino acid residues in the CH2-CH3 intercellular region of an antibody constituent unit (in other words, an immunoglobulin unit containing two heavy chains and, optionally, two light chains).

[0009] A further objective of the present invention is to develop antibody derivatives or antibody-functional substance conjugates into which two amino acid residues in the CH2-CH3 intercellular region of the heavy chain of an antibody constituent unit are simultaneously and regioselectively chemically modified, and bioorthogonal functional groups or the like are introduced.

[0010] As a result of diligent research, the inventors have found that by using (A) an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain, and (B) a compound or salt thereof containing two reactive groups for the antibody, two amino acid residues in the CH2-CH3 intercellular region of the antibody's heavy chain can be simultaneously and regioselectively chemically modified.

[0011] The compound or salt of the present invention can associate with the heavy chain of an antibody via an affinity polypeptide (A) having affinity for the CH2-CH3 intercellular region of the antibody heavy chain, and then react specifically with the side chains of two specific amino acid residues in the CH2-CH3 intercellular region of the antibody heavy chain via two reactive groups (R) for the antibody, thereby producing an affinity polypeptide-modified antibody or salt thereof in which two amino acid residues in the CH2-CH3 intercellular region of the antibody heavy chain are regioselectively modified.

[0012] Patent documents 1 to 5 disclose that antibodies can be regioselectively modified with functional substances by using affinity substances and compounds containing reactive groups for antibodies. However, selectively modifying antibodies at multiple positions requires sequential modification of each position, which is time-consuming and labor-intensive, and there is room for improvement in the yield of antibodies modified at multiple positions. Thus, patent documents 1 to 5 do not describe or suggest (1) the problem of developing a technology that can simultaneously chemically modify two amino acid residues in the CH2-CH3 intercellular region of the antibody heavy chain, nor do they describe (2) the use of affinity polypeptides that have affinity for the CH2-CH3 intercellular region of the antibody heavy chain, and that contain first and second reactive groups for antibodies as affinity substances.

[0013] The inventors have also succeeded in generating antibodies in which two amino acid residues in the CH2-CH3 intercellular region of the heavy chain of the antibody are chemically modified by using the compound of the present invention or a salt thereof. Such antibodies are characterized in that they (a) comprise an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a modification unit (e.g., the affinity polypeptide, bioorthogonal functional group, or functional substance), and (c) the modification unit is introduced via two amino acid residues in the CH2-CH3 intercellular region of the heavy chain in the immunoglobulin unit.

[0014] In other words, the present invention is as follows: [1] A compound or salt thereof comprising (A) an affinity polypeptide having an affinity moiety having affinity for the CH2-CH3 interchain region of an antibody, (B) a first antibody-reactive group, and (C) a second antibody-reactive group, wherein the affinity polypeptide comprises first and second amino acid residues having side-chain amino groups, and the first antibody-reactive group and the second antibody-reactive group are linked to the affinity polypeptide via linkers introduced to the side-chain amino groups of the first and second amino acid residues having side-chain amino groups, respectively. [2] The compound or salt thereof according to [1], wherein the first and second antibody-reactive groups are groups having responsiveness to the side-chain amino groups of lysine residues contained in the polypeptide chain of an antibody. [3] The compound or salt thereof according to [1], wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are each substituted with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence. Sequence ID 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC Sequence ID 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC [4] The compound or a salt thereof according to [3], wherein the two amino acid residues are either the 32nd E or D, the 31st A or the 30th I, and the 4th Q, the 3rd A or the 2nd N. [5] The compound or a salt thereof according to [3], wherein the two amino acid residues are the 31st A and the 3rd A. [6] The compound or a salt thereof according to [1], wherein the first and second amino acid residues having a side chain amino group are selected from a lysine residue, an ornithine residue, a diaminobutanoic acid, and a diaminopimelic acid residue, respectively. [7] The compound or a salt thereof according to [1], wherein the heavy chain CH2-CH3 intercellular region is a human heavy chain CH2-CH3 intercellular region. [8] The compound or a salt thereof according to [1], wherein the antibody is IgG. [9] The compound or a salt thereof according to [8], wherein the CH2-CH3 interregion is the region from position 230 to 439 of the human IgG heavy chain.

[10] The region between CH2-CH3 is the compound or its salt described in [8], which is the region from position 248 to position 288 of the heavy chain of human IgG.

[11] The compound is represented by the following formula (I): R. １ -L １ -A-L ２ -R ２ (I) [In the formula, R １ and R ２ represent the reactive group, L １ represents the first linker, L ２ represents the second linker, and A represents the affinity polypeptide.]. The compound or its salt described in [1].

[12] The compound or its salt further contains (iii) a cleavable moiety between (i) the affinity polypeptide and (ii) the reactive group. The compound or its salt described in [1].

[13] The cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side upon cleavage. The compound or its salt described in

[12] .

[14] The compound is represented by the following formula (Ia): R １ -L3-CLE(B) １ -L4-A-L5-CLE(B) ２ -L6-R ２ (Ia) [In the formula, R １ , R ２ represent the reactive group, L ３ represents the third linker, L ４ represents the fourth linker, L ５ represents the fifth linker, L ６ represents the sixth linker, CLE(B) １ , CLE(B) ２ each represent a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side upon cleavage, and A represents the affinity polypeptide.]. The compound or its salt described in

[13] .

[15] The compound is represented by the following formula (Ia-1): [In the formula, X １ , X ２ represent the leaving group, W １ , W ２ , W ３ , W ４ , W ５ and W ６Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L ８ This indicates the eighth linker, L ９ This indicates the 9th linker, L １０

[14] The compound or a salt thereof, represented by

[14] , wherein

[14] represents the 10th linker, S represents a sulfur atom, and A represents the affinity polypeptide.

[16] The compound or a salt thereof, represented by

[15] , wherein the leaving group is selected from the following: (a) R Ａ -S (here, R Ａ (b) R Ａ -O (Here, R Ａ (c)R Ａ - (R Ｂ -) N (Here, R Ａ and R Ｂ (i) represents a hydrogen atom, a optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom. ); or (d) a halogen atom.

[17] The compound or salt thereof according to

[12] , further comprising (ii) a bioorthogonal functional group between the reactive group and (iii) a cleavable moiety.

[18] The compound is of the following formula (ib): [In the formula, R １ , R ２ L represents the reactive group, １１ This indicates the 11th linker, L １２ This indicates the 12th linker, L １３ This indicates the 13th linker, L １４ This indicates the 14th linker, B １ , B ２ This indicates a group containing a bioorthogonal functional group, CLE １ CLE ２represents the cleavable portion, and A represents the affinity polypeptide. The compound or a salt thereof described in

[17] , represented by ].

[19] The compound is of the following formula (Ib-1): [In the formula, X ３ , X ４ This indicates a leaving group, W ７ , W ８ , W ９ , W １０ , W １１ and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １６ This indicates the 16th linker, L １７ This indicates the 17th linker, L １８ This indicates the 18th linker, B ３ , B ４ This indicates a group containing a bioorthogonal functional group, V １ , V ２[1] represents an oxygen atom or a sulfur atom, and A represents the affinity polypeptide.

[20] A compound or salt thereof according to

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

[21] A reagent for antibody derivatization comprising any compound or salt thereof from [1] to

[20] .

[22] An affinity polypeptide-modified antibody or salt thereof, wherein the affinity polypeptide having an affinity moiety having affinity for the heavy chain CH2-CH3 inter-region of the antibody is contained in the heavy chain CH2-CH3 inter-region of the antibody, wherein the affinity polypeptide is introduced into the heavy chain CH2-CH3 inter-region by modification of the amino groups in the side chains of amino acid residues having side chain amino groups present in two locations in the heavy chain CH2-CH3 inter-region.

[23] The affinity polypeptide-modified antibody or salt thereof according to

[22] , comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) the affinity polypeptide, and (c) the affinity polypeptide introduced into the CH2-CH3 inter-heavy chain region of the immunoglobulin unit via modification of amino groups in the side chains of lysine residues located at two locations in the CH2-CH3 inter-heavy chain region of the immunoglobulin unit.

[24] The affinity polypeptide-modified antibody or salt thereof according to

[23] , wherein the two locations in the CH2-CH3 inter-heavy chain region are K248 and K288 of a human IgG heavy chain according to EU numbering.

[25] The affinity polypeptide has an amino acid sequence in which two amino acid residues are replaced with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence, and is modified to the amino groups in the side chains of the amino acid residues having side-chain amino groups located in the two CH2-CH3 inter-heavy chain regions via a linker introduced to the side-chain amino groups of said amino acid residues, the affinity polypeptide modified antibody or salt thereof according to

[22] .SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC

[26] Affinity polypeptide-modified antibody is given by the following formula (II): [In the formula, Ig represents an immunoglobulin unit including the heavy chain and, optionally, the light chain, L １ This indicates the first linker, L ２

[22] The affinity polypeptide-modified antibody or a salt thereof, comprising a structural unit represented by [2], wherein (i) represents a second linker, (ii) represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%.

[27] The affinity polypeptide-modified antibody or a salt thereof, comprising the antibody or a salt thereof, further comprising (iii') a cleavable moiety between (i') the affinity polypeptide and (ii') the antibody.

[28] The affinity polypeptide-modified antibody or a salt thereof, comprising the antibody or a salt thereof, wherein the cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

[29] The affinity polypeptide-modified antibody comprises the following formula (IIa): [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ４ This indicates the fourth linker, L ５ This indicates the fifth linker, L ６ This indicates the sixth linker, CLE(B) １ and CLE(B) ２ [1] represents a cleavable portion that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%.

[28] The affinity polypeptide modified antibody or a salt thereof, comprising a structural unit represented by

[28] .

[30] The affinity polypeptide modified antibody is given by the following formula (IIa-1): [In the formula, Ig represents the immunoglobulin unit, W １ , W ２ , W３ and W ４ and W ５ and W ６ each independently represents an oxygen atom or a sulfur atom, L ７ represents a 7th linker, L ８ represents an 8th linker, L ９ represents a 9th linker, L １０ represents a 10th linker, S represents a sulfur atom, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody or a salt thereof according to

[29] , which contains a structural unit represented by.

[31] The affinity polypeptide-modified antibody or a salt thereof according to

[27] , wherein the antibody or a salt thereof further contains a bioorthogonal functional group (iv') between (ii') the antibody and (iii') the cleavable moiety.

[32] The affinity polypeptide-modified antibody is represented by the following formula (IIb): [In the formula, Ig represents the immunoglobulin unit, L １１ represents an 11th linker, L １２ represents a 12th linker, L １３ represents a 13th linker, L １４ represents a 14th linker, B １ and B ２ each represents a group containing a bioorthogonal functional group, CLE １ and CLE ２ represent a cleavable moiety, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody or a salt thereof according to

[31] , which contains a structural unit represented by.

[33] The affinity polypeptide-modified antibody is represented by the following formula (IIb-1): [In the formula, Ig represents the immunoglobulin unit, W ７ and W ８ and W ９ and W １０ and W １１ and W １２ each independently represents an oxygen atom or a sulfur atom, L １５This indicates the 15th linker, L １６ This indicates the 16th linker, L １７ This indicates the 17th linker, L １８ This indicates the 18th linker, B ３ and B ４ This indicates a group containing a bioorthogonal functional group, V １ and V ２A represents an oxygen atom or a sulfur atom, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%. The affinity polypeptide modified antibody or a salt thereof according to

[32] , comprising a structural unit represented by

[34] . A method for producing an affinity polypeptide modified antibody or a salt thereof, comprising reacting the antibody derivatization reagent according to

[21] with an antibody comprising an immunoglobulin unit comprising a heavy chain and optionally a light chain to produce an affinity polypeptide modified antibody or a salt thereof comprising the affinity polypeptide in the heavy chain CH2-CH3 interregion of the immunoglobulin unit.

[35] A method for producing an antibody or salt that does not contain an affinity polypeptide, comprising (A) an affinity polypeptide having an affinity moiety having affinity for the CH2-CH3 interchain region of an antibody, and (B) an antibody, wherein (C) (A) the affinity polypeptide and (B) the antibody are linked by first and second linkers, and the first and second linkers each have first and second cleavable moieties, respectively, and the affinity polypeptide-modified antibody or salt thereof is cleaved at the first and second cleavable moieties to produce an antibody or salt that does not contain an affinity polypeptide.

[36] The method for producing an antibody or salt that does not contain an affinity polypeptide, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are substituted with amino acid residues having side chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence. Sequence ID 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC Sequence ID 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC

[37] The method according to

[35] , wherein the first and second cleavable portions are cleavable portions that can generate bioorthothic functional groups on the antibody side by cleavage, and the antibody or salt that does not contain affinity polypeptides is an antibody derivative or salt that contains two bioorthothic functional groups per heavy chain.

[38] The method according to

[37] , wherein the antibody derivative or salt that contains two bioorthothic functional groups per heavy chain comprises a structural unit represented by the following formula (IIIa) or formula (IIIa-1). [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ６ This indicates the sixth linker, B ５ and B ６ This indicates a group containing a bioorthogonal functional group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ L represents an oxygen atom or a sulfur atom. ７ This indicates the seventh linker, L １０

[39] The method according to

[35] , wherein the antibody or salt thereof that does not contain affinity polypeptide further comprises first and second bioorthogonal functional groups between the antibody and the first and second cleavable moieties, and the antibody or salt thereof that does not contain affinity polypeptide is an antibody derivative or salt thereof that contains two bioorthogonal functional groups per heavy chain.

[40] The method according to

[39] , wherein the antibody derivative or salt thereof that contains two bioorthogonal functional groups per heavy chain comprises a structural unit represented by the following formula (IIIb) or the following formula (IIIb-1). [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, B １ and B ２ This indicates a group containing a bioorthogonal functional group, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L１５ represents the 15th linker, L １８ represents the 18th linker, B ３ and B ４ represent a group containing a bioorthogonal functional group, T ３ and T ４ represent a monovalent group. The modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65 to 135%.].

[41] A method for producing a conjugate containing an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) Producing an antibody derivative containing two bioorthogonal functional groups per heavy chain or a salt thereof by the method described in

[37] : and (2) Reacting an antibody derivative containing two bioorthogonal functional groups per heavy chain or a salt thereof with a functional substance to produce an antibody and a conjugate or a salt thereof containing two functional substances per heavy chain.

[42] The method according to

[41] , wherein the conjugate or a salt thereof contains a structural unit represented by the following formula (IVa) or (IVa-1). [In the formula, Ig represents the immunoglobulin unit, L ３ represents the 3rd linker, L ６ represents the 6th linker, Z １ and Z ２ represent a functional substance. The average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65 to 135%.] [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ each independently represent an oxygen atom or a sulfur atom, L ７ represents the 7th linker, L １０ represents the 10th linker, Z １ and Z ２∇ [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, Z １ and Z ２ This indicates a functional substance, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, Z １ and Z ２ This indicates a functional substance, T ３ and T ４The group is monovalent, and the percentage r of modification per heavy chain in the immunoglobulin unit by the functional substance is 65 to 135%.

[45] A method for producing an antibody and a conjugate or salt thereof containing an antibody and a functional substance, wherein the conjugate contains an antibody and a functional substance, wherein the conjugate contains an antibody and a functional substance, wherein the conjugate contains an antibody and a functional substance, wherein the conjugate contains an antibody and a functional substance, wherein the antibody derivative or salt containing two bioorthogonal functional groups per heavy chain is an antibody derivative or salt thereof, wherein the antibody derivative or salt containing two bioorthogonal functional groups per heavy chain is (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) a first and second bioorthogonal functional group, and (c) the first and second bioorthogonal functional groups are introduced into the CH2-CH3 interregion of the heavy chain in the immunoglobulin unit. A method comprising an antibody and functional substance conjugate or a salt thereof, wherein the conjugate comprises an antibody and functional substance containing two functional substances per heavy chain, wherein the conjugate comprises an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) two functional substances per heavy chain of the antibody, and (c) the functional substances are introduced at two locations in the CH2-CH3 interregion of the heavy chain in the immunoglobulin unit.

[46] The method according to

[45] , wherein the antibody derivative or a salt thereof comprising two bioorthogonal functional groups per heavy chain of the antibody has a structural unit represented by the following formulas (IIIa), (IIIa-1), (IIIb) and / or (IIIb-1). [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ６ This indicates the sixth linker, B ５ and B ６ This indicates a group containing a bioorthogonal functional group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ L represents an oxygen atom or a sulfur atom. ７represents the 7th linker, L １０ represents the 10th linker, SH represents a thiol group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65 to 135%.]. [In the formula, Ig represents the immunoglobulin unit, L １１ represents the 11th linker, L １４ represents the 14th linker, B １ and B ２ represent groups containing a bioorthogonal functional group, T １ and T ２ represent monovalent groups, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65 to 135%.] [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W<00,00173>, and W １２ each independently represent an oxygen atom or a sulfur atom, L １５ represents the 15th linker, L １８ represents the 18th linker, B ３ and B ４ [[ID=)34]]represent groups containing a bioorthogonal functional group, T ３ and T ４ represent monovalent groups, and the modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65 to 135%.].

[47] A conjugate or a salt thereof containing two functional substances per heavy chain of an antibody, wherein the conjugate or the salt thereof contains a structural unit represented by the following formula (IVa), (IVa-1), (IVb) and / or (IVb-1), according to the method described in

[45] . [In the formula, Ig represents the immunoglobulin unit, L ３ represents the 3rd linker, L ６ represents the 6th linker, Z １ and Z ２ represent functional substances, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65 to 135%.] [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L １０ This indicates the 10th linker, Z １ and Z ２ This indicates a functional substance, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, Z １ and Z ２ This indicates a functional substance, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, Z １ and Z ２ This indicates a functional substance, T ３ and T ４The group is monovalent, and the percentage of modification r per heavy chain in the immunoglobulin unit by the functional substance is 65 to 135%.

[48] An affinity polypeptide or a salt thereof comprising an affinity moiety having affinity for the CH2-CH3 interchain region of an antibody, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are replaced with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence. SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC

[49] A polynucleotide encoding an affinity polypeptide having affinity for the CH2-CH3 intercellular region of an antibody heavy chain, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are replaced with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence.

[0015] According to the present invention, two amino acid residues in the CH2-CH3 intercellular region of the heavy chain of an antibody (an immunoglobulin unit containing two heavy chains and optionally two light chains) can be simultaneously and regioselectively modified. Therefore, according to the present invention, an antibody in which two amino acid residues in the CH2-CH3 intercellular region of the antibody's heavy chain are regioselectively modified can be provided simply, i.e., with fewer steps and in high yield. Simultaneous modification also has the effect of suppressing aggregation of the antibody-functional substance conjugate obtained by the modification, compared to sequential modification of one site at a time.

[0016] Analysis results of DAR calculator for Trastuzumab ADC with DAR=4 synthesized using affinity reagent P1-2. Analysis results of DAR calculator for Trastuzumab ADC with DAR=4 synthesized using affinity reagent P2-2. Analysis results of DAR calculator for Trastuzumab ADC with DAR=4 synthesized using affinity reagent P4-2. Figure 4 shows the MS spectrum (measured value: m / z 952.23187, theoretical value: 952.22942, tetravalent) of the peptide fragment THTCPPCPAPELLGGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 15), which consists of 33 amino acid residues including a modification site to the lysine residue by trypsin digestion of Trastuzumab (a thiol-transformed compound that has undergone carboxylation with iodoacetamide (+145.019Da)) (Example 10-4). Figure 5 shows the HCD spectrum of the product ion with m / z 1167.12 (theoretical value: 1166.61), which corresponds to the divalent y20 and shows the modification of the lysine residue at position 248 of the human IgG heavy chain in EU numbering (Example 10-4). Figure 6 shows the MS spectrum (measured value: m / z 577.03757, theoretical value: 577.03557, tetravalent) of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 16), which consists of 18 amino acid residues including a modification site to the lysine residue by trypsin digestion of Trastuzumab (a thiol derivative that has undergone carboxylation with iodoacetamide (+145.019Da)) (Example 10-4). Figure 7 shows the HCD spectrum of the product ion with m / z 798.40 (theoretical value: 798.40), which corresponds to the divalent y13, showing the modification of the lysine residue at position 288 of the human IgG heavy chain in EU numbering (Example 10-4). Figure 8 shows the results of searching for peptide fragments containing thiol derivatives (+145.019Da) that have undergone modification of lysine residues (Carbamidemethylation with iodoacetamide) in the trypsin digest of Trastuzumab using BioPharma Finder (Example 10-4).The horizontal axis shows the identified lysine residues, and the vertical axis shows the intensity.

[0017] 1. General Definitions of Terms In this specification, terms and expressions used to describe a particular invention or subject may also be incorporated into descriptions of other inventions or subjects. Accordingly, the definitions, examples, and preferred examples of terms and expressions used to describe a particular invention or subject may also be incorporated into descriptions of other inventions or subjects described by such terms and expressions.

[0018] In this invention, the term "antibody" is defined as follows. The term "immunoglobulin unit" corresponds to the bivalent monomer unit that constitutes such an antibody, and is an immunoglobulin unit containing two heavy chains and optionally two light chains. Therefore, the definitions, examples, and preferred examples of terms and expressions for immunoglobulin units, such as their origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, amino acid residue (e.g., lysine residue) position, and regioselectivity, are the same as those for antibodies described below and are interchangeable with the expression "antibody".

[0019] The origin of the antibodies is not particularly limited and may be derived from animals such as mammals or birds (e.g., chickens). Preferably, the immunoglobulin units are derived from mammals. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g., dogs, cats), livestock (e.g., cattle, pigs, goats), and working animals (e.g., horses, sheep), with primates or rodents being preferred, and humans being more preferred.

[0020] The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a predetermined glycan has been attached (e.g., antibodies modified to have a glycan-binding consensus sequence such as an N-linked glycan-binding consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond-reducing antibodies. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, a full-length antibody or an antibody fragment containing a variable region and a CH2-CH3 interregion can be used as the monoclonal antibody, but a full-length antibody, an Fc region protein, or an Fc fusion protein is preferred. The antibody is preferably a human IgG monoclonal antibody, and more preferably a human IgG full-length monoclonal antibody.

[0021] Any antigen can be used as the antigen for the antibody. For example, such antigens include proteins (including oligopeptides and polypeptides; proteins modified with biomolecules such as sugars (e.g., glycoproteins)), glycans, nucleic acids, and small molecule compounds. Preferably, the antibody may be an antibody that uses a protein as its antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0022] More specifically, the antigen protein of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0023] (1) Oncology field PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesotheli n, CD138, c-Met, Ang2, VEGF- A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, maccrophase inhibitory factor, CD74, Notch1, Notch 2. Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM- CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEG F, RSPO, LIV-1, SLITRK6, Nect in-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissu Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73 , FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7 , EFNA4, FAP, DR5, CEA, Ly6E, CA6、CEACAM5、LAMP1、tissue factor, EPHA2, DR5, B7-H3, FGFR4 , FGFR2, α2-PI, A33, GDF15, CAIX, C D166, ROR1, GITR, BCMA, TBA, LAG- 3. EphA2, TIM-3, CD-200, EGFRvIII , CD16A, CD32B, PIGF, Axl, MICA / B , Thomsen-Friedenreich, CD39, CD 37、CD73、CLEC12A、Lgr3、トランスフェリンReceptor, TGFβ, IL-17, 5T4, RTK, Immune Supplier Protein, NaPi2b, ルイス blood type B antigen, A34, Lysil-Oxidase , DLK-1, TROP-2, α9インテグリン, TAG-72 (CA72-4), CD70,

[0024] (2) Autoimmune diseases / inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS , Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP (Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74 , CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIF HT

[0025] (3) Neurological diseases: CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containering 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0026] (4) Infectious Diseases: Clostridium Difficile toxin B, cytomegalovirus, RSV, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PCRV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0027] (5) Hereditary and rare diseases: Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0028] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0029] (7) Bone / orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0030] (8) Blood diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0031] (9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

[0032] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, ortatoxacimab) and certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, allentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, ecrizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab, ikesekizumab) Examples include iodine (IgG4), reslizumab (IgG4), atezolizumab), and specific human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, laxibakumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), pembrolizumab (IgG4), alirocumab, necitumumab, brodalumab (IgG2), olaratumab) (where the IgG subtype is not mentioned, it is assumed to be IgG1).

[0033] In this invention, two specific amino acid residues in the CH2-CH3 intercellular region of the heavy chain in an antibody can be regioselectively modified. Examples of such specific amino acid residues include those having a side-chain amino group, such as lysine residues. For example, in human IgG such as human IgG1, the following amino acid residues located in the CH2-CH3 intercellular region of the heavy chain can be exposed on the antibody surface, and these amino acid residues can be used to introduce specific cleavage sites (the positions of amino acid residues are according to EU numbering; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). Examples of exposed lysine residues: position 248 (K248), position 288 (K288).

[0034] The location of amino acid residues in antibodies and the location of the constant region of the heavy chain (e.g., the CH2-CH3 region) follows EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html).

[0035] Preferably, lysine residues (e.g., lysine residues at position 248 and 248) can be regioselectively modified as specific amino acid residues in the CH2-CH3 intercellular region of the heavy chain that are regioselectively modified. In this specification, "regioselectivity" means that even though specific amino acid residues are not eclectically distributed in specific regions of the antibody, a predetermined structural unit capable of binding to a specific amino acid residue in the antibody is eclectically distributed in specific regions of the antibody. Therefore, expressions related to regioselectivity such as "regioselectively present," "regioselective binding," and "regioselective binding" mean that the possession rate or binding rate of a predetermined structural unit in a target region containing one or more specific amino acid residues is significantly higher than the possession rate or binding rate of the same structural unit in a non-target region containing multiple amino acid residues of the same type as the specific amino acid residue in the target region. Such regioselectivity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%. According to the present invention, specific lysine residues in the heavy chain of an antibody can be regioselectively modified without using a linker containing a peptide. The peptide portion has potential immunogenicity and is readily hydrolyzed in the blood. Therefore, avoiding the use of a linker containing a peptide portion is desirable in clinical applications.

[0036] In this invention, as long as two specific amino acid residues in the heavy chain CH2-CH3 interregion (e.g., lysine residues at specific positions) are regioselectively modified, one or more other specific amino acid residues may be additionally regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in International Publications 2018 / 199337, 2019 / 240288, 2019 / 240287, and 2020 / 090979. Such specific amino acid residues can be amino acid residues having easily modifiable side chains (e.g., amino group, carboxyl group, amide group, hydroxyl group, thiol group) (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine ​​residues), but preferably lysine residues having a side chain containing an amino group, tyrosine residues having a side chain containing a hydroxyl group, serine residues, and threonine residues, or cysteine ​​residues having a side chain containing a thiol group, and more preferably lysine residues.

[0037] (Halogen atoms) Examples of halogen atoms include fluorine, chlorine, bromine, and iodine.

[0038] (Monovalent groups) Examples of monovalent groups include monovalent hydrocarbon groups and monovalent heterocyclic groups.

[0039] The monovalent group may be substituted with one or more substituents (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) as described below.

[0040] (Monovalent hydrocarbon groups and related terms) Examples of monovalent hydrocarbon groups include monovalent linear hydrocarbon groups, monovalent alicyclic hydrocarbon groups, and monovalent aromatic hydrocarbon groups.

[0041] The monovalent chain hydrocarbon group means a hydrocarbon group composed only of a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be straight-chain or branched-chain. Examples of the monovalent chain hydrocarbon group include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl, and alkynyl may be either straight-chain or branched-chain.

[0042] As alkyl, alkyl having 1 to 12 carbon atoms is preferable, alkyl having 1 to 6 carbon atoms is more preferable, and alkyl having 1 to 4 carbon atoms is even more preferable. When alkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl.

[0043] As alkenyl, alkenyl having 2 to 12 carbon atoms is preferable, alkenyl having 2 to 6 carbon atoms is more preferable, and alkenyl having 2 to 4 carbon atoms is even more preferable. When alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, n-butenyl.

[0044] As alkynyl, alkynyl having from 2 to 12 carbon atoms is preferable, alkynyl having 2 to 6 carbon atoms is more preferable, and alkynyl having 2 to 4 carbon atoms is even more preferable. When alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, n-butynyl.

[0045] As the monovalent chain hydrocarbon group, alkyl is preferable.

[0046] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as its ring structure and does not contain an aromatic ring. The alicyclic hydrocarbon may be monocyclic or polycyclic. However, it is not necessary to be composed solely of alicyclic hydrocarbons; it may contain a chain-like structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.

[0047] As for cycloalkyls, cycloalkyls having 3 to 12 carbon atoms are preferred, cycloalkyls having 3 to 6 carbon atoms are more preferred, and cycloalkyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkyls having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0048] As the cycloalkenyl, cycloalkenyls having 3 to 12 carbon atoms are preferred, cycloalkenyls having 3 to 6 carbon atoms are more preferred, and cycloalkenyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkenyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkenyls having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0049] As the cycloalkynyl, cycloalkynyls having 3 to 12 carbon atoms are preferred, cycloalkynyls having 3 to 6 carbon atoms are more preferred, and cycloalkynyls having 5 to 6 carbon atoms are even more preferred. If the cycloalkynyl has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of cycloalkynyls having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0050] As the monovalent alicyclic hydrocarbon group, cycloalkyl is preferred.

[0051] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not need to consist solely of an aromatic ring; it may also contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be monocyclic or polycyclic. Preferred monovalent aromatic hydrocarbon groups are aryl groups having 6 to 12 carbon atoms, more preferably aryl groups having 6 to 10 carbon atoms, and even more preferably aryl groups having 6 carbon atoms. If the monovalent aromatic hydrocarbon group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0052] Phenyl is preferred as the monovalent aromatic hydrocarbon group.

[0053] Among these, alkyl, cycloalkyl, and aryl groups are preferred as monovalent hydrocarbon groups.

[0054] (Monovalent heterocyclic groups and related terminology) A monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. A monovalent heterocyclic group is either a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heterocyclic group preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms.

[0055] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. If the monovalent aromatic heterocyclic group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of monovalent aromatic heterocyclic groups include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridadinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, prinyl, anthraquinolyl, carbazonal, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0056] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. If the monovalent non-aromatic heterocyclic group has substituents, the number of carbon atoms of the substituents is not included in the above carbon atom count. Examples of monovalent non-aromatic heterocyclic groups include oxylanil, azilidinil, azetidinil, oxetanil, thietanil, pyrrolidinil, dihydrofuranil, tetrahydrofuranil, dioxolanil, tetrahydrothiophenyl, pyrrolinil, imidazolidinil, oxazolidinil, piperidinil, dihydropyranil, tetrahydropyranil, tetrahydrothiopyranil, morpholinil, thiomorpholinil, piperazinil, dihydrooxazinil, tetrahydrooxazinil, dihydropyrimidinil, and tetrahydropyrimidinil.

[0057] Among these, a 5-membered or 6-membered heterocyclic group is preferred as the monovalent heterocyclic group.

[0058] (Divalent groups) Divalent groups include divalent linear hydrocarbon groups, divalent cyclic hydrocarbon groups, divalent heterocyclic groups, -C(=O)-, -C(=S)-, and -NR ｘ -, -C(=O)-NR ｘ -, -NR ｘ -C(=O)-, -C(=S)-NR ｘ -, -NR ｘ -C(=S)-, -O-, -S-, -(OR ｙ )n-, and -(S-R ｙ R is a group having a main chain structure comprising one group selected from the group consisting of m-, or two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, particularly preferably 2 or 3). ｘ R represents a hydrogen atom or a substituent as described later. ｙn represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. n and m are integers from 1 to 10, preferably from 1 to 8, more preferably from 1 to 6, even more preferably from 1 to 5, and particularly preferably from 1 to 3.

[0059] The divalent linear hydrocarbon group is a linear alkylene, linear alkenylene, or linear alkylene. Linear alkylenes are linear alkylenes having 1 to 6 carbon atoms, with linear alkylenes having 1 to 4 carbon atoms being preferred. Examples of linear alkylenes include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. Linear alkenylenes are linear alkenylenes having 2 to 6 carbon atoms, with linear alkenylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkenylenes include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. Linear alkylenes are linear alkylenes having 2 to 6 carbon atoms, with linear alkylenes having 2 to 4 carbon atoms being preferred. Examples of linear alkylenes include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. Linear alkylenes are preferred as the divalent linear hydrocarbon group.

[0060] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. As the arylene, arylenes having 6 to 14 carbon atoms are preferred, arylenes having 6 to 10 carbon atoms are more preferred, and arylenes having 6 carbon atoms are particularly preferred. Examples of arylenes include phenylene, naphthylene, and anthracenylene. As the divalent non-aromatic cyclic hydrocarbon group, a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms is preferred, a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms is more preferred, and a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms is particularly preferred. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. Arylene is preferred as the divalent cyclic hydrocarbon group.

[0061] A divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatoms constituting the heterocycle preferably include one or more selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably one or more selected from the group consisting of oxygen, sulfur, and nitrogen atoms. As for the divalent aromatic heterocyclic group, a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a divalent aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, franziyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluoradiyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As for divalent non-aromatic heterocyclic groups, non-aromatic heterocyclic groups having 3 to 15 carbon atoms are preferred, non-aromatic heterocyclic groups having 3 to 9 carbon atoms are more preferred, and non-aromatic heterocyclic groups having 3 to 6 carbon atoms are particularly preferred. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxylandiyl, aziridindiyl, azetidinediyl, oxetanediyl, thietandiyl, pyrrolidinediyl, dihydrofranziyl, tetrahydrofranziyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. Divalent aromatic heterocyclic groups are preferred as the divalent heterocyclic groups.

[0062] Preferably, the divalent group is alkylene, arylene, -C(=O)-, -NR ｘ -, -C(=O)-NR ｘ -, -NR ｘ -C(=O)-, -O-, and -(O-R) ２ A divalent group having a main chain structure containing one group selected from the group consisting of n-, or alkylene, arylene, -C(=O)-, -NR ｘ -, -C(=O)-NR ｘ -, -NR ｘ -C(=O)-, -O-, and -(O-R) ｙ A divalent group having a main chain structure containing two or more groups selected from the group consisting of n-, R ｘ However, it is a hydrogen atom or an alkyl group, R ｙ n may be an alkylene or arylene, and n may be an integer from 1 to 5 (i.e., 1, 2, 3, 4, or 5). Alkylene, arylene, and alkyl are as described above.

[0063] The main chain structure in the divalent group may be substituted with one or more substituents (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) described later.

[0064] (Substituents) Substituents include: (i) halogen atoms; (ii) monovalent hydrocarbon groups; (iii) monovalent heterocyclic groups; (iv) aralkyl groups; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C (=O)-, or R ａ -C(=O)-O-(R ａ (i)NR represents a hydrogen atom or a monovalent hydrocarbon group. 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(i) (vii) Nitro group, sulfate group, sulfonic acid group, cyano group, and carboxyl group.

[0065] The definitions, examples, and preferred examples of halogen atoms, monovalent hydrocarbon groups, and monovalent heterocyclic groups in the above substituents are the same as those described above.

[0066] An aralkyl refers to an arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyls are as described above. Preferred aralkyls have 3 to 15 carbon atoms. Examples of such aralkyls include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0067] Preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl, phenyl, or naphthyl atom having 1 to 12 carbon atoms; (iii) an aralkyl atom having 3 to 15 carbon atoms; (iv) a 5-membered or 6-membered heterocycle; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C (=O)-, or R ａ -C(=O)-O-(R ａ (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 (vii) The same or different groups listed in (vii) above.

[0068] More preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl group having 1 to 12 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, Rａ -O-C (=O)-, or R ａ -C(=O)-O-(R ａ (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 (v) The same or different hydrogen atoms or alkyl groups having 1 to 12 carbon atoms.

[0069] More preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl group having 1 to 6 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C (=O)-, or R ａ -C(=O)-O-(R ａ (iv)NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O-, or Rb-C(=O)-NR ｃ - (R ｂ and R ｃ (v) The same or different groups, which represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.

[0070] Particularly preferred, the substituents may be: (i) a halogen atom; (ii) an alkyl group having 1 to 4 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C (=O)-, or R ａ -C(=O)-O-(Rａ (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 (v) The same or different hydrogen atoms or alkyl groups having 1 to 4 carbon atoms.

[0071] (Bioorthogonal Functional Groups) Bioorthogonal functional groups are groups that do not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bioorthogonal functional groups are well known in the art (see, for example, Sharpless K.B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

[0072] In this invention, a bioorthogonal functional group for proteins is used as the bioorthogonal functional group. This is because the antibody to be derivatized by the reagent of this invention is a protein. A bioorthogonal functional group for proteins is a group that does not react with the side chains of the 20 natural amino acid residues that make up proteins, or reacts slowly with those side chains, but reacts with the target functional group. The 20 natural amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Of these 20 naturally occurring amino acids, glycine, which lacks a side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine, whose side chains are hydrocarbon groups (i.e., their side chains do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms), are inert to normal reactions. Therefore, bioorthogonal functional groups for proteins are those that do not react with the side chains of these amino acids, which have side chains that are inert to normal reactions, as well as those that react with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, or react slowly, but do react with the functional group of interest.

[0073] Examples of such bioorthogonal functional groups include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, any residue that has a vinylene (ethenylene) moiety, which is the smallest unit having an intercarbon double bond; the same applies hereafter), alkyne residues (in other words, any residue that has an ethynylene moiety, which is the smallest unit having an intercarbon triple bond; the same applies hereafter), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues (e.g., carbonyl residues having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position; the same applies hereafter), isonitrile residues, cydonone residues, and selenium residues.

[0074] Bioorthogonal functional groups may be protected or unprotected. A bioorthogonal functional group refers to either an unprotected or protected bioorthogonal functional group. An unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above. A protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleaving a protecting group. Cleavage of the protecting group can be performed by specific treatments under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes; (b) treatment with physicochemical stimuli selected from the group consisting of light; or (c) standing up with a cleavage linker containing a self-degrading cleavageable moiety. Such protecting groups and their cleavage conditions are common technical knowledge in the art (e.g., G. Lerice, 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); DeSimoné, J.M., Journal of American Chemical Society). Society. 132, 17928 (2010); Thompson, D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004). The reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are described below.

[0075] Examples of protected bioorthogonal functional groups include disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinaldiol residues.

[0076] Preferably, the bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0077] More preferably, the bioorthogonal functional group may be a specific bioorthogonal functional group that exhibits excellent reactivity (e.g., reaction level and / or reaction specificity) with other bioorthogonal functional groups. Examples of such bioorthogonal functional groups include azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with substituents as described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues. Examples of combinations of two bioorthogonal functional groups that can react with each other include combinations of azide residues and alkyne residues, combinations of tetrazine residues and alkene residues, combinations of tetrazine residues and alkyne residues, combinations of thiol residues and maleimide residues, combinations of furan residues and maleimide residues, combinations of thiol residues and halocarbonyl residues (where the halogen is replaced with a thiol by substitution reaction), and combinations of a thiol residue and another thiol residue (where a disulfide bond is formed).

[0078] (Functional Substances) Functional substances are not particularly limited as long as they are substances that confer any function to an antibody, and include, for example, drugs, labeling substances, affinity substances, transport substances, and stabilizers, but preferably they may be drugs, labeling substances, affinity substances, or transport substances. Functional substances may also be a single functional substance or a substance in which two or more functional substances are linked together.

[0079] The drug may be any drug for any disease. Such diseases include, for example, cancer (e.g., lung cancer, stomach 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 and inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), neurological diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infections, viral infections), hereditary and rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone and orthopedic diseases (e.g., osteoarthritis), blood disorders (e.g., leukemia, purpura), and other diseases (e.g., metabolic disorders such as diabetes and hyperlipidemia, liver diseases, kidney diseases, lung diseases, cardiovascular diseases, digestive system diseases). The drug may be a preventative or therapeutic agent for a disease, or a drug to alleviate side effects.

[0080] More specifically, the drug may be an anticancer agent. Examples of anticancer agents include chemotherapeutic agents, toxins, radioisotopes, or substances containing them. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalators, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., lysine). Examples of radioactive isotopes include the radioactive isotope of hydrogen (e.g., 3H), the radioactive isotope of carbon (e.g., 14C), the radioactive isotope of phosphorus (e.g., 32P), the radioactive isotope of sulfur (e.g., 35S), the radioactive isotope of yttrium (e.g., 90Y), the radioactive isotope of technetium (e.g., 99mTc), the radioactive isotope of indium (e.g., 111In), the radioactive isotopes of iodine (e.g., 123I, 125I, 129I, 131I), the radioactive isotope of samarium (e.g., 153Sm), the radioactive isotope of rhenium (e.g., 186Re), the radioactive isotope of astatine (e.g., 211At), and the radioactive isotope of bismuth (e.g., 212Bi). More specifically, the drugs include auristatin (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calichemycin, duocalmycin, eribulin, anthracycline, dmDNA31, and tubulcin.

[0081] Labeling substances are substances that enable the detection of targets (e.g., tissues, cells, materials). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those mentioned above), or substances containing them.

[0082] An affinity substance is a substance that has affinity for a target. Examples of affinity substances include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary chains to target nucleic acids. The affinity substance is preferably an affinity protein or affinity polypeptide, and more preferably an antibody. The type of animal from which the antibody used as a functional substance originates is the same as described above.

[0083] The type of antibody used as a functional substance may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies to which a predetermined sugar chain has been attached (e.g., antibodies modified to have a sugar chain binding consensus sequence such as an N-linked sugar chain binding consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide bond reduct antibodies. Examples of isotypes of monoclonal antibodies include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Examples of antibodies used as functional substances include full-length antibodies and their fragments (fragment antibodies). Fragment antibodies only need to maintain their ability to bind to the desired antigen, for example, Fab, Fab', F(ab') ２ Examples include scFv and VHH antibodies.

[0084] The antigenicity of the antibody used as a functional substance may be the same as or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, and it is preferable that it be different. Furthermore, the origin of the antibody used as a functional substance may be the same as or different from the origin of the immunoglobulin unit, and it is preferable that it be different. Therefore, the antibody used as a functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody, or an antibody derived therefrom, as mentioned in the specific examples of monoclonal antibodies described above. The antibody used as a functional substance may also be IgG1, IgG2, IgG3, or IgG4, or an antibody derived therefrom, as mentioned in the specific examples of monoclonal antibodies described above.

[0085] The transport material is a substance that has the ability to transport compounds. Preferred transport materials are substances that can encapsulate compounds within a protein shell (e.g., multimers) (e.g., ferritin, virus particles, virus-like particles).

[0086] Stabilizers are substances that enable the stabilization of antibodies. Examples of stabilizers include diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0087] Functional substances may also be peptides, proteins, nucleic acids, organic compounds, inorganic compounds, glycans, lipids, high molecular weight polymers, metals (e.g., gold), or chelators. Examples of peptides include cell membrane permeable peptides, blood-brain barrier permeable peptides, and peptide pharmaceuticals. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, antibodies, and ferritin. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Other examples of nucleic acids include RNA interference-inducible nucleic acids (e.g., siRNA), aptamers, and antisense compounds. Examples of organic compounds include low molecular weight organic compounds such as proteolytic chimeric molecules, dyes, and photodegradable compounds. Examples of inorganic compounds include silica, talc, and alumina.

[0088] (Salts) In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Examples of salts with inorganic acids include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of salts with inorganic bases include salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Examples of salts with organic bases include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and salts with acidic amino acids (e.g., aspartic acid, glutamic acid). The salts are preferably salts with inorganic acids (e.g., hydrogen chloride) or organic acids (e.g., trifluoroacetic acid).

[0089] 2. Affinity substances or salts thereof, and related inventions 2-1. Affinity substances or salts thereof The present invention provides affinity substances or salts thereof that include an affinity moiety having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody.

[0090] The affinity substance used in the present invention comprises an affinity moiety having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain. In one embodiment, the antibody is an immunoglobulin unit comprising two heavy chains and optionally two light chains. Examples of antibodies comprising an immunoglobulin unit comprising two heavy chains and two light chains include bivalent antibodies (e.g., IgG, IgD, IgE), or quadrivalent or higher antibodies (e.g., IgA antibody, IgM antibody), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and disulfide-bonded reductantibodies. Examples of antibodies comprising an immunoglobulin unit comprising two heavy chains include Fc region proteins and their fusion proteins.

[0091] Examples of affinity moieties include polymeric substances of a predetermined structural unit [e.g., peptides (including oligopeptides, polypeptides, and proteins), nucleic acids (including oligonucleotides and polysaccharides), sugars (including oligosaccharides and polysaccharides)], and non-polymeric substances (e.g., low molecular weight compounds). Numerous substances have been reported that can be used as affinity moieties that have affinity for the constant region of the heavy chain of antibodies, including peptides, nucleic acids, sugars, and small molecule compounds (e.g., published publications such as International Publication Nos. 2007 / 004748, 2008 / 054030, 2013 / 027796, 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 090979, etc.; Nomura Y et al., Nucleic Acids Res., 2010 Nov; 38(21):7822-9; Miyakawa S et al., RNA., 2008 See Jun;14(6):1154–63 and the scientific papers cited below.

[0092] Furthermore, substances that can be used as affinity moieties having affinity for the CH2-CH3 intercellular region of the antibody heavy chain can be obtained by any method. For example, such substances can be obtained by screening for substances having affinity for the constant region of the antibody heavy chain from a library of any substance (e.g., small molecule compound library, peptide library, aptamer library, sugar library, phage library, mRNA library, cDNA library) (e.g., high-throughput screening method, phage display method, SELEX method, mRNA display method, ribosome display method, cDNA display method, yeast display method). In addition, when screening for substances having affinity for a specific region in the constant region of an antibody (e.g., the region between the CH2 and CH3 regions), substances that can selectively bind to such regions can be efficiently obtained by using partial peptides present in specific regions (e.g., CH1) of various antibodies (e.g., IgG, IgA, IgM, IgD, IgE).

[0093] The affinity portion may have affinity for the CH2-CH3 intercellular region in the antibody heavy chain. Here, "having affinity for the CH2-CH3 intercellular region" is not particularly limited as long as it has affinity for at least a portion of the CH2-CH3 intercellular region, and may have affinity for a partial region of the CH2-CH3 intercellular region.

[0094] The CH2-CH3 intercellular region of the heavy chain of the antibody to which the affinity portion has affinity may be derived from the animals described above (e.g., mammals, birds). The CH2-CH3 intercellular region in the heavy chain of the antibody may preferably be a mammalian CH2-CH3 intercellular region, more preferably a primate CH2-CH3 intercellular region or a rodent CH2-CH3 intercellular region, and even more preferably a human CH2-CH3 intercellular region.

[0095] The CH2-CH3 intercellular region in the heavy chain of an antibody to which the affinity portion has affinity may be the CH2-CH3 intercellular region of a bivalent antibody (e.g., IgG, IgD, IgE) or a quadrivalent or higher antibody (e.g., IgA antibody, IgM antibody). Such a CH2-CH3 intercellular region is preferably the CH2-CH3 intercellular region of a bivalent antibody (e.g., IgG, IgD, IgE), and more preferably the CH2-CH3 intercellular region of IgG. The human CH2-CH3 intercellular region may be, for example, the region from positions 230 to 439 of the human IgG heavy chain, or the region from positions 248 to 288 of the human IgG heavy chain.

[0096] Preferably, the affinity substance may be an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain. Affinity polypeptides can be obtained by the screening methods described above (e.g., the method using the library described above, or the display method described above).

[0097] For affinity polypeptides, amino acid residues from the 20 common natural amino acids that make up proteins, or from non-natural amino acids, can be used. Examples of the 20 common natural amino acids that make up proteins include, for example, L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (the notation L is omitted below).

[0098] The affinity polypeptide or a salt thereof of the present invention can be used, for example, as a synthetic intermediate for the compound or a salt thereof of the present invention, which comprises the affinity polypeptide and the first and second antibody-reactive groups. Therefore, the affinity polypeptide or a salt thereof of the present invention preferably contains two specific reactive groups that enable specific reactions with the partial compound containing the antibody-reactive groups, in order to easily realize the uniform synthesis of the compound or a salt thereof of the present invention, which comprises the first and second antibody-reactive groups. As a result, the affinity polypeptide and the first and second antibody-reactive groups can be specifically reacted via specific reactive groups in the affinity polypeptide, so that the compound or a salt thereof of the present invention, which comprises the affinity polypeptide and the first and second antibody-reactive groups, can be easily produced as a uniform compound.

[0099] Examples of specific reactive groups for introducing the first and second antibody-reactive groups include the following: (1) Amino group (NH ２ NHR ３ , NR ３ R ４ . R ３ and R ４(1) a monovalent group as described above, preferably a monovalent hydrocarbon group, more preferably an alkyl group, and even more preferably an alkyl group having 1 to 6 carbon atoms; (2) a residue that can react with an amino group, for example, activated ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethyl residues, diazonium terephthalic acid residues; (3) a carboxyl group (COOH); (4) a residue that can react with a carboxyl group, for example, an amino group as described above; (5) a hydroxyl group (OH) (including alcoholic and phenolic hydroxyl groups); and (6) a residue that can react with a hydroxyl group, for example, a diazonium residue, a diazodicalboxate residue, or a 2,3-dihydro-1H-pyrazine-6-one residue. 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 is even more preferably (1), and particularly preferably an amino group (NH ２ ) is also acceptable.

[0100] Therefore, affinity polypeptides that have affinity for the CH2-CH3 intercellular region of the antibody's heavy chain and contain two of the specific reactive groups described above can be used.

[0101] Preferably, affinity polypeptides containing two specific reactive groups may be polypeptides containing two amino acid residues having side-chain amino groups. Examples of amino acid residues having side-chain amino groups include lysine, ornithine (Orn), diaminobutanoic acid (Dab), and diaminopimelic acid (Dap). That is, when affinity polypeptides are produced by organic synthesis systems (e.g., solid-phase synthesis), not only lysine residues, which are natural amino acids that make up proteins, but also other amino acid residues having side-chain amino groups (e.g., ornithine, diaminobutanoic acid, diaminopimelic acid, etc.) can be used. More preferably, affinity polypeptides containing two specific reactive groups can be easily produced not only by organic synthesis systems but also by polypeptide expression systems using host cells and cell-free synthesis systems, by including amino groups (NH ２ It may also be a polypeptide containing two lysine residues having ) in its side chain.

[0102] Furthermore, if the affinity polypeptide contains two amino acid residues having side-chain amino groups, it is preferable that the N-terminus of the affinity polypeptide is protected. Alternatively, the affinity polypeptide may contain one amino acid residue having a side-chain amino group and have an amino group at its N-terminus, thereby containing two amino groups as specific reactive groups.

[0103] Furthermore, affinity polypeptides or salts thereof containing two amino groups as specific reactive groups may also be affinity polypeptides or salts thereof that do not contain groups that can react with amino groups (e.g., carboxyl groups) in order to suppress undesirable reactions (e.g., intramolecular or intermolecular reactions). Therefore, affinity polypeptides may not contain amino acid residues having carboxyl groups in their side chains. The carboxyl terminus may also be protected.

[0104] Affinity polypeptides containing two specific reactive groups can be, for example, those having an amino acid sequence that has affinity for the CH2-CH3 intercellular region of the antibody's heavy chain, and that contains two amino acid residues with side-chain amino groups. However, if the amino acid sequence having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain does not contain amino acid residues with side-chain amino groups, the two amino acid residues in the same amino acid sequence can be substituted with amino acid residues that each have side-chain amino groups.

[0105] For example, affinity polypeptides that do not contain amino acid residues having side-chain amino groups and have affinity for the CH2-CH3 intercellular region of the antibody heavy chain include: (i) affinity polypeptides having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13; (ii) affinity polypeptides that contain an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more identity with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 (however, amino acid residues having side-chain amino groups are not included), and that have affinity for the CH2-CH3 intercellular region of the antibody heavy chain; or (iii) affinity polypeptides that contain an amino acid sequence in which one or several (e.g., 1 to 2) amino acid residues in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 are substituted with other amino acid residues other than amino acid residues having side-chain amino groups, and that have affinity for the CH2-CH3 intercellular region of the antibody heavy chain. Sequence ID 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC Sequence ID 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC

[0106] Furthermore, amino acid residue substitutions may be conservative substitutions. The term "conservative substitution" refers to the substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in this field. For example, such families include amino acids with basic side chains (e.g., arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Amino acids with uncharged polar side chains and amino acids with nonpolar side chains are sometimes collectively referred to as neutral amino acids. Preferably, the conservative amino acid substitutions may be substitutions between aspartic acid and glutamic acid, between arginine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine, between leucine, isoleucine and alanine, and between glycine and alanine.

[0107] Affinity polypeptides or salts thereof may contain the aforementioned natural or non-natural amino acid residues as constituent amino acid residues. If the affinity polypeptide contains only the aforementioned natural amino acid residues, it can be produced, for example, by a polypeptide expression system using host cells, a cell-free synthesis system, or an organic synthesis system (e.g., solid-phase synthesis). If the affinity polypeptide contains non-natural amino acid residues, it can be produced, for example, by an organic synthesis system (e.g., solid-phase synthesis). Preferably, the affinity polypeptide may contain only natural amino acid residues to enable the production of large quantities of affinity polypeptide by a polypeptide expression system using host cells or a cell-free synthesis system.

[0108] In the amino acid sequence of an affinity polypeptide, the position of the amino acid residue having two side-chain amino groups is not particularly limited, but it is preferable that the amino acid residue is located near the target lysine present in the CH2-CH3 intercellular region of the antibody's heavy chain in the stereochemistry of the affinity polypeptide when it is coexisting with the antibody. Examples of target lysine include the lysine at position 248 and the lysine at position 288. Nearby means, for example, within 30 Å.

[0109] In the amino acid sequence of SEQ ID NO: 5, the two specific amino acid residues that are substituted for an amino acid residue having a side-chain amino group may be, for example, the first being an amino acid located near lysine at position 248, specifically selected from position 32E, 31A, or 30I in SEQ ID NO: 5, and the second being an amino acid located near lysine at position 288, selected from position 4Q, 3A, or 2N in SEQ ID NO: 5. In the amino acid sequence of SEQ ID NO: 13, the two specific amino acid residues that are substituted for an amino acid residue having a side-chain amino group may be, for example, the first being an amino acid located near lysine at position 248, specifically selected from position 32D, 31A, or 30I in SEQ ID NO: 13, and the second being an amino acid located near lysine at position 288, selected from position 4Q, 3A, or 2N in SEQ ID NO: 5. If the affinity polypeptide is an amino acid sequence that has 60% or more identity with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, or an amino acid sequence that includes one to several amino acid substitutions, deletions, additions, or insertions in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13, then the specific amino acid residues are, for example, the first one which corresponds to the 32nd E (32nd D in SEQ ID NO: 13), the 31st A, or the 30th I in SEQ ID NO: 5, and the second one which corresponds to the 4th Q, the 3rd A, or the 2nd N in SEQ ID NO: 5. Here, the corresponding positions can be identified by amino acid alignment. Note that the nth in SEQ ID NO: 5 refers to the number of the amino acid residue counted from the N-terminus.

[0110] More preferably, when the target lysine is the lysine at position 248, the specific amino acid residue substituted for the amino acid residue having a side chain amino group is preferably the 31st A of SEQ ID NO: 5 or SEQ ID NO: 13, and when the target lysine is the lysine at position 288, the specific amino acid residue substituted for the amino acid residue having a side chain amino group is preferably the 3rd A of SEQ ID NO: 5 or SEQ ID NO: 13.

[0111] In other words, in a preferred embodiment, the affinity polypeptide may have one of the following amino acid sequences: Sequence ID No. 6: FNXQCQRRFYEALHDPNLNEEQRNARIRSIXEEC where X is an amino acid residue having a side-chain amino group, for example, lysine, ornithine, diaminobutanoic acid, or diaminopimelic acid. Sequence ID No. 14: FNXQCQRRFYEALHDPNLNEEQRNARIRSIXDDC where X is an amino acid residue having a side-chain amino group, for example, lysine, ornithine, diaminobutanoic acid, or diaminopimelic acid.

[0112] The amino and carboxyl groups at the ends of affinity polypeptides can be protected as appropriate. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., acetyl groups, propoxy groups, tert-butoxycarbonyl groups, etc.), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl group), aryloxycarbonyl groups, and arylalkyl (aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl group). Preferably, the N-terminal amino group may be alkylated, formylated, or acetylated. Examples of protecting groups for the C-terminal carboxyl group include groups capable of forming esters or amides. Examples of groups capable of forming esters or amides include alkyloxy groups (e.g., methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (e.g., phenyloxy, naphthyloxy), aralkyloxy groups (e.g., benzyloxy), and amino groups.

[0113] Furthermore, if the N-terminal amino acid of an affinity polypeptide is glutamic acid (E) or glutamine (Q), the N-terminus can be protected using their side chains. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid can have a pyroglutamic acid cyclic structure. Also, when the N-terminal amino acid is glutamine, the protected N-terminal glutamine can have a pyroglutamic acid cyclic structure. ２The amide group present in its side chain reacts with the pyroglutamyl group (pyroglutamylation) to form a pyroglutamic acid-type cyclic structure. Therefore, the N-terminal amino acid may preferably be glutamic acid or glutamine.

[0114] The affinity polypeptide may further contain a tripeptide consisting of Gln-Glu-Thr (QET) at its N-terminus. In this case, a polypeptide expression system using host cells enables large-scale and simple secretion production of the affinity polypeptide, along with protection of the N-terminal amino group by pyroglutamylation of Q (see International Publication Nos. 2013 / 062029 and 2020 / 090979). In this case, a signal peptide such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 7) can be added to the N-terminus of QET (see International Publication Nos. 2013 / 062029 and 2020 / 090979).

[0115] 2-2. Related Inventions of Affinity Polypeptides or Salts The affinity polypeptides of the present invention can be prepared by chemical synthesis, or, if they consist only of natural amino acids, they can be prepared using host cells containing an expression unit comprising a polynucleotide encoding the affinity polypeptide and a promoter operably linked thereto, or using cell-free systems, etc. The present invention also provides such polynucleotides and host cells, as well as expression vectors that can be used to prepare host cells.

[0116] The polynucleotide of the present invention is a polynucleotide that encodes the affinity polypeptide of the present invention. The polynucleotide of the present invention may be DNA or RNA, but DNA is preferred.

[0117] The host cells of the present invention can be prepared, for example, by a method using an expression vector containing the polynucleotide of the present invention (e.g., competent cell method, electroporation method), or by genome modification technology. If the expression vector is an integrative vector that undergoes homologous recombination with the host cell's genomic DNA, the expression units can be incorporated into the host cell's genomic DNA by transformation. On the other hand, if the expression vector is a non-integrative vector that does not undergo homologous recombination with the host cell's genomic DNA, the expression units are not incorporated into the host cell's genomic DNA by transformation and can exist independently of the genomic DNA within the host cell in the state of the expression vector. Alternatively, genome editing technology (e.g., CRISPR / Cas system, Translation Activator-Like Effector Nucleases (TALEN)) makes it possible to incorporate expression units into the host cell's genomic DNA and to modify expression units inherently present in the host cell.

[0118] The present invention also provides an expression vector comprising the polynucleotide of the present invention and a promoter operably linked thereto. The expression vector of the present invention may further include elements such as a terminator that functions in a host cell, a ribosome binding site, and a drug resistance gene. Examples of drug resistance genes include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0119] The expression vector may also further include a region that enables homologous recombination with the host cell's genome for homologous recombination with the host cell's genomic DNA. For example, the expression vector may be designed so that the expression units contained therein are located between a pair of homologous regions (e.g., homology arms, or loxP, or FRT, homologous to a specific sequence in the host cell's genome). The host cell genomic region to which the expression units should be introduced (the target of the homologous region) is not particularly limited, but may be a locus of a gene that is highly expressed in the host cell.

[0120] The expression vector may be a plasmid, viral vector, phage, or artificial chromosome. The expression vector may also be an integrative or non-integrative vector. An integrative vector may be a type in which the entire vector is integrated into the host cell's genome. Alternatively, an integrative vector may be a type in which only a portion of it (e.g., an expression unit) is integrated into the host cell's genome. The expression vector may further be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and their derivatives.

[0121] Host cells for expressing the affinity polypeptide of the present invention include, for example, various prokaryotic cells including Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (e.g., Corynebacterium glutamicum), and Bacillus bacteria (e.g., Bacillus subtilis), as well as Saccharomyces bacteria (e.g., Saccharomyces cerevisiae), Pichia bacteria (e.g., Pichia stipitis), and Aspergillus bacteria (e.g., Aspergillus oryzae). Various eukaryotic cells, including *Euphyllia 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. A strain lacking a specific gene may also be used as a host. Examples of host cells include host cells that possess an expression vector in the cytoplasm, and host cells into which the target gene has been introduced into the genome.

[0122] When the affinity polypeptide of the present invention contains a tripeptide consisting of Gln-Glu-Thr (QET) at its N-terminus, it is preferable to use an affinity polypeptide that can be prepared by a polypeptide secretion production method using Corynebacterium as a host (International Publication No. 2013 / 062029). This method is suitable for preparing affinity polypeptides because it can add the three N-terminal residues Gln-Glu-Thr (QET) of the mature Csp protein to the N-terminus of the target polypeptide, and moreover, it can easily and in large quantities prepare polypeptides containing a glutamine residue (Q) at the N-terminus. In this case, various signal peptides such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 7) can be added to the N-terminal side of QET (see Examples, International Publication No. 2013 / 062029, and International Publication No. 2020 / 090979). Examples of coryneform bacteria that can be used in this method include bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum, Corynebacterium statyonis) and bacteria of the genus Brevibacterium.

[0123] The host cells of the present invention can be cultured in a culture medium having, for example, the composition described below, using a predetermined culture apparatus (e.g., test tubes, flasks, jar fermenters). Culture conditions can be set as appropriate. Specifically, the culture temperature may be 10°C to 37°C, the pH may be 6.5 to 7.5, and the culture time may be 1 h to 100 h. Culture may also be performed while controlling the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture medium may be used as a control indicator. The aeration and stirring conditions can be controlled so that the relative dissolved oxygen concentration (DO value), assuming an atmospheric oxygen concentration of 21%, does not fall below, for example, 1 to 10%, preferably 3 to 8%. Furthermore, the culture may be performed as batch culture or fed-batch culture. In the case of fed-batch culture, the culture can be continued by continuously or discontinuously adding a solution that serves as a sugar source or a solution containing phosphate to the culture medium.

[0124] As promoters for expressing the polynucleotides of the present invention, promoters commonly used for heterologous protein production in E. coli can be used. Examples of strong promoters include PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and T5 promoter, with PhoA, PhoC, and lac being preferred. In addition, as vectors, for example, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and their derivatives may be used.

[0125] Furthermore, a terminator, which is a transcription termination sequence, 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.

[0126] As the culture medium, you may use a medium commonly used for culturing E. coli, such as M9-casamino acid medium or LB medium. The medium may contain a specified carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, peptone, yeast extract, NaCl, glucose, MgSO4. ４ Ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc., may also be used. Furthermore, the culture conditions and production induction conditions are appropriately selected according to the type of marker, promoter, and host bacteria of the vector used.

[0127] The affinity polypeptide of the present invention can be recovered by the following methods. The affinity polypeptide of the present invention can be obtained as a lysate and lysate by recovering the transformed cells of the present invention and then disrupting (e.g., sonication, homogenization) or lysating (e.g., lysozyme treatment) the bacterial cells. If the affinity polypeptide is secreted or leaked outside the cell, a disinfectant solution containing the affinity polypeptide can be obtained from the culture medium by centrifugation or membrane filtration. The affinity polypeptide of the present invention can be obtained by subjecting such lysates, lysates and disinfectant solutions to extraction, precipitation, filtration, column chromatography, or other methods.

[0128] 3. Compounds or Salts thereof The compounds or salts thereof of the present invention comprise (A) an affinity polypeptide having an affinity moiety having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody, and (B) first and second antibody-reactive groups (antibody-reactive groups). The definition, examples, and preferred examples of affinity polypeptides are as described above.

[0129] As an antibody-reactive group, a reactive group can be used that reacts to amino acid residues with readily reactive side chains among the amino acid residues that make up the antibody (polypeptide). Of the 20 natural amino acids that make up proteins as described above, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, which have hydrocarbon side chains, are inactive to normal reactions. Therefore, the antibody-reactive group can be a group that can react to the side chain of any of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine.

[0130] The antibody-reactive group is more preferably a reactive group specific to the side chain of one of the amino acids lysine, tyrosine, tryptophan, or cysteine, and even more preferably a reactive group specific to the side chain of one of the amino acids lysine, tyrosine, or tryptophan, and particularly preferably a reactive group specific to the side chain of lysine or tyrosine, and especially to the amino group of the side chain of lysine. For details of such reactive groups, see, for example, International Publication Nos. 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979.

[0131] Groups that can specifically react with the amino group in the side chain of a lysine residue include, for example, activated ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethyl residues, diazonium terephthalic acid residues, α-halogen-substituted acetamides, α-halogen-substituted methyl ketones, and thioester groups. These reactive groups are specific to the side chain of a lysine residue, and the amino group (NH) present in the side chain of the lysine residue reacts with the amino group (NH) present in the side chain of the lysine residue. ２ Reactions between ) can produce, for example, amide residues, urea residues, pyridine residues, carbamate residues, and sulfonamide residues as linking portions.

[0132] In certain embodiments, the compound or salt of the present invention is given by the following formula (I): R １ -L １ -A-L ２ -R ２ (I) [wherein, R １ and R ２ These each indicate a reactive group for antibodies, L １ This indicates the first linker, L ２ represents the second linker, and A represents an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody heavy chain. It may also be represented as ].１ and R ２ The definitions, examples, and preferred examples of the reactive group for antibodies indicated by A, and the affinity polypeptide indicated by A, are as described above.

[0133] L １ The first linker shown by, and L ２ The second linker indicated by may be the same or different divalent group. The divalent group may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above.

[0134] In certain embodiments, the compound or salt of the present invention may further contain a cleavage moiety between the affinity polypeptide and the antibody-reactive group. In this case, the compound or salt represented by formula (I) may contain a linker containing the cleavage moiety.

[0135] The cleavable portion is a site that can be cleaved by a specific treatment under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). Therefore, the cleavable portion can be said to be a site (bonds other than amide bonds) that can be cleaved by a specific cleavage treatment under mild conditions. Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli such as light, or (c) incubation when using a cleavable linker containing a self-degradable cleavable portion. Such cleavable linkers and their cleavage conditions are common technical knowledge in the field (e.g., G. Lerice, L. Chisholm, A. Wagner, Bioorganic & Medical 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); DeSimonone, J.M., Journal of American Chemical Society). Society. 132, 17928 (2010); Thompson, D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004). The reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are described below.Examples of cleavable residues include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues (e.g., tert-butyloxycarbamate residues), silane residues, hydrazone-containing residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoamidate residues, aconityl residues, trityl residues, azo residues, vicinaldiol residues, selenium residues, aromatic ring-containing residues with electron-withdrawing groups, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0136] The aromatic ring group having an electron-withdrawing group is preferably an aromatic ring group selected from the group consisting of aryl, aralkyl, aromatic heterocyclic, and alkyl having an aromatic heterocyclic group, with aralkyl and alkyl having an aromatic heterocyclic group being more preferred. The electron-withdrawing group is preferably bonded to the 2-position of the ring. Even more preferably, the aromatic ring-containing residue having an electron-withdrawing group is, for example, an aralkyl (e.g., benzyl) having an electron-withdrawing group at the 2-position. Examples of electron-withdrawing groups include halogen atoms, alkyl groups substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl, and keto groups (e.g., acyl).

[0137] The definitions, examples, and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, and aralkyl, which are found as prefixes, suffixes, and other terms related to the names of residues as cleavable parts, are the same as those described above.

[0138] Examples of ester residues include ordinary ester residues composed of carbon atoms and oxygen atoms [e.g., alkyl esters (e.g., tert-butyloxycarbonyl and other tertiary alkyloxycarbonyls), aryl esters (e.g., phenacyl esters, 2-(diphenylphosphin)benzoate), glycosyl ester residues, orthoester residues], ester residues containing sulfur and oxygen atoms (e.g., thioester residues such as α-thiophenyl ester residues and alkylthioester residues), ester residues containing phosphorus and oxygen atoms (e.g., phosphodiester residues, phosphotriester residues), and activated ester residues (e.g., N-hydroxysuccinimide residues).

[0139] Examples of sulfone-containing residues include sulfone residues and quinolinylbenzenesulfonate residues.

[0140] The silane residue is preferably a silane residue having a group selected from the group consisting of alkyl, aryl, aralkyl, and alkoxy. Examples of such silane residues include dialkyldialkoxysilane residues (e.g., dimethyldialkoxysilane, diethyldialkoxysilane) or diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane).

[0141] Alkoxyalkyl (i.e., alkyloxyalkyl) residues are groups formed by combining alkyloxy and alkyl as described above, and include, but are not limited to, methoxymethyl, ethoxymethyl, methoxyethyl, and ethoxyethyl residues.

[0142] Unsaturated bond-containing chain residues are residues that contain an unsaturated bond portion consisting only of carbon atoms [e.g., vinyl (ethenyl), the smallest unit with a carbon-carbon double bond, or acetylenyl (ethynyl), the smallest unit with a carbon-carbon triple bond], or residues that contain an unsaturated bond portion consisting of a carbon atom and a heteroatom (e.g., nitrogen atom, sulfur atom, oxygen atom) [e.g., aldehyde, cyano]. Examples of unsaturated bond-containing chain residues include vinyl ether residues, cyanoethyl residues, ethylene residues, and malondialdehyde residues.

[0143] Examples of acidic substances (also called electrophiles) include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acidic substances such as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3-morpholinopropanesulfonic acid, sodium dihydrogen phosphate, citric acid, dodecyl sulfate, N-dodecanoylsarcosinate, and trifluoroacetic acid. Examples of sites that can be cleaved by acidic substances include alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, β-thiopropionate residues, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, and 2-(diphenylphosphinone)benzoate residues.

[0144] Examples of basic substances (also called nucleophiles) include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, and ammonium acetate, and organic basic substances such as hydroxylamine, triethylamine, and N,N'-diisopropylamine. Examples of sites that can be cleaved by basic substances include silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyl disuccinate residues, α-thiophenyl ester residues, unsaturated vinyl sulfide residues, malondialdehyde residues, acylhydrazone residues, and alkylthioester residues.

[0145] Examples of reducing agents include cysteine, dithiothreitol, reduced glutathione, and β-mercaptoethanol. Examples of sites that can be cleaved by reducing agents include disulfide residues, alkoxyalkyl residues, and azo residues.

[0146] Examples of oxidizing agents include sodium periodate and oxidized glutathione. Examples of sites that can be cleaved by oxidizing agents include vicinaldiol residues and selenium residues.

[0147] Examples of enzymes include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloproteinase, lipase, endoglycosidase, and PN gauze F. Examples of sites that can be cleaved by these enzymes include ester residues, phosphodiester residues, and glycosyl residues.

[0148] Examples of sites that can be cleaved by light include 2-nitrobenzyl residues, phenacyl ester residues, 8-quinolinebenzenesulfonate residues, coumarin residues, phosphotriester residues, bisarylhydrazone residues, and bimandithiopropionic acid residues.

[0149] Examples of self-degrading cleavable moieties include activated ester residues (e.g., N-hydroxysuccinimide residues).

[0150] If the compound or salt of the present invention contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthothic functional group on the reactive group side upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including ordinary ester residues and other ester residues such as thioester residues), acetal residues (including ordinary ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinaldiol residues.

[0151] If the compound or salt of the present invention contains a cleavable moiety that can generate a bioorthothic functional group on the reactive group side upon cleavage, then the formula is as follows: (Ia): R １ -L3-CLE(B) １ -L4-A-L5-CLE(B) ２ -L6-R ２ (Ia) [wherein, R １ and R ２ These each indicate a reactive group for antibodies, L ３ This indicates the third linker, L ４ This indicates the fourth linker, L ５ This indicates the fifth linker, L ６ This indicates the sixth linker, CLE(B)１ and CLE(B) ２ Each of the following represents a cleavable portion capable of generating a bioorthogonal functional group on the reactive group side by cleavage, and A represents an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain. It may also be represented as ]. １ and R ２ The definitions, examples, and preferred examples of the reactive group for the antibody indicated by A, and the affinity polypeptide indicated by A are as described above. CLE(B) １ and CLE(B) ２ This represents a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side through cleavage, and specific examples of this are as described above.

[0152] L ３ The third linker, L, is shown by ４ The fourth linker, L, is shown by ５ The fifth linker indicated by, and L ６ The sixth linker indicated by may be the same or different divalent group. The divalent group may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above.

[0153] In certain embodiments, the total number of atoms constituting the main chain in the third and fourth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7. The total number of atoms constituting the main chain in the fifth and sixth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0154] The number of atoms constituting the main chain in the third and fourth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6. The number of atoms constituting the main chain in the fifth and sixth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0155] The main chains in the third and fourth linkers (and similarly in the fifth and sixth linkers) consist of a chain-like structure, a cyclic structure, or a combination thereof. If the main chain is a chain-like structure that does not include a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain-like structure. On the other hand, if the main chain has a structure that includes a cyclic structure, the number of atoms in the main chain can be determined by counting a predetermined number of atoms that constitute the cyclic structure as the number of atoms in the main chain. Specifically, the number of atoms in the main chain in a cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (see, for example, the bolded paths in (a) to (d) below). If the main chain has a structure that includes a combination of chain-like and cyclic structures, the number of atoms in the main chain can be determined by adding the number of atoms in the chain-like structure that does not include a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure. The method for counting the number of atoms in the main chain is the same for the other linkers. • represents a bonding bond. In case (a), the shortest path is the bolded path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 2. In case (b), the shortest path is the bolded path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 3. In case (c), both paths are the shortest paths (equidistance), so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4. In case (d), the path at the condensation site is the shortest path, so the number of atoms in the divalent cyclic structure that can be counted as the number of atoms in the main chain is 4.

[0156] In certain embodiments, the compound or salt of the present invention is given by the following formula (Ia-1): [In the formula, X １ and X ２ These each represent a leaving group, W １ , W ２ , W ３ , W ４ , W ５ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L ８ This indicates the eighth linker, L ９ This indicates the 9th linker, L １０ represents the 10th linker, S represents a sulfur atom, and A represents an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain. The compound represented by ] or a salt thereof may also be used. The definition, examples, and preferred examples of the affinity polypeptide represented by A are as described above.

[0157] X １ and X ２ The leaving groups shown by are, respectively, X １ and X ２ Adjacent to C=W １ Or C = W ６ This is a group that can be eliminated by a reaction between the carbon atom and the amino group. Those skilled in the art can appropriately design such a leaving group. Examples of such leaving groups include: (a) R Ａ -S (here, R Ａ(b) R Ａ -O (Here, R Ａ (c)R Ａ - (R Ｂ -) N (Here, R Ａ and R Ｂ (d) A halogen atom.

[0158] Preferably, X １ and X ２ The leaving group represented by may be one of the following: (a) R Ａ -S (here, R Ａ (b) R Ａ -O (Here, R Ａ (c)R Ａ - (R Ｂ -) N (Here, R Ａ and R Ｂ Each of these independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom.

[0159] Comfortably, X １ and X ２ The leaving group represented by may be one of the following: (a) R Ａ -S (here, R Ａ (b) R Ａ-O (Here, R Ａ (wherein ∫ represents a hydrogen atom, a optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom.)

[0160] More more, X １ and X ２ The leaving group represented by may be one of the following: (a) R Ａ -S (here, R Ａ (wherein is a hydrogen atom, a optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S is a sulfur atom.)

[0161] Particularly preferred, X １ and X ２ The leaving group shown by may be the following: (a')R Ａ -S (here, R Ａ (wherein is a monovalent aromatic hydrocarbon group which may have substituents (e.g., phenyl), and S is a sulfur atom.)

[0162] W １ , W ２ , W ３ , W ４ , W ５ and W ６ Each independently represents either an oxygen atom or a sulfur atom. Preferably, W １ , W ２ , W ３ , W ４ , W ５ and W ６ This could be an oxygen atom.

[0163] L ７ The seventh linker, L, is shown as shown. ８ The eighth linker, L, is shown by ９ The ninth linker indicated by, and L １０ The tenth linker indicated by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above.

[0164] In certain embodiments, the total number of atoms constituting the main chain in the seventh and eighth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7. The total number of atoms constituting the main chain in the ninth and tenth linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0165] The number of atoms constituting the main chain in the seventh and eighth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6. The number of atoms constituting the main chain in the ninth and tenth linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0166] If the compound or salt of the present invention contains a cleavable moiety, it may further contain a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety. The bioorthogonal functional group is as described above. Preferably, the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with substituents as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, and a halocarbonyl residue.

[0167] An example of a compound of formula (Ia-1) is shown below.

[0168] If the compound or salt of the present invention further contains a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety, then the formula (Ib) is as follows: [In the formula, R １ and R ２ These each indicate a reactive group for antibodies, L １１ This indicates the 11th linker, L １２ This indicates the 12th linker, L １３ This indicates the 13th linker, L １４ This indicates the 14th linker, B １ and B ２ These each indicate a group containing a bioorthogonal functional group, CLE １ and CLE ２ Each of the following represents a cleavage portion, and A represents an affinity polypeptide that has affinity for the CH2-CH3 intercellular region of the antibody's heavy chain. It may also be represented as ]. １ and R ２ The definitions, examples, and preferred examples of the reactive group for the antibody indicated by A, and the affinity polypeptide indicated by A are as described above. CLE １ and CLE ２ The term indicates a cleavage portion, whose definition, examples, and preferred examples are as described above.

[0169] L １１ The 11th linker, L １２ The 12th linker, L １３ The 13th linker, indicated by L １４ The 14th linker indicated by may be the same or different divalent group. The divalent group may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above.

[0170] In certain embodiments, the total number of atoms constituting the main chain in the 11th and 12th linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7. The total number of atoms constituting the main chain in the 13th and 14th linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0171] The number of atoms constituting the main chain in the 11th and 12th linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6. The number of atoms constituting the main chain in the 13th and 14th linkers may be 1 to 9, respectively. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0172] B １ and B ２ The group containing the bioorthogonal functional group indicated by may be a group consisting of a bioorthogonal functional group, or a group containing a bioorthogonal functional group and other parts. Other parts include, for example, the linkage between the bioorthogonal functional group and the linker. The linkage is, for example, a divalent group. The divalent group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the bioorthogonal functional group, the divalent group, and the substituent when the divalent group is substituted are as described above.

[0173] In certain embodiments, the compound or salt of the present invention is given by the following formula (Ib-1): [In the formula, X ３ and X ４ These each represent a leaving group, W ７ , W ８ , W ９ , W １０ , W １１ and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １６ This indicates the 16th linker, L １７ This indicates the 17th linker, L １８ This indicates the 18th linker, B ３ and B ４ Each of these indicates a group containing a bioorthogonal functional group, V １ and V ２ A represents an oxygen atom or a sulfur atom, and A represents an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain. The compound may be represented by [ ] or a salt thereof. X ３ and X ４ The leaving group indicated by is X １ and X ２ Examples of leaving groups similar to those shown by are B. ３ and B ４ The group containing the bioorthogonal functional group indicated by A may be a group consisting of a bioorthogonal functional group, or a group containing a bioorthogonal functional group and other parts. Other parts include, for example, the linking part between the bioorthogonal functional group and the linker. The linking part is, for example, a divalent group. The divalent group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the bioorthogonal functional group, the divalent group, and the substituent when the divalent group is substituted are as described above. The definitions, examples, and preferred examples of the affinity polypeptide indicated by A are as described above.

[0174] W ７ , W ８ , W ９ , W １０ , W １１ and W １２Each independently represents either an oxygen atom or a sulfur atom. Preferably, W ７ , W ８ , W ９ , W １０ , W １１ and W １２ This could be an oxygen atom.

[0175] L １５ The 15th linker, L １６ The 16th linker, L １７ The 17th linker, indicated by L １８ The 18th linker, indicated by , may be the same or different divalent group. The divalent group may be substituted or unsubstituted. Examples of divalent groups include those listed above. Examples of substituents when the divalent group is substituted include those listed above.

[0176] In certain embodiments, the total number of atoms constituting the main chain in the 15th and 16th linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7. The total number of atoms constituting the main chain in the 17th and 18th linkers may be 2 to 10. Such a total number of atoms may be 3 or more, or 4 or more. Such a total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, such a total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0177] The number of atoms constituting the main chain in the 15th linker and the 16th linker may be 1 to 9, each. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6. The number of atoms constituting the main chain in the 17th linker and the 18th linker may be 1 to 9, each. Such a number of atoms may be 2 or more, or 3 or more. Such a number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such a number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0178] V １ and V ２ Each independently represents either an oxygen atom or a sulfur atom. Preferably, V １ and V ２ It may also be a sulfur atom.

[0179] An example of a compound of formula (Ib-1) is shown below.

[0180] The compounds of the present invention or their salts can readily modify two specific amino acid residues in the heavy chain CH2-CH3 intercellular region of an antibody component. The compounds of the present invention or their salts can also provide a regioselectively modified antibody while readily modifying two specific amino acid residues in the heavy chain CH2-CH3 intercellular region of an antibody component.

[0181] The above-described series of compounds or salts thereof can be produced by reacting the affinity polypeptide of the present invention (a polypeptide having affinity for the heavy chain CH2-CH3 intercellular region and comprising first and second amino acid residues having side-chain amino groups) with a partial compound containing a reactive group for antibodies. For example, such a reaction can be carried out using a suitable organic solvent system (e.g., CH3). ２ Cl ２The reaction can be carried out at a suitable temperature (e.g., about -10 to 30°C) in an organic solvent containing alkyl halides (e.g., methyl halide) and amines such as triethylamine. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0182] The formation of the above-mentioned series of compounds or their salts can be confirmed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific molecular weights of the raw materials and products. Such compounds or their salts can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0183] 4. Affinity Polypeptide-Modified Antibodies or Salts thereof The present invention provides affinity polypeptide-modified antibodies or salts thereof, wherein an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody's heavy chain is contained within the CH2-CH3 intercellular region of the antibody's heavy chain. Definitions, examples, and preferred examples of affinity polypeptides, antibodies, and their constituent elements are as described above.

[0184] Preferably, the affinity polypeptide-modified antibody or a salt thereof comprises (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) an affinity polypeptide, and (c) the affinity polypeptide may be introduced via two amino acid residues in the CH2-CH3 intercellular region of the heavy chain in the immunoglobulin unit. Definitions, examples, and preferred examples of the antibody, immunoglobulin unit, affinity polypeptide, and their constituent elements (e.g., constant region) are as described above.

[0185] Affinity polypeptide-modified antibodies or salts thereof may contain affinity polypeptides through modification of functional groups in the side chains of two amino acid residues selected from asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are present in the CH2-CH3 intercellular region of the heavy chain. That is, since affinity polypeptides have two antibody-reactive groups, they can chemically bind to antibodies through the reaction of these two antibody-reactive groups with the functional groups in the side chains of the two amino acid residues of the antibody. Affinity polypeptide-modified antibodies or salts thereof may contain affinity polypeptides, preferably through modification of functional groups in the side chains of two amino acids selected from lysine, tyrosine, tryptophan, or cysteine ​​present in the CH2-CH3 interchain region of the heavy chain; more preferably through modification of functional groups in the side chains of two amino acids selected from lysine, tyrosine, or tryptophan; even more preferably through modification of functional groups in the side chains of two amino acids selected from lysine or tyrosine; and particularly preferably through modification of amino groups in the side chains of two lysine residues. The positions of these amino acid residues in the CH2-CH3 interchain region are as described above. The modification sites of antibodies or salts thereof by affinity polypeptides can be confirmed by peptide mapping. The modifications may be regioselective, as described above. Therefore, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described later, the immunoglobulin unit may regioselectively have the corresponding modified unit via the functional groups in the side chains of the two amino acid residues.

[0186] Preferably, affinity polypeptide-modified antibodies may include the affinity polypeptide chemically bound via modification of amino groups in the side chains of two lysine residues in the CH2-CH3 inter-heavy chain region of the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of the two lysine residues may be positions 248 and 288 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Therefore, in formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modified unit via the amino groups in the side chains of the two lysine residues.

[0187] The production of affinity polypeptide-modified antibodies or salts thereof can be carried out by reacting the compound or salt thereof of the present invention with an antibody (including an antibody fragment) or salt thereof that contains an immunoglobulin unit comprising two heavy chains and optionally two light chains. The equivalent amount of the compound or salt thereof of the present invention relative to the antibody in the reaction (compound or salt thereof / antibody) is not particularly limited as it varies depending on factors such as the compound or salt thereof of the present invention and the type of antibody, but is for example 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 40, and particularly preferably 6 to 20.

[0188] Such reactions can be carried out under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). For example, such mild reactions can be carried out at room temperature (e.g., about 15-30°C) in a suitable reaction system, such as a buffer. The pH of the buffer is, for example, 5-9, preferably 5.5-8.5, and more preferably 6.0-8.0. The buffer may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such reactions, see, for example, G. J. L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G. J. L. Bernardes et al., Chem. Asian. J. See also 4, 630 (2009); B. G. Davies et al., Nat. Commun., 5, 4740 (2014); and A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0189] In certain embodiments, the affinity polypeptide-modified antibody or its salt is of the following formula (II): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L １ and L ２ represents a linker, A represents an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody heavy chain, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, L １ and L ２ The definitions, examples, and preferred examples of the linker indicated by A, the affinity polypeptide indicated by A, and the antibody are as described above (for example, L １ and L ２ For the linker shown, L is present in the compound of formula (I). １ and L ２ (See the linker shown).

[0190] The average modification percentage r per heavy chain in the above immunoglobulin units by affinity polypeptides is 65 to 135%. The average modification percentage r may be 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 82% or more, 84% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more, or 96% or more. The average modification percentage r may also be 130% or less, 125% or less, 120% or less, 115% or less, 110% or less, 105% or less, 100% or less, 98% or less, 96% or less, 94% or less, 92% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, or 80% or less. The average modification percentage r can be determined by mass spectrometry (DAR calculator (Agilent software) can be used in conjunction; see Examples).

[0191] The average modification percentage r may also preferably be 65-100%, more preferably 70-100%, even more preferably 75-100%, particularly preferably 80-100%, 85-100%, 90-100%, or 95-100%. In these average modification percentages, the upper limit may be a value less than or equal to the above-mentioned percentages, such as 98% or 96% or less. Alternatively, the average modification percentage r may be 96-100%, 97-100%, 98-100%, 99-100%, or 100%.

[0192] The degree of the average modification percentage r described above can be similarly applied to other average modification percentages r. That is, the degree of the average modification percentage r described above can be similarly applied not only to the average modification percentage r by affinity polypeptides described later, but also to the average modification percentage r by any modification (e.g., bioorthothic functional groups, functional substances) described later.

[0193] The production of an antibody or a salt thereof containing a structural unit represented by formula (II) can be carried out by reacting a compound or a salt thereof represented by formula (I) with an antibody (including an antibody fragment) or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0194] In certain embodiments, affinity polypeptide-modified antibodies or salts thereof may further contain cleavage moieties between the affinity polypeptide and the antibody (immunoglobulin unit). In this case, the antibody or salt thereof containing the structural unit represented by formula (II) above may contain cleavage moieties in the two linker portions that bind the affinity polypeptides. The definition, examples, and preferred examples of cleavage moieties are as described above.

[0195] If an affinity polypeptide-modified antibody or a salt thereof contains a cleavable moiety, the cleavable moiety may be capable of generating bioorthogonal functional groups on the antibody (immunoglobulin unit) side upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including ordinary ester residues and other ester residues such as thioester residues), acetal residues (including ordinary ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinaldiol residues.

[0196] If an affinity polypeptide-modified antibody or its salt contains a cleavable moiety that can generate bioorthogonal functional groups on the antibody (immunoglobulin unit) side upon cleavage, then the following formula (IIa): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L ３ This indicates the third linker, L ４ This indicates the fourth linker, L ５ This indicates the fifth linker, L ６ This indicates the sixth linker, CLE(B) １ and CLE(B) ２ The expression represents a cleavable portion that can generate bioorthogonal functional groups on the immunoglobulin unit side by cleavage, A represents an affinity polypeptide having affinity for the CH2-CH3 interchain region of the antibody heavy chain, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65-135%. The antibody may also contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, L ３The third linker, L, is shown by ４ The fourth linker, L, is shown by ５ The fifth linker, L, is shown by ６ The sixth linker, CLE(B), is indicated by １ and CLE(B) ２ The cleavable moiety indicated by , the affinity polypeptide indicated by A, and the average modification percentage indicated by r, as well as the definition, examples, and preferred examples of antibodies, are as described above.

[0197] The production of an antibody or a salt thereof containing a structural unit represented by formula (IIa) can be carried out by reacting a compound or a salt thereof represented by formula (Ia) with an antibody (including an antibody fragment) or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0198] In certain embodiments, the affinity polypeptide-modified antibody or its salt is defined by the following formula (IIa-1): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, W １ , W ２ , W ３ , W ４ , W ５ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L ８ This indicates the eighth linker, L ９ This indicates the 9th linker, L １０ The antibody may contain a structural unit represented by ], where represents the 10th linker, S represents a sulfur atom, and A represents an affinity polypeptide having affinity for the CH2-CH3 interchain region of the antibody heavy chain, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65-135%. The antibody may also contain a salt thereof. The immunoglobulin unit represented by Ig, W １ , W ２ , W ３ , W ４ , W ５ and W ６ Atoms represented by, L ７ The seventh linker, L, is shown as shown.８ The eighth linker, L, is shown by ９ The ninth linker, L, is indicated by １０ The tenth linker indicated by , the affinity polypeptide indicated by A, and the average modification percentage indicated by r, as well as the definition, examples, and preferred examples of the antibody, are as described above. Note that in the figures showing the structures of affinity polypeptide-modified antibodies in the examples described later, the structure of the affinity polypeptide is omitted.

[0199] An example of an affinity polypeptide-modified antibody represented by formula (IIa-1) is shown below.

[0200] The production of an antibody or a salt thereof containing a structural unit represented by formula (IIa-1) can be carried out by reacting a compound or a salt thereof represented by formula (Ia-1) with an antibody (including an antibody fragment) or a salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0201] If the affinity polypeptide-modified antibody or its salt also contains a cleavable moiety, it may further contain a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety. The bioorthogonal functional group is as described above. Preferably, the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with substituents as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, and a halocarbonyl residue.

[0202] If an affinity polypeptide-modified antibody or its salt further contains a bioorthogonal functional group between the antibody (immunoglobulin unit) and the affinity polypeptide, and a cleavable moiety in two linkers that bind the antibody and the affinity polypeptide, then the formula is as follows (IIb): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L １１ This indicates the 11th linker, L １２ This indicates the 12th linker, L １３ This indicates the 13th linker, L １４ This indicates the 14th linker, B１ and B ２ This indicates a group containing a bioorthogonal functional group, CLE １ and CLE ２ The cleavable portion is indicated, A represents an affinity polypeptide having affinity for the CH2-CH3 interchain region of the antibody heavy chain, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, L １１ The 11th linker, L １２ The 12th linker, L １３ The 13th linker, L, is shown as shown. １４ The 14th linker, B, is shown as shown. １ and B ２ Groups containing bioorthogonal functional groups as shown, CLE １ and CLE ２ The cleavage site indicated by , the affinity polypeptide indicated by A, and the average modification percentage indicated by r, as well as the definition, examples, and preferred examples of antibodies, are as described above.

[0203] The production of an antibody or salt thereof containing a structural unit represented by formula (IIb) can be carried out by reacting a compound or salt thereof represented by formula (Ib) with an antibody (including an antibody fragment) or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0204] In certain embodiments, the affinity polypeptide-modified antibody or its salt is the following formula (IIb-1): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, W ７ , W ８ , W ９ , W １０ , W １１ and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １６ This indicates the 16th linker, L １７ This indicates the 17th linker, L １８This indicates the 18th linker, B ３ and B ４ This indicates a group containing a bioorthogonal functional group, V １ and V ２ represents an oxygen atom or a sulfur atom, A represents an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the antibody heavy chain, and the modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65-135%. The antibody may also contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, W ７ , W ８ , W ９ , W １０ , W １１ and W １２ Atoms represented by, L １５ The 15th linker, L １６ The 16th linker, L １７ The 17th linker, L １８ The 18th linker, B, is shown as shown. ３ and B ４ A group containing a bioorthogonal functional group as shown by V １ and V ２ The atoms indicated by , the affinity polypeptide indicated by A, and the average modification percentage indicated by r, as well as the definition, examples, and preferred examples of antibodies, are as described above. Note that in the diagrams showing the structures of affinity polypeptide-modified antibodies in the examples described later, the structure of the affinity polypeptide is omitted.

[0205] An example of an affinity polypeptide-modified antibody represented by formula (IIb-1) is shown below.

[0206] The production of an antibody or salt containing a structural unit represented by formula (IIb-1) can be carried out by reacting a compound or salt represented by formula (Ib-1) with an antibody (including an antibody fragment) or salt containing an immunoglobulin unit comprising two heavy chains and optionally two light chains.

[0207] Affinity polypeptide-modified antibodies or salts thereof may further contain additional modification moieties. Various methods are known for modifying antibodies. Therefore, in the present invention, affinity polypeptide-modified antibodies or salts thereof can be modified to further contain additional modification moieties. The additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0208] In certain embodiments, the additional modification portion may be an additional affinity substance (for example, an additional affinity polypeptide containing an additional affinity portion having affinity for the constant region of the antibody heavy chain) that has affinity for the constant region of the antibody heavy chain. The additional affinity portion may be the same as or different from the affinity portion described above, but it is preferable that it be different. That is, the additional affinity portion may be an additional affinity substance containing an additional affinity portion having affinity for a region of the constant region of the antibody heavy chain other than the CH2-CH3 interregion, for example, the CH1 region, CH2 region, or CH3 region.

[0209] In certain embodiments, additional modification moieties having affinity for the constant region of the antibody heavy chain may be introduced into the constant region of the heavy chain via modification of amino groups in the side chains of lysine residues located at one or more positions in the constant region of the heavy chain. Affinity-modified antibodies or salts thereof may contain additional modification moieties via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant region of the heavy chain (e.g., the CH1, CH2, or CH3 region) of the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be selected from positions 246, 274, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979).

[0210] Numerous peptides have been reported as affinity polypeptides that have affinity for the constant region of the antibody heavy chain and contain one lysine residue (see, e.g., International Publication Nos. 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, and 2020 / 090979). Therefore, in the present invention, such peptides can be used as additional affinity polypeptides.

[0211] Furthermore, additional modification moieties may be introduced into the steady region of the heavy chain via cysteine ​​generated by reducing disulfide bonds. Such cysteine ​​may be located at positions 220, 226, or 229 of the human IgG heavy chain according to EU numbering. Additionally, additional modification moieties may be introduced into the steady region of the light chain via cysteine ​​generated by reducing disulfide bonds. Such cysteine ​​may be located at position 214 of the human IgG light chain according to EU numbering.

[0212] Affinity polypeptide-modified antibodies can have affinity polypeptides depending on the number of heavy chains. That is, the compounds of the present invention or their salts can introduce affinity polypeptides via two specific amino acid residues in the CH2-CH3 intercellular region of the antibody's heavy chain, so that one affinity polypeptide can be introduced per heavy chain. For example, by using an antibody with two antibody heavy chains (e.g., IgG, IgD, IgE), it is possible to obtain an antibody in which each of the two antibody heavy chains regioselectively has a structural unit (or multiple types of structural units of a sub-concept) containing an affinity polypeptide in the same target region. In other words, an antibody with two antibody heavy chains can contain one affinity polypeptide in each heavy chain, for a total of two affinity polypeptides. On the other hand, for example, by using an antibody with one antibody heavy chain (e.g., single-chain Fc), it is possible to obtain an antibody in which one antibody heavy chain regioselectively has a structural unit (or multiple types of structural units of a sub-concept) containing one affinity polypeptide in the target region of that one antibody heavy chain. If additional modification portions are included, each heavy chain can have one affinity polypeptide.

[0213] Confirmation of the production of the desired affinity polypeptide-modified antibody or its salt can be performed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, depending on the specific molecular weight of the raw materials and product. Regioselectivity can be confirmed by peptide mapping. Peptide mapping can be performed by protease treatment and mass spectrometry, for example. Endoproteases are preferred as proteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of affinity polypeptide molecules can be confirmed by mass spectrometry (DAR calculator (Agilent software) can be used in combination). The affinity polypeptide-modified antibody or its salt can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0214] 5. Antibodies or salts thereof that do not contain affinity polypeptides 5-1. Overview Antibodies or salts thereof that do not contain affinity polypeptides can be manufactured using an affinity polypeptide-modified antibody or a salt thereof, comprising (A) an affinity polypeptide having affinity for the CH2-CH3 interchain region of the antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and (C) the affinity polypeptide (A) and the antibody (immunoglobulin unit) are linked by two linkers, each linker containing a cleavable moiety.

[0215] More specifically, a method for producing an antibody or a salt thereof that does not contain affinity polypeptides may be the following methods 1-1 or 1-2.

[0216] (Method 1-1) A method for producing an antibody or salt that does not contain an affinity polypeptide, comprising (A) an affinity polypeptide having affinity for the CH2-CH3 interchain region of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), wherein (C) the affinity polypeptide and (B) the antibody (immunoglobulin unit) are linked by two linkers, each linker containing a cleavable moiety, and the affinity polypeptide-modified antibody or salt thereof is cleaved at each cleavable moiety to produce an antibody or salt thereof that does not contain an affinity polypeptide.

[0217] (Method 1-2) A method for producing an antibody or salt thereof that does not contain affinity polypeptide, comprising the following (1) and (2): (1) Reacting a compound or salt thereof, comprising (A) an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody (immunoglobulin unit), and (B) a reactive group for the antibody (immunoglobulin unit), and (C) the affinity polypeptide and (B) the reactive group are linked by two linkers, each linker containing a cleavable moiety, with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an affinity polypeptide-modified antibody or salt thereof containing two cleavable moieties between the affinity polypeptide and the antibody; and (2) Cleaving the affinity polypeptide-modified antibody or salt thereof containing two cleavable moieties between the affinity polypeptide and the antibody at each cleavable moiety to produce an antibody or salt thereof that does not contain affinity polypeptide.

[0218] Cleavage treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes as described above; (b) treatment with physicochemical stimuli such as light; or (c) incubation when using a cleavage linker containing a self-degradable cleavage portion. For more information on these cleavage treatments, please refer to International Publication Nos. 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979.

[0219] Such cleavage reactions can be carried out under conditions that do not cause protein denaturation or degradation (e.g., cleavage of amide bonds) (mild conditions). For example, such mild conditions are as described above. Also, if the cleavage site is an ester (e.g., a normal ester, or other esters such as a thioester), the cleavage reaction can be carried out by incubation in a hydroxylamine hydrochloride solution (e.g., pH 4.0–8.0, 10 mM–10 M) for an appropriate time (e.g., 1 hour) (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148–160).

[0220] Confirmation of the production of antibodies or salts

[0221] Incidentally, if an affinity polypeptide modified antibody or salt containing (A) an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody (immunoglobulin unit) and (B) an antibody (immunoglobulin unit), and the affinity polypeptide and the antibody are linked by two linkers, each linker containing a cleavable moiety, then (a) the cleavable moiety includes a cleavable moiety that can generate a bioorthothic functional group on the antibody (immunoglobulin unit) side upon cleavage, or (b) the antibody (immunoglobulin unit) includes a bioorthothic functional group between the antibody (immunoglobulin unit) and the cleavable moiety, then an antibody derivative or salt containing two bioorthothic functional groups per heavy chain can be produced as an antibody or salt that does not contain an affinity polypeptide.

[0222] Furthermore, by reacting an antibody derivative or a salt thereof containing two bioorthogonal functional groups per heavy chain with a functional substance, it is possible to produce an antibody or a salt thereof that does not contain affinity polypeptides, and that contains two functional substances per heavy chain of the antibody, as an antibody and functional substance conjugate or salt thereof.

[0223] The following describes in detail antibodies or salts thereof that do not contain affinity polypeptides, including (1) antibody derivatives or salts thereof containing two bioorthogonal functional groups per heavy chain, and (2) conjugates of antibodies and functional substances or salts thereof containing an antibody and two functional substances per heavy chain of the antibody.

[0224] 5-2. Antibody derivatives or salts thereof containing two bioorthogonal functional groups By using the compounds or salts thereof of the present invention, an antibody derivative or salt thereof containing two bioorthogonal functional groups is provided, comprising (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) first and second bioorthogonal functional groups, and (c) the first and second bioorthogonal functional groups introduced at two specific locations in the CH2-CH3 interregion of the heavy chain in the immunoglobulin unit. Definitions, examples, and preferred examples of the antibody, immunoglobulin unit, and bioorthogonal functional groups, and the elements constituting them (e.g., constant regions) are as described above.

[0225] Antibody derivatives or salts thereof may contain two bioorthogonal functional groups per heavy chain by modifying the functional groups in the side chains of two amino acids selected from asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are present in the CH2-CH3 intercellular region of the heavy chain. Preferably, antibody derivatives or salts thereof may contain two bioorthogonal functional groups per heavy chain by modifying the functional groups in the side chains of two amino acids selected from lysine, tyrosine, tryptophan, or cysteine, which are present in the CH2-CH3 intercellular region of the heavy chain; more preferably, by modifying the functional groups in the side chains of two amino acids selected from lysine, tyrosine, or tryptophan; even more preferably, by modifying the functional groups in the side chains of two amino acids selected from lysine or tyrosine; and particularly preferably, by modifying the amino groups in the side chains of two lysine molecules. The positions of these amino acid residues in the CH2-CH3 intercellular region of the heavy chain are as described above. The modification sites of antibodies or their salts by bioorthogonal functional groups can be confirmed by peptide mapping. The modifications may be regioselective, as described above. Therefore, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modification unit via the functional group in the side chain of the above amino acid residue.

[0226] Preferably, the antibody derivative may contain two bioorthogonal functional groups per heavy chain through modification of amino groups in the side chains of two lysine residues in the CH2-CH3 region of the heavy chain within the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be at positions 248 and 288 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Therefore, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modified unit via the amino groups in the side chains of the lysine residues.

[0227] In certain embodiments, the antibody derivative or its salt is defined by the following formula (IIIa): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L ３ This indicates the third linker, L ６ This indicates the sixth linker, B ５ and B ６ Each of these groups represents a group containing a bioorthogonal functional group, and the average modification percentage r per heavy chain in the above immunoglobulin unit by the bioorthogonal functional group is 65 to 135%. The antibody may also contain a structural unit represented by [ ] or a salt thereof. B ５ and B ６ The group containing the bioorthogonal functional group represented by may be a group consisting of a bioorthogonal functional group, or a group containing a bioorthogonal functional group and other parts. Examples of other parts include the linking portion between the bioorthogonal functional group and the linker. The linking portion is, for example, a divalent group. The divalent group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the bioorthogonal functional group, the divalent group, and the substituent when the divalent group is substituted are as described above. Ig represents an immunoglobulin unit, L ３ The third linker, L, is shown by ６The sixth linker, indicated by , the average modification percentage, indicated by r, and the definition, examples, and preferred examples of antibodies are as described above. A particularly preferred bioorthogonal functional group is the thiol group.

[0228] In equation (IIIa), L ３ -B ５ and L ６ -B ６ The molecular weights of the substructures shown may each be 700 or less. ３ -B ５ and L ６ -B ６ When the molecular weights of the substructures shown are 700 or less, antibody derivatives or salts having bioorthogonal functional groups are relatively difficult to purify based on molecular weight differences because the ratio of the molecular weight of the substructures to the molecular weight of the entire antibody is very small. However, according to the present invention, which enables advanced control of DAR, antibody derivatives exhibiting the desired DAR can be obtained in high purity without necessarily requiring purification based on molecular weight differences. ３ -B ５ and L ６ -B ６ The molecular weights of the substructures shown are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0229] In another specific embodiment, the antibody derivative or a salt thereof is given by the following formula (IIIa-1): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, W １ and W ６ These represent either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L １０ represents the 10th linker, SH represents a thiol group, and the average modification percentage r per heavy chain in the above immunoglobulin unit by bioorthogonal functional groups is 65 to 135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, W １and W ６ Atoms represented by, L ７ The seventh linker, L, is shown as shown. １０ The tenth linker, indicated by , the average modification percentage, indicated by r, and the definition, examples, and preferred examples of antibodies are as described above.

[0230] In equation (IIIa-1), C(=W) １ )-L ７ -SH and C(=W ６ )-L １０ The molecular weights of the substructures represented by -SH may be 700 or less. C(=W) １ )-L ７ -SH and C(=W) ６ )-L １０ The molecular weight of each substructure represented by -SH is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[0231] In equation (IIIa-1), L ７ The seventh linker, indicated by (CH ２ ) ｎ１ It may also be an integer n1 between 1 and 10. Preferably, n1 may be an integer greater than or equal to 2. n1 may also be an integer less than or equal to 9, 8, 7, 6, 5, 4, 3, or 2. Particularly preferably, n1 is 2. L １０ The tenth linker indicated by (CH ２ ) ｎ１ It is also possible that n1 is an integer between 1 and 10. Preferably, n1 is an integer greater than or equal to 2. n1 may also be an integer less than or equal to 9, 8, 7, 6, 5, 4, 3, or 2. Particularly preferably, n1 is 2.

[0232] In yet another specific embodiment, the antibody derivative or a salt thereof is of the following formula (IIIb): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L １１This indicates the 11th linker, L １４ This indicates the 14th linker, B １ and B ２ Each of these indicates a group containing a bioorthogonal functional group, T １ and T ２ Each of these represents a monovalent group, and the average modification percentage r per heavy chain in the above immunoglobulin unit by bioorthogonal functional groups is 65 to 135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, L １１ The 11th linker, L １４ The 14th linker, B, is shown as shown. １ and B ２ The groups containing bioorthogonal functional groups indicated by , the average modification percentage indicated by r, and the definition, examples, and preferred examples of antibodies are as described above. Particularly preferred bioorthogonal functional groups are azide groups.

[0233] T １ and T ２ This is a monovalent group and can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of monovalent groups include those mentioned above. Examples of substituents when the monovalent group is substituted include those mentioned above.

[0234] In a particular embodiment, T １ and T ２ The monovalent group represented by may be a substituted hydroxyamino group. A substituted hydroxyamino group can be represented by the following formula (α): NR ｉ -OR ｉｉ　 (α) [wherein, R ｉ , R ｉｉ Each of these independently represents a hydrogen atom or a monovalent hydrocarbon group.) Here, the monovalent hydrocarbon group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the monovalent hydrocarbon group and the substituent when the monovalent hydrocarbon group is substituted are as described above. Preferably, the hydroxyamino group which may be substituted is NH-OR ｉｉ (Here, Rｉｉ represents an alkyl group. ) It may also be substituted. More preferably, the substituted hydroxyamino group is NH-OR ｉｉ (Here, R ｉｉ (This may also represent an alkyl group having 1 to 6 carbon atoms.)

[0235] In equation (IIIb), L １１ (-B １ )-T １ and L １４ (-B ２ )-T ２ The molecular weights of the substructures shown may each be 700 or less. １１ (-B １ )-T １ and L １４ (-B ２ )-T ２ The molecular weights of the substructures shown are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0236] In yet another specific embodiment, the antibody derivative or a salt thereof is of the following formula (IIIb-1): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, B ３ and B ４ Each of these indicates a group containing a bioorthogonal functional group, T ３ and T ４ Each of these represents a monovalent group, and the modification percentage r per heavy chain in the above immunoglobulin unit by bioorthogonal functional groups is 65-135%. The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, W ７ , W ８ , W１１ , and W １２ Atoms represented by, L １５ The 15th linker, L １８ The 18th linker, B, is shown as shown. ３ and B ４ The groups containing bioorthogonal functional groups indicated by , the average modification percentage indicated by r, and the definition, examples, and preferred examples of antibodies are as described above. Particularly preferred bioorthogonal functional groups are azide groups.

[0237] T ３ and T ４ This is a monovalent group and can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of monovalent groups include those listed above. Examples of substituents when the monovalent group is substituted include those listed above. T ３ and T ４ The monovalent group indicated by may be a substituted hydroxyamino group. For details on the substituted hydroxyamino group, see T １ and T ２ This is similar to what was described earlier.

[0238] In equation (IIIb-1), C(=W ７ )-L １５ (-B ３ ) - C (= W ８ )-T ３ and C(=W １２ )-L １８ (-B ４ ) - C (= W １１ )-T ４ The molecular weights of the substructures shown may each be 700 or less. C(=W ７ )-L １５ (-B ３ ) - C (= W ８ )-T ３ and C(=W １２ )-L １８ (-B ４ ) - C (= W １１ )-T ４The molecular weights of the substructures shown are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0239] In equation (IIIb-1), L １５ The 15th linker and L are shown as indicated. １８ The 18th linker, indicated by (CH), is as follows: ２ ) ｎ２ It is also possible that n2 is an integer between 1 and 10. Preferably, n2 may be an integer greater than or equal to 2, or greater than or equal to 3. n2 may also be an integer less than or equal to 9, less than or equal to 8, less than or equal to 7, less than or equal to 6, less than or equal to 5, less than or equal to 4, or less than or equal to 3. Particularly preferably, n2 may be 3.

[0240] In formula (IIIb-1), the group containing the bioorthogonal functional group is NH-C(=O)-(CH ２ ) ｎ３ -N ３ It is also possible that n3 is an integer between 1 and 10. Preferably, n3 may be an integer of 2 or more, 3 or more, or 4 or more. n3 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. Particularly preferably, n3 may be 4.

[0241] The production of antibody derivatives or salts thereof containing bioorthogonal functional groups can be carried out using an affinity polypeptide-modified antibody or salt thereof, comprising (A) an affinity polypeptide having affinity for the CH2-CH3 interchain region of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), wherein (C) the affinity polypeptide (A) and the antibody (immunoglobulin unit) (B) are linked by two linkers, and each linker contains a cleavable moiety.

[0242] In certain embodiments, if the affinity polypeptide-modified antibody or a salt thereof contains a cleavable moiety that can generate bioorthogonal functional groups on the antibody (immunoglobulin unit) side by cleavage, an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain can be produced.

[0243] More specifically, examples of such manufacturing methods include (2-1) to (2-6) below.

[0244] (Method 2-1) A method for producing an antibody derivative or salt containing two bioorthogonal functional groups per heavy chain, comprising: (A) an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody (immunoglobulin unit); and (B) an antibody (immunoglobulin unit); and (C) an affinity polypeptide-modified antibody or salt containing an affinity polypeptide having two linkers, where each linker is a cleavable moiety (where the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side by cleavage at each cleavable moiety to produce an antibody derivative or salt containing a bioorthogonal functional group.

[0245] (Method 2-2) A method for producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain, comprising the following (1) and (2): (1) A compound or salt thereof containing (A) an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody (immunoglobulin unit), and (B) two reactive groups for the antibody (immunoglobulin unit), and further comprising (C) a cleavable moiety between (A) the affinity polypeptide and (B) each reactive group (where the cleavable moiety is a cleavable moiety that can generate bioorthogonal functional groups on the reactive group side for the antibody (immunoglobulin unit) by cleavage), reacting this compound or salt thereof with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains, to produce an affinity polypeptide-modified antibody or salt thereof containing two cleavable moieties between the affinity polypeptide and the antibody (where the cleavable moiety is a cleavable moiety that can generate bioorthogonal functional groups on the antibody side by cleavage); and (2) An affinity polypeptide-modified antibody or a salt thereof, which contains two cleavable moieties between the affinity polypeptide and the antibody, is subjected to cleavage at each cleavable moiety to produce an antibody derivative or a salt thereof containing two bioorthogonal functional groups per heavy chain.

[0246] (Method 2-3) A method for producing an antibody derivative or salt containing two bioorthogonal functional groups per heavy chain, comprising cleaving an antibody or salt containing the structural unit represented by formula (IIa) at a cleavable portion to produce an antibody derivative or salt containing the structural unit represented by formula (IIIa) above.

[0247] (Method 2-4) A method for producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain, comprising (1) and (2) below: (1) Reacting a compound or salt thereof represented by formula (Ia) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIa); and (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIa) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa).

[0248] (Method 2-5) A method for producing an antibody derivative or salt containing two bioorthogonal functional groups per heavy chain, comprising cleaving an antibody or salt containing the structural unit represented by the above formula (IIa-1) at a cleavable portion to produce an antibody derivative or salt containing the structural unit represented by the above formula (IIIa-1).

[0249] (Method 2-6) A method for producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain, comprising (1) and (2) below: (1) Reacting a compound or salt thereof represented by formula (Ia-1) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIa-1); and (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIa-1) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa-1).

[0250] Method 2-1 may be carried out by Method 2-3 or 2-5. Method 2-2 may be carried out by Method 2-4 or 2-6. Methods 2-2, 2-4, and 2-6 may further include reacting the affinity polypeptide of the present invention with a partial compound containing a reactive group for an antibody to produce the compound of the present invention or a salt thereof.

[0251] In another specific embodiment, if the affinity polypeptide-modified antibody or salt thereof contains (b) a bioorthogonal functional group between the antibody (immunoglobulin unit) and each cleavable moiety, an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain can be produced.

[0252] More specifically, examples of such manufacturing methods include (2-7) to (2-12) below.

[0253] (Method 2-7) A method for producing an antibody derivative or salt containing two bioorthogonal functional groups per heavy chain, comprising: (A) an affinity polypeptide having affinity for the CH2-CH3 interchain region of an antibody (immunoglobulin unit); (B) an antibody (immunoglobulin unit); and (C) the affinity polypeptide and (B) the antibody (immunoglobulin unit) are linked by two linkers, each linker containing a cleavable moiety; and (D) a bioorthogonal functional group further comprising a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety; and cleaving the affinity polypeptide-modified antibody or salt containing a bioorthogonal functional group at the cleavable moiety to produce an antibody derivative or salt containing a bioorthogonal functional group.

[0254] (Method 2-8) A method for producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain, comprising (1) and (2) below: (1) A compound or salt thereof comprising (A) an affinity polypeptide having affinity for the CH2-CH3 intercellular region of the heavy chain of an antibody (immunoglobulin unit), and (B) a reactive group for the antibody (immunoglobulin unit), and (C) the affinity polypeptide and (B) the reactive group are linked by two linkers, each linker containing a cleavable moiety, and (D) a bioorthogonal functional group further comprising between the reactive group and the cleavable moiety; reacting the compound or salt thereof with an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an affinity polypeptide-modified antibody or salt thereof containing a cleavable moiety between the affinity polypeptide and the antibody, and a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety; and (2) An affinity polypeptide-modified antibody or a salt thereof, which contains a cleavable moiety between the affinity polypeptide and the antibody, is cleaved at the cleavable moiety to produce an antibody derivative or a salt thereof containing two bioorthogonal functional groups per heavy chain.

[0255] (Method 2-9) A method for producing an antibody derivative or salt containing two bioorthogonal functional groups, comprising cleaving an antibody or salt containing the structural unit represented by formula (IIb) at a cleavable portion to produce an antibody derivative or salt containing the structural unit represented by formula (IIIb).

[0256] (Method 2-10) A method for producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain, comprising the following (1) and (2): (1) Reacting the compound represented by formula (Ib) or a salt thereof with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIb); and (2) Cutting the antibody or salt thereof containing the structural unit represented by formula (IIb) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb).

[0257] (Method 2-11) A method for producing an antibody derivative or salt containing two bioorthogonal functional groups per heavy chain, comprising cleaving an antibody or salt containing the structural unit represented by formula (IIb-1) at a cleavable portion to produce an antibody derivative or salt containing the structural unit represented by formula (IIIb-1)

[0258] (Method 2-12) A method for producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain, comprising (1) and (2) below: (1) Reacting a compound or salt thereof represented by formula (Ib-1) with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIb-1); and (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIb-1) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb-1).

[0259] Method 2-7 may be carried out by Method 2-9 or 2-11. Method 2-8 may be carried out by Method 2-10 or 2-11. Methods 2-8, 2-10, and 2-12 may further include reacting the affinity polypeptide of the present invention with a moiety containing a reactive group for an antibody to produce the compound of the present invention or a salt thereof.

[0260] The antibody derivative or a salt thereof may further contain additional modification moieties. Various methods are known for modifying antibodies. Therefore, in the present invention, the antibody derivative or a salt thereof can be modified to further contain additional modification moieties. The additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0261] In certain embodiments, the additional modification portion may include a bioorthogonal functional group. The bioorthogonal functional group is the same as that described above. The bioorthogonal functional group included in the additional modification portion may be the same as or different from the bioorthogonal functional group of (b) above, but it is preferable that it be different.

[0262] In certain embodiments, additional modification moieties containing bioorthogonal functional groups may be introduced into the constant region of the heavy chain via modification of amino groups in the side chains of lysine residues located at one or more positions in the constant region of the heavy chain. Antibody derivatives or salts thereof may contain additional modification moieties via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant region of the heavy chain (preferably the CH1 region, CH2 region, or CH3 region) of the antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be selected from positions 246, 274, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979).

[0263] Furthermore, additional modification moieties may be introduced into the steady region of the heavy chain via cysteine ​​generated by reducing disulfide bonds. Such cysteine ​​may be located at positions 220, 226, or 229 of the human IgG heavy chain according to EU numbering. Additionally, additional modification moieties may be introduced into the steady region of the light chain via cysteine ​​generated by reducing disulfide bonds. Such cysteine ​​may be located at position 214 of the human IgG light chain according to EU numbering.

[0264] Antibody derivatives having bioorthogonal functional groups obtained using the compound or salt of the present invention can also have bioorthogonal functional groups depending on the number of heavy chains. That is, since the compound or salt of the present invention can introduce bioorthogonal functional groups via two specific amino acid residues in the CH2-CH3 region of the antibody heavy chain, two bioorthogonal functional groups can be introduced per heavy chain, and when using an antibody having multiple antibody heavy chains, the modification pattern by bioorthogonal functional groups can be made identical across multiple (e.g., two) heavy chains.

[0265] Therefore, by using an antibody having multiple antibody heavy chains (for example, 2 to 8, preferably 2 to 4, more preferably 2) in the production method of the present invention, it is possible to produce an antibody having two regioselective structural units (or multiple structural units of a sub-concept) containing bioorthogonal functional groups in the same target region of the multiple antibody heavy chains. For example, an antibody having two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab')) ２ By using an antibody in the production method of the present invention, it is possible to produce an antibody having two regioselectively located structural units (or multiple types of structural units with a sub-concept) containing bioorthogonal functional groups in the same target region of two antibody heavy chains, i.e., an antibody having four bioorthogonal functional groups. On the other hand, by using an antibody having one antibody heavy chain (e.g., a single-chain Fc antibody) in the production method of the present invention, it is possible to produce an antibody having two regioselectively located structural units (or multiple types of structural units with a sub-concept) containing bioorthogonal functional groups in the target region of one antibody heavy chain. If the antibody derivative includes additional modification portions, the antibody derivative may contain two or more bioorthogonal functional groups per heavy chain.

[0266] The formation of antibody derivatives or salts containing bioorthogonal functional groups can be confirmed by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reversed-phase column chromatography, HPLC), or mass spectrometry, depending on the specific molecular weight of the raw materials and product. Regioselectivity can be confirmed by peptide mapping as described above. The number of introduced bioorthogonal functional groups can be confirmed by mass spectrometry (DAR calculator (Agilent software) can be used in combination). Antibody derivatives or salts containing bioorthogonal functional groups can be purified as appropriate by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0267] 5-4. Conjugate or Salt of Antibody and At least Two Functional Substances By using the compounds or salts thereof of the present invention, a conjugate or salt of antibody and functional substances is provided, wherein (a) an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains), and (b) two functional substances comprising each heavy chain, and (c) the two functional substances being introduced at two amino acid residues in the CH2-CH3 intercellular region of the heavy chain in the immunoglobulin unit. Definitions, examples, and preferred examples of antibody, immunoglobulin unit, and conjugate of antibody and functional substances, as well as the elements constituting them (e.g., constant regions), are as described above.

[0268] The conjugate or a salt thereof may contain two functional substances through modification of the functional groups in the side chains of two amino acid residues selected from asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, which are located in the region between the CH2 and CH3 regions. Preferably, the conjugate or a salt thereof may contain functional substances through modification of the functional groups in the side chains of two amino acid residues selected from lysine, tyrosine, tryptophan, or cysteine, which are located in the region between the CH2 and CH3 regions; more preferably, through modification of the functional groups in the side chains of two amino acid residues selected from lysine, tyrosine, or tryptophan; even more preferably, through modification of the functional groups in the side chains of two amino acid residues selected from lysine or tyrosine; and particularly preferably, through modification of the amino groups in the side chains of two lysine residues. The positions of these amino acid residues in the region between the CH2 and CH3 regions are as described above. The modification sites of antibodies or their salts by functional substances can be confirmed by peptide mapping. As described above, the modifications may be regioselective. Therefore, in formulas (IVa), (IVa-1), (IVb), and (IVb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modification unit via the functional group in the side chain of the amino acid residue.

[0269] Preferably, the conjugate may contain two functional substances via modification of amino groups in the side chains of two lysine residues in the region between the CH2 and CH3 regions of the heavy chain in the antibody's constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of the two lysine residues may be positions 248 and 288 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Therefore, in formulas (IVa), (IVa-1), (IVb), and (IVb-1) described later, the immunoglobulin unit may regioselectively possess the corresponding modified unit via the amino groups in the side chains of the lysine residues.

[0270] In certain embodiments, the conjugate or its salt is of the following formula (IVa): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L ３ This indicates the third linker, L ６ This indicates the sixth linker, B ５ and B ６ This represents a group containing a bioorthogonal functional group, and the average modification percentage r per heavy chain in the above immunoglobulin unit by the functional substance is 65 to 135%. This may also be an antibody or a salt thereof containing a structural unit represented by ]. The immunoglobulin unit represented by Ig, L ３ The third linker, L, is shown by ６ The sixth linker, B, is shown as shown. ５ and B ６ The bioorthogonal functional groups indicated by , the average modification percentage indicated by r, and the definition, examples, and preferred examples of antibodies are as described above. Particularly preferred bioorthogonal functional groups are thiol groups.

[0271] In equation (IVa), L ３ -Z １ , L ６ -Z ２ The molecular weights of the substructures shown may each be 700 or less. ３ -Z １ , L ６ -Z ２ When the molecular weights of the substructures shown are 700 or less, the conjugate or its salt is relatively difficult to purify based on molecular weight differences because the ratio of the molecular weights of the substructures to the total molecular weight of the antibody is very small. However, according to the present invention, which enables high-level control of DAR, it is possible to obtain a conjugate exhibiting the desired DAR in high purity without necessarily requiring purification based on molecular weight differences. ３ -Z １ , L ６ -Z ２The molecular weights of the substructures shown are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0272] In another specific embodiment, the conjugate or its salt is of the following formula (IVa-1): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, W １ and W ６ Each represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L １０ This indicates the 10th linker, Z １ and Z ２ represents a functional substance, and the average modification percentage r per heavy chain in the above immunoglobulin unit by the functional substance is 65 to 135%. It may also be an antibody or a salt thereof containing a structural unit represented by ]. The immunoglobulin unit represented by Ig, W １ and W ６ Atoms represented by, L ７ The seventh linker, L, is shown as shown. １０ The 10th linker, Z １ and Z ２ The functional substances indicated by , the average modification percentage indicated by r, and the definition, examples, and preferred examples of antibodies are as described above.

[0273] In equation (IVa-1), C(=W) １ )-L ７ -Z １ and C(=W ６ )-L １０ -Z ２ The molecular weights of the substructures shown may each be 700 or less. C(=W １ )-L ７ -Z １ and C(=W ６ )-L １０ -Z ２The molecular weights of the substructures indicated by are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[0274] In equation (IVa-1), L ７ and L １０ The substructures shown are (CH ２ ) ｎ１ This is also acceptable. The definition, examples, and preferred examples of n1 are as described above.

[0275] In yet another specific embodiment, the conjugate or its salt is of the following formula (IVb): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, Z １ and Z ２ This indicates a functional substance, T １ and T ２ The antibody may contain a structural unit represented by ] or a salt thereof. The immunoglobulin unit represented by Ig, L １１ The 11th linker, L １４ The 14th linker, Z １ and Z ２ Functional substances indicated by T １ and T ２ The monovalent group indicated by , the average modification percentage indicated by r, and the definition, examples, and preferred examples of antibodies are as described above.

[0276] In equation (IVb), L １１ (-Z １ )-T １ and L １４ (-Z ２ )-T ２ The molecular weights of the substructures shown may each be 700 or less. １１ (-Z１ )-T １ and L １４ (-Z ２ )-T ２ The molecular weights of the substructures shown are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0277] In yet another specific embodiment, the conjugate or its salt is the following formula (IVb-1): [In the formula, Ig represents an immunoglobulin unit containing two heavy chains and optionally two light chains, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, Z １ and Z ２ This indicates a functional substance, T ３ and T ４ This represents a monovalent group, and the modification percentage r per heavy chain in the above immunoglobulin unit by the functional substance is 65-135%. This may also be an antibody or a salt thereof containing a structural unit represented by ]. The immunoglobulin unit represented by Ig, W ７ , W ８ , W １１ , and W １２ Atoms represented by, L １５ The 15th linker, L １８ The 18th linker, Z １ and Z ２ Functional substances indicated by T ３ and T ４ The monovalent group indicated by , the average modification percentage indicated by r, and the definition, examples, and preferred examples of antibodies are as described above.

[0278] In equation (IVb-1), C(=W) ７ )-L １５ (-Z １ ) - C (= W ８)-T ３ and C(=W １２ )-L １８ (-Z ２ ) - C (= W １１ )-T ４ The molecular weights of the substructures shown may each be 700 or less. C(=W ７ )-L １５ (-Z １ ) - C (= W ８ )-T ３ and C(=W １２ )-L １８ (-Z ２ ) - C (= W １１ )-T ４ The molecular weights of the substructures shown are preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0279] In equation (IVb-1), L １５ and L １８ The substructure shown is (CH ２ ) ｎ２ This is also acceptable. The definition, examples, and preferred examples of n2 are as described above.

[0280] In formula (IVb-1), the group containing the bioorthogonal functional group is NH-C(=O)-(CH ２ ) ｎ３ -N ３ This is also acceptable. The definition, examples, and preferred examples of n3 are as described above.

[0281] A method for producing a conjugate or a salt thereof may be carried out by reacting an antibody derivative or a salt thereof containing two bioorthogonal functional groups per heavy chain with a functional substance to produce a conjugate or a salt thereof containing an antibody and a functional substance, each containing two functional substances per heavy chain.

[0282] Alternatively, a method for producing a conjugate or salt thereof containing two functional substances per heavy chain may be carried out by a method comprising the following (1) and (2): (1) producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain by the method described above; and (2) reacting an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain with a functional substance to produce a conjugate or salt thereof containing an antibody and a functional substance, each containing two functional substances per heavy chain.

[0283] More specifically, examples of methods for producing a conjugate or a salt thereof containing two functional substances per heavy chain include the following (3-1) to (3-12).

[0284] (Method 3-1) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising reacting an antibody derivative or a salt thereof containing the structural unit represented by formula (IIIa) with a functional substance to produce a conjugate or a salt thereof containing the structural unit represented by formula (IVa) above.

[0285] (Method 3-2) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising (1) and (2) below: (1) cleaving an antibody or salt of a functional substance containing the structural unit represented by formula (IIa) at a cleavable portion to produce an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIa); and (2) reacting an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIa) with a functional substance to produce a conjugate or salt of a functional substance containing the structural unit represented by formula (IVa).

[0286] (Method 3-3) A method for producing antibodies and functional substance conjugates or salts thereof, comprising (1) to (3) below, each containing two functional substances per heavy chain: (1) Reacting a compound represented by formula (Ia) or a salt thereof with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIa); (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIa) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa); and (3) Reacting an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa) with a functional substance to produce a conjugate or salt thereof containing a structural unit represented by formula (IVa).

[0287] (Method 3-4) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising reacting an antibody derivative or salt thereof containing the structural unit represented by formula (IIIa-1) with a functional substance to produce a conjugate or salt thereof containing the structural unit represented by formula (IVa-1) above.

[0288] (Method 3-5) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising (1) and (2) below: (1) cleaving an antibody or salt of a functional substance containing the structural unit represented by formula (IIa-1) at a cleavable portion to produce an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIa-1); and (2) reacting an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIa-1) with a functional substance to produce a conjugate or salt of a functional substance containing the structural unit represented by formula (IVa-1).

[0289] (Method 3-6) Methods for producing antibodies and functional substance conjugates or salts thereof, comprising (1) to (3) below, each containing two functional substances per heavy chain: (1) Reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIa-1); (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIa-1) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa-1); and (3) Reacting an antibody derivative or salt thereof containing a structural unit represented by formula (IIIa-1) with a functional substance to produce a conjugate or salt thereof containing a structural unit represented by formula (IVa-1).

[0290] (Method 3-7) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising reacting an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb) with a functional substance to produce a conjugate or salt thereof containing a structural unit represented by formula (IVb) above.

[0291] (Method 3-8) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising (1) and (2) below: (1) cleaving an antibody or salt of a functional substance containing the structural unit represented by formula (IIb) at a cleavable portion to produce an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIb); and (2) reacting an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIb) with a functional substance to produce a conjugate or salt of a functional substance containing the structural unit represented by formula (IVb).

[0292] (Method 3-9) Methods for producing antibodies and functional substance conjugates or salts thereof, comprising (1) to (3) below, each containing two functional substances per heavy chain: (1) Reacting a compound represented by formula (Ib) or a salt thereof with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIb); (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIb) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb); and (3) Reacting an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb) with a functional substance to produce a conjugate or salt thereof containing a structural unit represented by formula (IVb).

[0293] (Method 3-10) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising reacting an antibody derivative or a salt thereof containing the structural unit represented by formula (IIIb-1) with a functional substance to produce a conjugate or a salt thereof containing the structural unit represented by formula (IVb-1) above.

[0294] (Method 3-11) A method for producing an antibody and a conjugate or salt of a functional substance containing two functional substances per heavy chain, comprising the following (1) and (2): (1) cleaving an antibody or salt of a functional substance containing the structural unit represented by formula (IIb-1) at a cleavable portion to produce an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIb-1); and (2) reacting an antibody derivative or salt of a functional substance containing the structural unit represented by formula (IIIb-1) with a functional substance to produce a conjugate or salt of a functional substance containing the structural unit represented by formula (IVb-1).

[0295] (Method 3-12) Method for producing antibodies and functional substance conjugates or salts thereof, comprising two functional substances per heavy chain, including the following (1) to (3): (1) Reacting a compound represented by formula (IB-1) or a salt thereof with an antibody or salt thereof containing an immunoglobulin unit comprising two heavy chains and optionally two light chains to produce an antibody or salt thereof containing a structural unit represented by formula (IIb-1); (2) Cutting an antibody or salt thereof containing a structural unit represented by formula (IIb-1) at a cleavable portion to produce an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb-1); and (3) Reacting an antibody derivative or salt thereof containing a structural unit represented by formula (IIIb-1) with a functional substance to produce a conjugate or salt thereof containing a structural unit represented by formula (IVb-1).

[0296] Methods 3-1 to 3-12 described above may further include reacting the affinity polypeptide of the present invention with a partial compound containing a reactive group for an antibody to produce the compound of the present invention or a salt thereof.

[0297] The conjugate or its salt may further contain additional modification moieties. Various methods are known for modifying antibodies. Therefore, in the present invention, the conjugate or its salt can be modified to further contain additional modification moieties. The additional modification moieties may be introduced into the heavy chain or light chain of the antibody, preferably the heavy chain of the antibody (particularly in the CH1, CH2, or CH3 region of the heavy chain).

[0298] In certain embodiments, the additional modification portion may include a functional substance. The functional substance is the same as that described above. The functional substance included in the additional modification portion may be the same as or different from the functional substance in (b) above, but it is preferable that it be different.

[0299] In certain embodiments, additional modification moieties containing functional substances may be introduced into the constant regions of the two heavy chains via modification of amino groups in the side chains of lysine residues located at one or more positions in the constant regions of the heavy chains. The conjugate or a salt thereof may include additional modification moieties via modification of amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the constant regions (preferably CH1, CH2, or CH3 regions) of the heavy chains in an antibody constituent unit (an immunoglobulin unit comprising two heavy chains and optionally two light chains). More specifically, the positions of one or more (preferably one or two, more preferably one) lysine residues may be selected from positions 246, 274, 290, 317, 320, and 322 of the human IgG heavy chain according to EUnumbering (see, e.g., International Publications 2016 / 186206, 2018 / 199337, 2019 / 240287, 2019 / 240288, 2020 / 009165, and 2020 / 090979). The position where the additional modification moiety containing the functional substance is introduced is preferably different from the position where the functional substance described in (b) above is introduced.

[0300] Furthermore, additional modification moieties may be introduced into the steady region of the heavy chain via cysteine ​​generated by reducing disulfide bonds. Such cysteine ​​may be located at positions 220, 226, or 229 of the human IgG heavy chain according to EU numbering. Additionally, additional modification moieties may be introduced into the steady region of the light chain via cysteine ​​generated by reducing disulfide bonds. Such cysteine ​​may be located at position 214 of the human IgG light chain according to EU numbering.

[0301] The antibody having a functional substance produced by the manufacturing method of the present invention (a conjugate of an antibody and a functional substance) may also have functional substances depending on the number of heavy chains. Specifically, each heavy chain may have two functional substances (structural units containing functional substances). This is because an antibody having a functional substance can be produced from an antibody having two structural units containing bioorthogonal functional groups per heavy chain. Therefore, by using an antibody having multiple antibody heavy chains (for example, 2 to 8, preferably 2 to 4, more preferably 2) in the manufacturing method of the present invention, it is possible to produce an antibody that regioselectively has structural units (or multiple structural units of a sub-concept) containing functional substances at two locations in the same target region of multiple antibody heavy chains, specifically in the CH2-CH3 region.

[0302] For example, antibodies that have two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab')) ２ By using an antibody in the production method of the present invention, it is possible to produce an antibody having regioselectively L-Z structural units at two specific locations in the same target region of two antibody heavy chains, specifically in the CH2-CH3 inter-region. That is, in an antibody containing a functional substance, the modification mode by the functional substance can be made identical across multiple (e.g., two) heavy chains, and an antibody with a total of four functional substances introduced can be obtained. On the other hand, by using an antibody having one antibody heavy chain in the production method of the present invention, it is possible to produce an antibody having two regioselectively L-Z structural units in the same target region of one antibody heavy chain. If the antibody-functional substance conjugate includes additional modification portions, the antibody-functional substance conjugate may contain two or more functional substances per heavy chain.

[0303] 6. Applications The compound or salt of the present invention can simultaneously modify two sites in the CH2-CH3 intercellular region of the heavy chain in an antibody constituent unit (an immunoglobulin unit containing two heavy chains and optionally two light chains) (the average modification percentage r per heavy chain in the immunoglobulin unit is 65-135%). The compound or salt of the present invention can also regioselectively modify two specific amino acid residues (preferably lysine residues) in the heavy chain of an immunoglobulin unit. Accordingly, the present invention provides an antibody derivatization reagent comprising the compound or salt of the present invention.

[0304] The reagent of the present invention may be provided in the form of a composition further comprising other components. Such other components include, for example, solutions and stabilizers (e.g., antioxidants, preservatives). As the solution, an aqueous solution is preferred. Examples of aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline) and buffer solutions (e.g., aqueous phosphoric acid solution, Tris-hydrochloride buffer, carbonic acid-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer), but buffer solutions are preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5. The reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[0305] The affinity polypeptide-modified antibodies, antibody derivatives, or salts thereof of the present invention are useful, for example, as intermediates for the preparation of the conjugate or salts thereof of the present invention.

[0306] The conjugate or salt of the present invention is useful, for example, as a pharmaceutical or reagent (e.g., diagnostic agent, research reagent). In particular, the conjugate or salt of the present invention, in which the heavy chain of the antibody constituent unit is modified (the average modification percentage r per heavy chain in the immunoglobulin unit is 65-135%) and is regioselectively modified with a functional substance, is useful as a pharmaceutical. It has been reported that changing the number and binding position of drugs bound to an antibody-drug conjugate (ADC) alters the pharmacokinetics, drug release rate, and efficacy. For these reasons, it is required to control the number and position of the conjugated drug in next-generation ADCs. It is believed that if the number and position are constant, the expected effectiveness, variations in conjugated drugs, and lot-to-lot differences, or so-called regulation problems, will be resolved. Therefore, the conjugate or salt of the present invention can solve such regulation problems.

[0307] The conjugate or salt thereof of the present invention may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate or salt thereof of the present invention, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizine ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersants such as surfactants; diluents such as water, physiological saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and kerosene. The conjugate or salt of the present invention may also have any modifications (e.g., PEGylation) that provide stability.

[0308] Suitable formulations for oral administration include liquid preparations in which an effective amount of ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of ligand as a solid or granule; suspensions in which an effective amount of the active ingredient is suspended in a suitable dispersion medium; and emulsions in which a solution of an effective amount of the active ingredient is dispersed in a suitable dispersion medium and emulsified.

[0309] Pharmaceutical compositions are suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Suitable pharmaceutical compositions for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Also, aqueous and non-aqueous sterile suspensions are examples, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0310] The dosage of a pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the target animal species, the target animal's drug tolerance, body weight, age, etc., but can be set as appropriate.

[0311] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0312] [Example 1] Synthesis of peptide reagent (1-1) Synthesis of peptides The peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 4 were synthesized in solid phase according to the method described in International Publication No. 2018 / 199337 (the same applies to peptide synthesis hereafter). However, the two cysteine ​​residues in the sequence form an intramolecular disulfide bond.

[0313] Sequence ID 1: Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Sequence ID 2: Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Sequence ID 3: Ac-FN(Dab)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Sequence ID 4: Ac-FN(Dap)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２

[0314] (1-2) Synthesis of peptide reagents (1-2-1) Synthesis of peptide reagent (P1-1) Following the previously reported method (WO2019 / 0240287), the Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH synthesized in Example (1-1) ２Peptide reagent (P1-1) was prepared by reacting (SEQ ID NO: 1) with linker (L-1). MS (ESI) m / z: z=4,1209.8 [M+4H] ４＋

[0315] (1-2-2) Synthesis of peptide reagent (P1-2) According to previously reported information (WO2019 / 0240287), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P1-2) was prepared by reacting (SEQ ID NO: 1) with linker (L-2). MS (ESI) m / z: z=4,1217.2 [M+4H] ４＋

[0316] (1-2-3) Synthesis of peptide reagent (P2-1) According to the previously reported (WO2019 / 0240287), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P2-1) was prepared by reacting (SEQ ID NO: 2) with linker (L-1). MS (ESI) m / z: z=4,1206.2 [M+4H] ４＋

[0317] (1-2-4) Synthesis of peptide reagent (P2-2) According to the previously reported (WO2019 / 0240287), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P2-2) was prepared by reacting (SEQ ID NO: 2) with linker (L-2). MS (ESI) m / z: z=5,971.05 [M+5H] ５＋

[0318] (1-2-5) Synthesis of peptide reagent (P3-1) According to the previously reported (WO2019 / 0240287), Ac-FN(Dab)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２Peptide reagent (P3-1) was prepared by reacting (SEQ ID NO: 3) with linker (L-1). MS (ESI) m / z: z=4,11194.2 [M+4H] ４＋

[0319] (1-2-6) Synthesis of peptide reagent (P3-2) According to the previously reported (WO2019 / 0240287), Ac-FN(Dab)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P3-2) was prepared by reacting (SEQ ID NO: 3) with linker (L-2). MS (ESI) m / z: z=4,1210.0.02 [M+4H] ４＋

[0320] (1-2-7) Synthesis of peptide reagent (P4-1) According to the previously reported (WO2019 / 0240287), Ac-FN(Dap)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P4-1) was prepared by reacting (SEQ ID NO: 4) with linker (L-1). MS (ESI) m / z: z=4,1199.2 [M+4H] ４＋

[0321] (1-2-8) Synthesis of peptide reagent (P4-2) According to the previously reported (WO2019 / 0240287), Ac-FN(Dap)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P4-2) was prepared by reacting (SEQ ID NO: 4) with linker (L-2). MS (ESI) m / z: z=4,1133.2 [M+4H] ４＋

[0322] [Example 2] Modification of antibodies using affinity reagents (2-1) Modification of Trastuzumab using affinity reagent P2-2 (2-1-1) Synthesis of Trastuzumab with two molecules of affinity reagent bonded together

[0323] To a solution of 4 mg of the anti-human HER2 monoclonal antibody Trastuzumab (Chugai Pharmaceutical) in borate buffer (20 mM Borate, pH 8.2) (1 mg / mL), a DMF solution of affinity reagent P2-2 synthesized in Example (1-2-4) (10 equivalents relative to the antibody) was added and the mixture was shaken for 1 hour. The reaction solution was purified using Nap25 (Cytiva) to obtain 4 mg of Trastuzumab with two molecules of affinity reagent bound to it (yield 93%). (Note: In the figure above, for simplification, the affinity reagent is shown only on one of the heavy chains.) Following a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was identified at 157497, where two molecules of affinity reagent were bound to Trastuzumab.

[0324] (2-1-2) Synthesis of a Trustzumab containing four thiol groups

[0325] The 4 mg antibody solution obtained in Example (2-1-1) was subjected to a thioester group cleavage reaction (treatment with hydroxylamine) according to a previously reported (WO2019 / 240287) to obtain 3.4 mg of thiol-modified antibody (T-S) with four thiol groups introduced (yield 92%). (Note that in the figure above, for simplification, four thiol groups are shown on one heavy chain side of T-S, but in reality, two thiol groups are attached to each heavy chain, for a total of four thiol groups.) Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using HIC-HPLC analysis to confirm the introduction of four thiol groups.

[0326] (2-1-3) Synthesis of ADC with DAR=4

[0327] To a solution of 3.4 mg of the antibody (T-S) containing four thiol groups obtained in Example (2-1-2) in acetate buffer (20 mM sodium acetate, 10 mM EDTA, pH 5.5), a dimethylacetamide solution (8 equivalents, dimethylacetamide (DMA) (8% v / v) solution) of the known MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) was added, and the mixture was shaken at room temperature for 12 hours. The reaction solution was purified using a NAP-25 desalting column (Cytiva), and 3.2 mg of ADC (T-M) with a DAR of 2-4 was obtained (91%). The analysis followed a previously reported method (Anal. Chem., 2019, 91, 20, 12724-12732), performing ESI-TOFMS analysis. A peak was identified at 154018, where four molecules of the drug were bound to Trastuzumab. (Note that in the T-M diagram above, for simplification, four molecules of the drug are shown on one heavy chain side, but in reality, two molecules of the drug are bound to each heavy chain, for a total of four molecules.)

[0328] (2-2) Modification of Trastuzumab using affinity reagents P1-1, P1-2, P2-1, P3-1, P3-2, P4-1, and P4-2 (2-2-1) Synthesis of Trastuzumab with two molecules of affinity reagent bonded together

[0329] Following Example (2-1-1), Trastuzumab with two molecules of affinity reagent (P1-1, P1-2, P2-1, P3-1, P3-2, P4-1, P4-2) synthesized in Example (1-2) was used to obtain Trastuzumab with two molecules of affinity reagent bound to it. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and the peaks shown in Table 1, indicating that two molecules of affinity reagent were bound to Trastuzumab, were confirmed (note that, for simplification, the affinity reagent is only shown on one of the heavy chains in the figure above).

[0330]

[0331] (2-2-2) Synthesis of Trastuzumab having four thiol groups Following the example (2-1-2), a thiol-modified antibody was obtained from the antibody obtained in Example (2-2-1) with four thiol groups introduced. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that four thiol groups had been introduced into the Trastuzumab.

[0332] (2-2-3) Synthesis of Trastuzumab ADC with DAR=4 Following the examples (2-1-3), an ADC with DAR=4 was obtained using the antibody having four thiol groups obtained in Example (2-2-2) and the known substance MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 154018, where four molecules of the drug were bound to Trastuzumab.

[0333] (2-3) Confirmation of drug / antibody binding ratio of Trastuzumab ADC with DAR=4 using DAR calculator The results of confirming the peptide / antibody binding ratio using DAR calculator (Agilent software) for the MS data analyzed in (2-1-3) are shown in Tables 2 to 9. When affinity reagents P1-1, 1-2, 2-1, 2-2 and 4-2 were used, the average drug / antibody binding ratio calculated from the DAR peak and %Area in Tables 2 to 9 was 3 to 4. Figures 1 to 3 show the analysis results when affinity reagents P1-2, 2-2 and 4-2 were used.

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342] [Comparative Example 1] Synthesis of DAR=4 ADC using two types of affinity reagents (1-1) Synthesis of antibody (Q1) in which two thiol groups are attached to Lys288 of the antibody

[0343] Using 3.2 mg of the anti-human HER2 monoclonal antibody Trastuzumab (Chugai Pharmaceutical) and the peptide reagent (R1) described in the previously published report (WO2022 / 154116A1), 1.89 mg of antibody with two thiol groups introduced was obtained (yield 59%) by following the method described in the previously published report (WO2022 / 154116A1) (Note that in Q1 in the above figure, for simplification, two thiol groups are shown on one of the heavy chains, but in reality, one thiol group is attached to each heavy chain, for a total of two molecules). HIC-HPLC analysis was performed according to the previously published report (Anal. Chem., 2019, 91, 20, 12724-12732) to confirm that two thiol groups had been introduced.

[0344] (1-2) Synthesis of antibody (Q2) in which two molecules of thiol groups are attached to each Lys288 of the antibody.

[0345] Using 1.89 mg of antibody (Q1) synthesized in Comparative Example 1-1 with two thiol groups attached, and the peptide reagent (P11) described in the previously published report (WO2019 / 240287A1), 1.26 mg of antibody with four thiol groups attached was obtained (yield 66%) by following the method described in the previously published report (WO2019 / 240287A1) (Note that in the figure above, for simplification, four thiol groups are shown on one heavy chain side of Q2, but in reality, two thiol groups are attached to each heavy chain, for a total of four molecules). HIC-HPLC analysis was performed according to the previously published report (Anal. Chem., 2019, 91, 20, 12724-12732) to confirm that four thiol groups had been attached.

[0346] (1-3) Synthesis of ADC(Q4) with DAR=4

[0347] Following Example (2-1-3), an ADC with DAR=4 was obtained using 1.26 mg of the antibody containing four thiol groups synthesized in Example 2-1-2 and the known MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported method (Anal. Chem., 2019, 91, 20, 12724-12732), and ESI-TOFMS analysis was performed. A peak was confirmed at 153871, where four molecules of the drug were bound to Trastuzumab (Note that in Q4 in the figure above, for simplification, four molecules of the drug are shown on one heavy chain side, but in reality, two molecules of the drug are bound to each heavy chain, for a total of four molecules).

[0348] [Example 3] Reaction with human IgG antibody using affinity reagent (3-1) Synthesis of DAR=4 ADC of Rituximab (3-1-1) Modification of Rituximab using affinity reagent 2-2 Following Example (2-1-1), Rituximab with two molecules of affinity reagent was obtained by reacting the anti-CD20 monoclonal antibody Rituximab (Zenyaku Kogyo) with a DMF solution of affinity reagent 2-2 synthesized in Example (1-2-4). Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 156728, where two molecules of affinity reagent were bound to Rituximab, was confirmed.

[0349] (3-1-2) Synthesis of Rituximab having four thiol groups: Following the example (2-1-2) of Rituximab having four thiol groups, a thiol-modified antibody was obtained from the antibody obtained in Example (3-1-1) with four thiol groups introduced. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 147868, where four thiol groups were bonded to Rituximab, was confirmed.

[0350] (3-1-3) Synthesis of Rituximab ADC with DAR=4 Following the example (2-1-3), an ADC with DAR=4 was obtained using the antibody having four thiol groups obtained in Example (3-1-2) and the known substance MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 153217, where four molecules of the drug were bound to Rituximab.

[0351] (3-2) Synthesis of Infliximab ADC with DAR=4 (3-2-1) Modification of Infliximab using affinity reagent 2-2 Following the example (2-1-1) of the modification of Infliximab using affinity reagent 2-2, an Infliximab with two molecules of affinity reagent bound was obtained by reacting the anti-human TNF-α monoclonal antibody Infliximab (Tanabe Mitsubishi Pharma) with a DMF solution of affinity reagent 2-2 synthesized in Example (1-2-4). Following the previously reported (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 158260, where two molecules of affinity reagent were bound to Infliximab, was confirmed.

[0352] (3-2-2) Synthesis of Infliximab having four thiol groups Following the example (2-1-2) of Infliximab, a thiol-modified antibody was obtained from the antibody obtained in Example (3-2-1) with four thiol groups introduced. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 149415, where four thiol groups were bonded to Infliximab, was confirmed.

[0353] (3-2-3) Synthesis of Infliximab ADC with DAR=4 Following the example (2-1-3), an ADC with DAR=4 was obtained using the antibody having four thiol groups obtained in Example (3-2-2) and the known substance MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 154618, where four molecules of the drug were bound to Infliximab.

[0354] (3-3) Synthesis of Pembrolizumab ADC with DAR=4 (3-3-1) Modification of Pembrolizumab using affinity reagent 2-2 Following the example (2-1-1) of the modification of Pembrolizumab using affinity reagent 2-2, a Pembrolizumab with two molecules of affinity reagent bound was obtained by reacting the anti-human PD-1 monoclonal antibody Pembrolizumab (MSD) with a DMF solution of affinity reagent 2-2 synthesized in Example (1-2-4). Following the previously reported (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 158321, where two molecules of affinity reagent were bound to Pembrolizumab, was confirmed.

[0355] (3-3-2) Synthesis of Pembrolizumab having four thiol groups: Following the example (2-1-2), a thiol-modified antibody was obtained from the antibody obtained in Example (3-3-1) with four thiol groups introduced. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 149440, where four thiol groups were bonded to Pembrolizumab, was confirmed.

[0356] (3-3-3) Synthesis of Pembrolizumab ADC with DAR=4 Following the synthesis examples (2-1-3), an ADC with DAR=4 was obtained using the antibody having four thiol groups obtained in Example (3-3-2) and the known substance MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to previously reported data (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 154715, where four molecules of the drug were bound to Pembrolizumab.

[0357] [Example 4] Evaluation of aggregation rate by size exclusion chromatography (SEC-HPLC) Table 10 shows the results of evaluating the aggregation rate of DAR=4 ADC synthesized in Example (2-1-3) and Comparative Example (1-3) by size exclusion chromatography (SEC-HPLC).

[0358]

[0359] The ADC(T-M) synthesized in Example 2-1-3 was found to have a lower aggregation rate compared to the ADC(Q4) synthesized in Comparative Example 1-3.

[0360] [Example 5] Comparison of synthesis yield of DAR=4 ADC Table 11 shows a comparison of the number of steps and synthesis yield of DAR=4 ADC synthesized in Example (2-3-1) and Comparative Example (1-3). As this result shows, it was confirmed that the DAR=4 ADC synthesized with affinity reagent 2-2 had fewer steps and a higher yield.

[0361]

[0362] [Example 6] Synthesis of Peptide Reagents (6-1) Peptide Synthesis The peptides consisting of the amino acid sequences of SEQ ID NOs. 8 to 10 were synthesized in solid phase according to the method described in International Publication No. 2018 / 199337 (the same applies to peptide synthesis hereafter). However, the two cysteine ​​residues in the sequence form an intramolecular disulfide bond.

[0363] Sequence ID 8: Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH２ Sequence ID 9: Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Sequence ID 10: Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH ２

[0364] (6-2) Synthesis of peptide reagents (6-2-1) Synthesis of peptide reagent (P5-1) According to the previously reported (WO2019 / 0240287), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH was synthesized in Example (6-1). ２ Peptide reagent (P5-1) was prepared by reacting (SEQ ID NO: 1) with linker (L-1). MS (ESI) m / z: z=4,1206.2 [M+4H] ４＋

[0365] (6-2-2) Synthesis of peptide reagent (P5-2) According to the previously reported (WO2019 / 0240287), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH synthesized in Example (6-1) ２ Peptide reagent (P5-2) was prepared by reacting (SEQ ID NO: 1) with linker (L-2). MS (ESI) m / z: z=5,971.05 [M+5H] ５＋

[0366] (6-2-3) Synthesis of peptide reagent (P6-1) According to the previously reported (WO2019 / 0240287), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Peptide reagent (P6-1) was prepared by reacting (SEQ ID NO: 9) with linker (L-1). MS (ESI) m / z: z=4,1202.6 [M+4H] ４＋

[0367] (6-2-4) Synthesis of peptide reagent (P6-2) According to the previously reported (WO2019 / 0240287), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Peptide reagent (P6-2) was prepared by reacting (SEQ ID NO: 9) with linker (L-2). MS (ESI) m / z: z=4,1209.9 [M+4H] ４＋

[0368] (6-2-5) Synthesis of peptide reagent (P7-1) According to the previously reported (WO2019 / 0240287), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH ２ Peptide reagent (P7-1) was prepared by reacting (SEQ ID NO: 10) with linker (L-1). MS (ESI) m / z: z=4,1199.2 [M+4H] ４＋

[0369] (6-2-6) Synthesis of peptide reagent (P7-2) According to the previously reported (WO2019 / 0240287), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKDDC-NH ２ Peptide reagent (P7-2) was prepared by reacting (SEQ ID NO: 10) with linker (L-2). MS (ESI) m / z: z=4,1206.5 [M+4H] ４＋

[0370] (6-2-7) Synthesis of peptide reagent (P1-3) According to the previously reported (WO2022 / 191283), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P1-3) was prepared by reacting (SEQ ID NO: 1) with linker (L-3). MS (ESI) m / z: z=4,1280.2 [M+4H] ４＋

[0371] (6-2-8) Synthesis of peptide reagent (P1-4) According to the previously reported (WO2022 / 191283), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P1-4) was prepared by reacting (SEQ ID NO: 1) with linker (L-4). MS (ESI) m / z: z=4,1328.8 [M+4H] ４＋

[0372] (6-2-9) Synthesis of peptide reagent (P2-3) According to the previously reported (WO2022 / 191283), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P2-3) was prepared by reacting (SEQ ID NO: 2) with linker (L-3). MS (ESI) m / z: z=4,1276.7 [M+4H] ４＋

[0373] (6-2-10) Synthesis of peptide reagent (P2-4) According to the previously reported (WO2022 / 191283), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSIKEEC-NH ２ Peptide reagent (P2-4) was prepared by reacting (SEQ ID NO: 2) with linker (L-4). MS (ESI) m / z: z=4,1325.2 [M+4H] ４＋

[0374] (6-2-11) Synthesis of peptide reagent (P5-3) According to the previously reported (WO2022 / 191283), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Peptide reagent (P5-3) was prepared by reacting (SEQ ID NO: 8) with linker (L-3). MS (ESI) m / z: z=4,1276.7 [M+4H] ４＋

[0375] (6-2-12) Synthesis of peptide reagent (P5-4) According to the previously reported (WO2022 / 191283), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Peptide reagent (P5-4) was prepared by reacting (SEQ ID NO: 8) with linker (L-4). MS (ESI) m / z: z=4,1325.2 [M+4H] ４＋

[0376] (6-2-13) Synthesis of peptide reagent (P6-3) According to the previously reported (WO2022 / 191283), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Peptide reagent (P6-3) was prepared by reacting (SEQ ID NO: 9) with linker (L-3). MS (ESI) m / z: z=4,1273.2 [M+4H] ４＋

[0377] (6-2-14) Synthesis of peptide reagent (P6-4) According to the previously reported (WO2022 / 191283), Ac-FN(Orn)QCQRRFYEALHDPNLNEEQRNARIRSI(Orn)EEC-NH ２ Peptide reagent (P6-4) was prepared by reacting (SEQ ID NO: 9) with linker (L-4). MS (ESI) m / z: z=4,1321.7 [M+4H] ４＋

[0378] [Example 7] Modification of antibodies using affinity reagents (7-1) Modification of Trastuzumab using affinity reagent P5-2 (7-1-1) Synthesis of Trastuzumab with two molecules of affinity reagent bonded together

[0379] To a borate buffer solution (20 mM Borate, pH 8.2) (1 mg / mL) of the anti-human HER2 monoclonal antibody Trastuzumab (Chugai Pharmaceutical), a DMF solution of affinity reagent P5-2 synthesized in Example (6-2-2) (10 equivalents relative to the antibody) was added and the mixture was shaken for 1 hour. By purifying the reaction solution, Trastuzumab with two molecules of affinity reagent bound to it was obtained. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 157506, where two molecules of affinity reagent were bound to Trastuzumab, was confirmed.

[0380] (7-1-2) Synthesis of a Trustzumab containing four thiol groups

[0381] The antibody solution obtained in Example (7-1-1) was subjected to a thioester group cleavage reaction (treatment with hydroxylamine) according to a previously reported (WO2019 / 240287) to obtain a thiol-modified antibody (T-S) with four thiol groups introduced. Analysis was performed by HIC-HPLC analysis according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) to confirm the introduction of four thiol groups.

[0382] (7-1-3) Synthesis of ADC with DAR=4

[0383] To a solution of the antibody (T-S) containing four thiol groups obtained in Example (7-1-2) in acetate buffer (20 mM sodium acetate, 10 mM EDTA, pH 5.5), a dimethylacetamide solution (8 equivalents, dimethylacetamide (DMA) (8% v / v) solution) of the known MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) was added, and the mixture was shaken at room temperature for 12 hours. The reaction solution was purified using a NAP-25 desalting column (Cytiva), and ADCs (T-M) with DAR = 2 to 4 were obtained. Following a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was identified at 153890, where four molecules of the drug were bound to Trastuzumab.

[0384] (7-2) Modification of Trastuzumab using affinity reagents P5-1, P6-1, and P6-2 (7-2-1) Synthesis of Trastuzumab with two molecules of affinity reagent bonded together

[0385] Following Example (2-1-1), Trastuzumab with two molecules of affinity reagent (P5-1, P6-1, and P6-2) synthesized in Example (6-2) was used to obtain Trastuzumab with two molecules of affinity reagent bound to it. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and the peaks shown in Table 12, indicating that two molecules of affinity reagent were bound to Trastuzumab, were confirmed.

[0386]

[0387] (7-2-2) Synthesis of Trastuzumab having four thiol groups Following the example (2-1-2) of Trastuzumab, a thiol-modified antibody was obtained from the antibody obtained in Example (7-2-1) with four thiol groups introduced. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that four thiol groups had been introduced into the Trastuzumab.

[0388] (7-2-3) Synthesis of Trastuzumab ADC with DAR=4 Following the example (2-1-3), a DAR=4 ADC was obtained using the antibody having four thiol groups obtained in Example (7-2-2) and the known substance MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 153890, where four molecules of the drug were bound to Trastuzumab.

[0389] (7-3) Confirmation of drug / antibody binding ratio of Trastuzumab ADC with DAR=4 using DAR calculator The results of confirming the peptide / antibody binding ratio using DAR calculator (Agilent software) for the MS data analyzed in (7-1-3) are shown in Tables 13-14. When affinity reagents P5-2 and 6-2 were used, the average drug / antibody binding ratio calculated from the DAR peak and %Area in Tables 13-14 was 3-4.

[0390]

[0391]

[0392] (7-4) Modification of Trastuzumab using affinity reagent P2-4 (7-4-1) Synthesis of Trastuzumab with two molecules of affinity reagent bonded together

[0393] To a borate buffer solution (20 mM Borate, pH 8.2) (1 mg / mL) of the anti-human HER2 monoclonal antibody Trastuzumab (Chugai Pharmaceutical), a DMF solution of affinity reagent P2-4 synthesized in Example (6-2-2) (10 equivalents relative to the antibody) was added and the mixture was shaken for 1 hour. By purifying the reaction solution, Trastuzumab with two molecules of affinity reagent bound was obtained. Following a previously reported study (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak at 158621, where two molecules of affinity reagent were bound to Trastuzumab, was confirmed.

[0394] (7-4-2) Synthesis of a Trustzumab having four azide groups

[0395] The antibody solution obtained in Example (7-4-1) was subjected to a thioester group cleavage reaction (treatment with methoxyamine) according to a previously reported (WO2022 / 191283) to obtain an azide-modified antibody (T-A) with four azide groups introduced. Analysis was performed by ESI-TOFMS analysis according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was confirmed at 149047, where two molecules of affinity reagent were bound to Trastuzumab.

[0396] (7-4-3) Synthesis of ADC with DAR=4

[0397] In Example (7-4-2), an antibody (T-A) containing four azide groups was obtained. To this solution, an acetate buffer solution (20 mM sodium acetate, 10 mM EDTA, pH 5.5) was added to a dimethylacetamide solution of the known DBCO-PEG4-MMAF (8 equivalents, dimethylacetamide (DMA) (8% v / v) solution), and the mixture was shaken at room temperature for 12 hours. The reaction solution was purified using a NAP-25 desalting column (Cytiva), and ADCs (T-M) with DAR = 2-4 were obtained. Analysis was performed according to a previously reported method (Anal. Chem., 2019, 91, 20, 12724-12732), and ESI-TOFMS analysis was performed. A peak was identified at 155276, where four molecules of the drug were bound to Trastuzumab.

[0398] [Comparative Example 2] Synthesis of DAR=4 ADC using two types of affinity reagents (2-1) Peptide synthesis The peptide consisting of the amino acid sequences of SEQ ID NOs. 11-12 was synthesized in solid phase according to the method described in International Publication No. 2018 / 199337 (the same applies to peptide synthesis hereafter). However, the two cysteine ​​residues in the sequence form an intramolecular disulfide bond.

[0399] Sequence ID 11: Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC-NH ２Sequence ID 12: Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH ２

[0400] (2-2) Synthesis of peptide reagents (2-2-1) Synthesis of peptide reagent (P12) Following the previously reported method (WO2019 / 0240287), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIREEC-NH was synthesized in comparative example (2-1). ２ Peptide reagent (P12) was prepared by reacting (SEQ ID NO: 11) with linker (L-1).

[0401]

[0402] (2-2-2) Synthesis of peptide reagent (P13) According to the previously reported (WO2019 / 0240287), Ac-FNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-NH synthesized in comparative example (2-1) ２ Peptide reagent (P13) was prepared by reacting (SEQ ID NO: 12) with linker (L-1).

[0403] (2-3) Synthesis of DAR=4 ADC using two types of affinity reagents (2-3-1) Synthesis of antibody (Q2) in which two molecules each of thiol groups are attached to Lys248 and 288 of the antibody

[0404] Using the antibody (Q1) synthesized in Comparative Example 1-1 with two thiol groups attached, and the peptide reagent (P12), an antibody with four thiol groups introduced was obtained by following the method described in a previous report (WO2019 / 240287A1). (Note that in the figure above, Q2 shows four thiol groups on one heavy chain for simplification, but in reality, two thiol groups are attached to each heavy chain, for a total of four molecules.) Following a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that four thiol groups had been introduced.

[0405] (2-3-2) Synthesis of ADC(Q4) with DAR=4

[0406] Following Example (2-1-3), an ADC with DAR=4 was obtained using the antibody having four thiol groups synthesized in Comparative Example 2-3-1 and the known MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 153871, where four molecules of the drug were bound to Trastuzumab (Note that in Q4 in the figure above, for simplification, four molecules of the drug are shown on one heavy chain side, but in reality, two molecules of the drug are bound to each heavy chain, for a total of four molecules).

[0407] (2-3-3) Synthesis of antibody (Q2) in which two molecules each of thiol groups are attached to Lys248 and 288 of the antibody.

[0408] Using the antibody (Q1) with two thiol groups attached, synthesized in Comparative Example 1-1, and the peptide reagent (P13), an antibody with four thiol groups attached was obtained by following the method described in a previous report (WO2019 / 240287A1). (Note that in the figure above, Q2 shows four thiol groups attached to one of the heavy chains for simplification, but in reality, two thiol groups are attached to each heavy chain, for a total of four molecules.) Following a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that four thiol groups had been attached.

[0409] (2-3-4) Synthesis of ADC(Q4) with DAR=4

[0410] Following Example (2-1-3), an ADC with DAR=4 was obtained using the antibody having four thiol groups synthesized in Comparative Example 2-3-3 and the known MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518). Analysis was performed according to a previously reported (Anal. Chem., 2019, 91, 20, 12724-12732) using ESI-TOFMS analysis, and a peak was confirmed at 153871, where four molecules of the drug were bound to Trastuzumab (Note that in Q4 in the figure above, for simplification, four molecules of the drug are shown on one heavy chain side, but in reality, two molecules of the drug are bound to each heavy chain, for a total of four molecules).

[0411] [Example 8] Evaluation of aggregation rate by size exclusion chromatography (SEC-HPLC) Table 15 shows the results of evaluating the aggregation rate of DAR=4 ADC synthesized in Example (2-1-3) and Comparative Example (2-3) by size exclusion chromatography (SEC-HPLC).

[0412]

[0413] The ADC(T-M) synthesized in Example 2-1-3 was found to have a lower aggregation rate compared to the ADC(Q4) synthesized in Comparative Example 2-3.

[0414] [Example 9] Comparison of synthesis yield of DAR=4 ADC Table 16 shows a comparison of the number of steps and synthesis yield of DAR=4 ADC synthesized in Example (2-3-1) and Comparative Example (2-3). As this result shows, it was confirmed that the DAR=4 ADC synthesized with affinity reagent 2-2 had fewer steps and a higher yield.

[0415]

[0416] [Example 10] Peptide mapping of a Trastuzumab thiol derivative prepared using an antibody affinity substance (P2-2) by trypsin treatment (10-1) Trypsin treatment of a Trastuzumab thiol derivative Peptide mapping was performed on the Trastuzumab thiol derivative obtained in Example (2-1-2) above. The sample solution and 0.25 M Tris-HCl buffer (pH 7.5) / 7.5 M guanidine hydrochloride were added to a 0.6 mL low-adsorption microtest tube so that the sample volume and liquid volume were 10 μg / 77.5 μL. 1.5 μL of 500 mM dithiothreitol aqueous solution was added and the mixture was heated at room temperature for 30 minutes, then 3.5 μL of 500 mM iodoacetamide aqueous solution was added and the mixture was reacted at room temperature for 30 minutes under light protection. After the reaction, 2 μL of 500 mM dithiothreitol aqueous solution was added to quench the iodoacetamide. The buffer was replaced with 1 M Tris-HCl buffer (pH 7.5) using a desalting column, and 5 μL of 400 ng / μL trypsin / Lys-C aqueous solution was added, followed by enzymatic digestion at 37°C for 1 hour. After digestion, the reaction was stopped by adding 1% formic acid / 2% acetonitrile to a final concentration, and the solution was diluted 10-fold with 0.1% formic acid / 2% acetonitrile aqueous solution and subjected to LC-MS / MS analysis.

[0417] (10-2) LC-MS / MS measurement conditions for Trastuzumab (Analytical instrument) Nano HPLC: Vanquish Neo (Thermo Fisher Scientific) Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

[0418] (HPLC analysis conditions) Trap column: Acclam PepMap (registered trademark) 100, 75 μm x 2 cm (Thermo Fisher Scientific) Analysis column: ESI-column (NTCC-360 / 75-3-125, 75 μm x 12.5 cm, 3 μm (Nikkyo Technos Co., Ltd.)) Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, 80% acetonitrile solution Flow rate: 300 nL / min Sample injection volume: 2 μL Gradient conditions (B%): 0% → 2.5% (0.0-0.5 min), 2.5% → 44% (0.5-28.0 min), 44% → 95% (28.0-28.5 min), 95% (28.5-33.0 min)

[0419] (Mass Spectrometer Analysis Conditions) Ionization Method: ESI, Positive Mode Scan Type: Data Dependent Aquisition Activation Type: Higher Energy Collisional Dissociation (HCD) Data acquisition was performed using the accompanying software Xcalibur 4.5 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 3.5 (Thermo Fisher Scientific).

[0420] (10-3) Analysis conditions for the modification site of Trastuzumab The analysis of the modification site of the LC-MS / MS measurement results was performed using BioPharma Finder 5.1 (Thermo Fisher Scientific).

[0421] The analysis using BioPharma Finder was performed with the S / N Threhold set to 224 and the MS Noise Level set to 4000. The digestive enzyme was set to Trypsin, and the Specification was set to High. For Static Modification, Carbamidemethyl (+57.021 Da) was selected as the modification of cysteine ​​residues by iodoacetamide. For Dynamic Modifications, we defined oxidation of methionine residues (+15.995 Da), addition of lysine residues to the C-terminus (+128.095), deamidation of asparagine and glutamine residues (+0.984 Da), and a modified lysine residue (thiol introduced after carboxymethylation with iodoacetamide (+145.019 Da)). Furthermore, we set filters to include only those with a Confidence Score of 80 or higher, a Mass Accuracy of 5 ppm or less during peptide identification, and for which MS / MS was observable. Regarding the residue numbers of lysine residues, those on the heavy chain VH domain and light chain are denoted by the sequence number (i.e., the N-terminal amino acid is considered the first; the same applies hereafter), while those on the heavy chain CH1, CH2, and CH3 domains are denoted using EU numbering.

[0422] Furthermore, the following SEQ ID NOs. 17 and SEQ ID NOs. 18 were used as data for the amino acid sequences targeted for searching for modified sites. (Sequence ID 17: Heavy chain amino acid sequence of Trastuzumab) EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG (Sequence ID 18: Light chain amino acid sequence of Trastuzumab) DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYQQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[0423] (10-4) Analysis of the modification site of Trastuzumab by LC-MS / MS Analysis using LC-MS / MS revealed that the MS spectrum (measured value: m / z 952.23187, theoretical value: 952.22942, quadrivalent) of the peptide fragment THTCPPCPAPELLGGGPSVFLFPPKPKDTLMISR (SEQ ID NO: 15), consisting of 33 amino acid residues, including the modification site of the lysine residue by trypsin digestion of Trastuzumab (thiol introduced product that has undergone carboxylation with iodoacetamide (+145.019Da)), was observed (Figure 4). The HCD spectrum showed modification of the lysine residue at position 248 in the EU numbering of the heavy chain, corresponding to the divalent y20 m / z A product ion of 1167.12 (theoretical value: 1166.61) was confirmed (Figure 5). In addition, the MS spectrum (measured value: m / z 577.03757, theoretical value: 577.03557, tetravalent) of the peptide fragment FNWYVDGVEVHNAKTKPR (SEQ ID NO: 16), which consists of 18 amino acid residues including a modification site to the lysine residue by trypsin digestion of Trastuzumab (a thiol-introduced product that has undergone carboxylation with iodoacetamide (+145.019Da)), was observed (Figure 6). From the HCD spectrum, a product ion of m / z 798.40 (theoretical value: 798.40), corresponding to divalent y13, indicating modification of the lysine residue at position 288 in the EU numbering of the heavy chain, was confirmed (Figure 7). Furthermore, analysis using BioPharma Finder showed that modifications to the lysine residues at positions 246 or 248 and at positions 288 or 290 occurred with high selectivity (Figure 8). This result indicates that conjugation is progressing regioselectively to Lys248 and Lys288 in the EU numbering on the antibody heavy chain.

Claims

1. A compound or salt thereof comprising (A) an affinity polypeptide having an affinity moiety having affinity for the CH2-CH3 intercellular region of an antibody heavy chain, (B) a first antibody-reactive group, and (C) a second antibody-reactive group, wherein the affinity polypeptide comprises first and second amino acid residues having side-chain amino groups, and the first and second antibody-reactive groups are linked to the affinity polypeptide via linkers introduced to the side-chain amino groups of the first and second amino acid residues having side-chain amino groups, respectively.

2. The compound or salt thereof according to claim 1, wherein the first and second antibody-reactive groups are groups that are reactive to the side-chain amino groups of lysine residues contained in the polypeptide chain of the antibody.

3. The affinity polypeptide has an amino acid sequence in which two amino acid residues are each replaced by amino acid residues having a side-chain amino group in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence, according to claim 1, or a salt thereof. SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC 4. The compound or salt thereof according to claim 3, wherein the two amino acid residues are either the 32nd E or D, the 31st A or the 30th I, and either the 4th Q, the 3rd A or the 2nd N.

5. The compound or salt thereof according to claim 3, wherein the two amino acid residues are the 31st A and the 3rd A.

6. The compound or salt thereof according to claim 1, wherein the first and second amino acid residues having a side chain amino group are selected from a lysine residue, an ornithine residue, a diaminobutanoic acid residue, and a diaminopimelic acid residue, respectively.

7. The compound or salt thereof according to claim 1, wherein the heavy chain CH2-CH3 intercellular region is a human heavy chain CH2-CH3 intercellular region.

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

9. The compound or salt thereof according to claim 8, wherein the CH2-CH3 intercellular region is the region from position 230 to 439 of the human IgG heavy chain.

10. The compound or salt thereof according to claim 8, wherein the CH2-CH3 intercellular region is the region from position 248 to 288 of the human IgG heavy chain.

11. The compound is of the following formula (I): R １ -L １ -A-L ２ -R ２ (I) [wherein, R １ and R ２ L represents the reactive group, １ This indicates the first linker, L ２ The compound or a salt thereof according to claim 1, represented by [ ], wherein represents the second linker, and A represents the affinity polypeptide.

12. The compound or salt thereof according to claim 1, wherein the compound or salt thereof further comprises (i) the affinity polypeptide and (ii) the reactive group, and (iii) a cleavable moiety between them.

13. The compound or salt thereof according to claim 12, wherein the cleavable portion is a cleavable portion capable of generating a bioorthogonal functional group on the reactive group side by cleavage.

14. The compound is represented by the following formula (Ia): R １ -L3-CLE(B) １ -L4-A-L5-CLE(B) ２ -L6-R ２ (Ia) [In the formula, R １ , R ２ each represent the reactive group, L ３ represents the third linker, L ４ represents the fourth linker, L ５ represents the fifth linker, L ６ represents the sixth linker, CLE(B) １ , CLE(B) ２ each represent a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side by cleavage, and A represents the affinity polypeptide.], the compound according to claim 13 or a salt thereof.

15. The compound is given by the following formula (Ia-1): [In the formula, X １ , X ２ This indicates a leaving group, W １ , W ２ , W ３ , W ４ , W ５ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L ８ This indicates the eighth linker, L ９ This indicates the 9th linker, L １０ The compound or a salt thereof according to claim 14, represented as follows: where represents the 10th linker, S represents a sulfur atom, and A represents the affinity polypeptide.

16. The compound or salt of claim 15, wherein the leaving group is selected from the following: (a) R Ａ -S (here, R Ａ (b) R Ａ -O (Here, R Ａ (c)R Ａ - (R Ｂ -) N (Here, R Ａ and R Ｂ (d) A halogen atom.

17. The compound or salt thereof according to claim 12, wherein the compound or salt thereof further comprises (ii) a bioorthogonal functional group between (iv) the reactive group and (iii) the cleavable moiety.

18. The compound is given by the following formula (Ib): [In the formula, R １ , R ２ L represents the reactive group, １１ This indicates the 11th linker, L １２ This indicates the 12th linker, L １３ This indicates the 13th linker, L １４ This indicates the 14th linker, B １ , B ２ This indicates a group containing a bioorthogonal functional group, CLE １ CLE ２ The compound or a salt thereof according to claim 17, represented as follows: where indicates a cleavable portion, and A indicates the affinity polypeptide.

19. The compound is given by the following formula (Ib-1): [In the formula, X ３ , X ４ This indicates a leaving group, W ７ , W ８ , W ９ , W １０ , W １１ and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １６ This indicates the 16th linker, L １７ This indicates the 17th linker, L １８ This indicates the 18th linker, B ３ , B ４ This indicates a group containing a bioorthogonal functional group, V １ , V ２ The compound or a salt thereof according to claim 18, represented as [wherein is an oxygen atom or a sulfur atom, and A is the affinity polypeptide].

20. The compound or salt thereof according to claim 13, wherein the bioorthogonal functional group is an azide residue, an alkyne residue, a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue.

21. A reagent for antibody derivatization comprising a compound or a salt thereof according to any one of claims 1 to 20.

22. An affinity polypeptide-modified antibody or a salt thereof, comprising an affinity polypeptide having an affinity moiety for the CH2-CH3 intercellular region of the heavy chain of the antibody, wherein the affinity polypeptide is introduced into the CH2-CH3 intercellular region of the heavy chain by modification of amino groups in the side chains of amino acid residues having side chain amino groups located at two positions in the CH2-CH3 intercellular region of the heavy chain.

23. The affinity polypeptide-modified antibody or salt thereof according to claim 22, comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) the affinity polypeptide, and (c) the affinity polypeptide being introduced into the CH2-CH3 inter-heavy chain region of the immunoglobulin unit by modification of amino groups in the side chains of lysine residues located at two positions in the CH2-CH3 inter-heavy chain region of the heavy chain.

24. The affinity polypeptide-modified antibody or salt thereof according to claim 23, wherein two locations in the CH2-CH3 interregion of the heavy chain are K248 and K288 of a human IgG heavy chain according to EU numbering.

25. The affinity polypeptide modified antibody or salt thereof according to claim 22, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are replaced with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence, and is modified to the amino groups in the side chains of the amino acid residues having side-chain amino groups located in the two CH2-CH3 inter-heavy chain regions via a linker introduced to the side-chain amino groups of said amino acid residues. SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC 26. Affinity polypeptide-modified antibody is expressed by the following formula (II): [In the formula, Ig represents an immunoglobulin unit including the heavy chain and, optionally, the light chain, L １ This indicates the first linker, L ２ A represents a second linker, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody or a salt thereof according to claim 22, comprising a structural unit represented by [ ].

27. The affinity polypeptide-modified antibody or salt thereof according to claim 26, wherein the antibody or salt thereof further comprises (i') the affinity polypeptide and (ii') the antibody, and (iii') a cleavable moiety between them.

28. The affinity polypeptide-modified antibody or salt thereof according to claim 27, wherein the cleavable portion is a cleavable portion capable of generating a bioorthogonal functional group on the immunoglobulin unit side by cleavage.

29. Affinity polypeptide-modified antibody is given by the following formula (IIa): [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ４ This indicates the fourth linker, L ５ This indicates the fifth linker, L ６ This indicates the sixth linker, CLE(B) １ and CLE(B) ２ A represents a cleavable portion that can generate a bioorthogonal functional group on the immunoglobulin unit side by cleavage, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody or salt thereof according to claim 28, comprising a structural unit represented by [ ].

30. Affinity polypeptide-modified antibody is given by the following formula (IIa-1): [In the formula, Ig represents the immunoglobulin unit, W １ , W ２ , W ３ , W ４ , W ５ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L ８ This indicates the eighth linker, L ９ This indicates the 9th linker, L １０ The affinity polypeptide-modified antibody or a salt thereof according to claim 29, comprising a structural unit represented by , wherein represents a 10th linker, S represents a sulfur atom, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%.

31. The affinity polypeptide-modified antibody or salt thereof according to claim 27, further comprising (ii') a bioorthogonal functional group between the antibody and (iii') a cleavable moiety.

32. The affinity polypeptide-modified antibody has the following formula (IIb): [In the formula, Ig represents the immunoglobulin unit, L １１ represents the 11th linker, L １２ represents the 12th linker, L １３ represents the 13th linker, L １４ represents the 14th linker, B １ and B ２ represent a group containing a bioorthogonal functional group, CLE １ and CLE ２ represent a cleavable moiety, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%.]. The affinity polypeptide-modified antibody or a salt thereof according to claim 31, which contains a structural unit represented by the formula.

33. The affinity polypeptide-modified antibody has the following formula (IIb-1): [wherein, Ig represents the immunoglobulin unit, and W ７ , W ８ , W ９ , W １０ , W １１ and W １２ each independently represents an oxygen atom or a sulfur atom, L １５ represents a 15th linker, L １６ represents a 16th linker, L １７ represents a 17th linker, L １８ represents a 18th linker, B ３ and B ４ represent groups containing a bioorthogonal functional group, V １ and V ２ each independently represents an oxygen atom or a sulfur atom, A represents the affinity polypeptide, and the average modification percentage r per heavy chain in the immunoglobulin unit by the affinity polypeptide is 65 to 135%.]. The affinity polypeptide-modified antibody or a salt thereof according to claim 32, which contains a structural unit represented by the above formula.

34. A method for producing an affinity polypeptide-modified antibody or a salt thereof, comprising reacting the antibody derivatization reagent described in claim 21 with an antibody comprising an immunoglobulin unit comprising a heavy chain and, optionally, a light chain, to produce an affinity polypeptide-modified antibody or a salt thereof comprising the affinity polypeptide in the CH2-CH3 interregion of the heavy chain in the immunoglobulin unit.

35. A method for producing an antibody or salt that does not contain an affinity polypeptide, comprising (A) an affinity polypeptide having an affinity moiety having affinity for the CH2-CH3 interchain region of the antibody, and (B) an antibody, wherein (C) the affinity polypeptide-modified antibody or a salt thereof, wherein (A) the affinity polypeptide and (B) the antibody are linked by first and second linkers, and the first and second linkers each have first and second cleavable moieties, respectively, and the antibody or salt thereof is cleaved at the first and second cleavable moieties to produce an antibody or salt thereof that does not contain an affinity polypeptide.

36. The method for producing the affinity polypeptide according to claim 35, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are substituted with amino acid residues having a side chain amino group in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence. SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC 37. The method according to claim 35, wherein the first and second cleavable portions are cleavable portions that can generate bioorthogonal functional groups on the antibody side by cleavage, and the antibody or salt thereof that does not contain affinity polypeptides is an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain.

38. The method according to claim 37, wherein the antibody derivative or salt thereof, which contains two bioorthogonal functional groups per heavy chain, comprises a structural unit represented by the following formula (IIIa) or formula (IIIa-1). [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ６ This indicates the sixth linker, B ５ and B ６ This indicates a group containing a bioorthogonal functional group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ L represents an oxygen atom or a sulfur atom. ７ This indicates the seventh linker, L １０ [where represents the 10th linker, SH represents a thiol group, and the average modification percentage r per heavy chain in the immunoglobulin unit by bioorthogonal functional groups is 65-135%.] 39. The method according to claim 35, wherein the antibody or salt thereof that does not contain affinity polypeptide further comprises first and second bioorthogonal functional groups between the antibody and the first and second cleavable moieties, and the antibody or salt thereof that does not contain affinity polypeptide is an antibody derivative or salt thereof that contains two bioorthogonal functional groups per heavy chain.

40. The method according to claim 39, wherein the antibody derivative or salt thereof, which contains two bioorthogonal functional groups per heavy chain, comprises a structural unit represented by the following formula (IIIb) or the following formula (IIIb-1). [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, B １ and B ２ This indicates a group containing a bioorthogonal functional group, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, B ３ and B ４ This indicates a group containing a bioorthogonal functional group, T ３ and T ４ This represents a monovalent group, and the modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%.

41. Methods for producing an antibody and a conjugate or salt thereof containing a functional substance, comprising the following (1) and (2): (1) producing an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain by the method described in claim 37; and (2) reacting an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain with a functional substance to produce an antibody and a conjugate or salt thereof containing two functional substances per heavy chain.

42. The method according to claim 41, wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (IVa) or (IVa-1). [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ６ This indicates the sixth linker, Z １ and Z ２ This indicates a functional substance, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L １０ This indicates the 10th linker, Z １ and Z ２ This indicates a functional substance, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%.

43. A method for producing an antibody and a conjugate containing a functional substance or a salt thereof, comprising (1) and (2) below, wherein the conjugate contains an antibody and a functional substance, comprising two functional substances per heavy chain: (1) producing an antibody derivative or a salt thereof comprising two bioorthogonal functional groups per heavy chain of the antibody by the method described in claim 39; and (2) reacting an antibody derivative or a salt thereof comprising two bioorthogonal functional groups per heavy chain of the antibody with a functional substance to produce an antibody and a conjugate containing a functional substance or a salt thereof, comprising two functional substances per heavy chain.

44. The method according to claim 43, wherein the conjugate or salt thereof comprises a structural unit represented by the following formula (IVb) or (IVb-1). [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, Z １ and Z ２ This indicates a functional substance, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, Z １ and Z ２ This indicates a functional substance, T ３ and T ４ This represents a monovalent group, and the modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%.

45. A method for producing an antibody and functional substance conjugate or salt thereof, comprising reacting an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain with a functional substance to produce an antibody and functional substance conjugate or salt thereof containing two functional groups per heavy chain, wherein the antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain is an antibody derivative or salt thereof comprising (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) first and second bioorthogonal functional groups, and (c) the first and second bioorthogonal functional groups introduced into the CH2-CH3 interregion of the heavy chain in the immunoglobulin unit. A method comprising an antibody and functional substance conjugate or salt thereof, wherein the conjugate contains two functional substances per heavy chain, and the conjugate contains (a) an immunoglobulin unit comprising two heavy chains and optionally two light chains, and (b) two functional substances per heavy chain of the antibody, and (c) the functional substances are introduced at two locations in the CH2-CH3 interregion of the heavy chain in the immunoglobulin unit.

46. ​​The method according to claim 45, wherein an antibody derivative or salt thereof containing two bioorthogonal functional groups per heavy chain of the antibody has a structural unit represented by the following formula (IIIa), (IIIa-1), (IIIb) and / or (IIIb-1). [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ６ This indicates the sixth linker, B ５ and B ６ This indicates a group containing a bioorthogonal functional group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ L represents an oxygen atom or a sulfur atom. ７ This indicates the seventh linker, L １０ [where represents the 10th linker, SH represents a thiol group, and the average modification percentage r per heavy chain in the immunoglobulin unit by bioorthogonal functional groups is 65-135%.] [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, B １ and B ２ This indicates a group containing a bioorthogonal functional group, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, B ３ and B ４ This indicates a group containing a bioorthogonal functional group, T ３ and T ４ This represents a monovalent group, and the modification percentage r per heavy chain in the immunoglobulin unit by the bioorthogonal functional group is 65-135%.

47. The method according to claim 45, wherein the conjugate or salt thereof, which contains two functional substances per heavy chain of antibody, comprises structural units represented by the following formulas (IVa), (IVa-1), (IVb) and / or (IVb-1). [In the formula, Ig represents the immunoglobulin unit, L ３ This indicates the third linker, L ６ This indicates the sixth linker, Z １ and Z ２ This indicates a functional substance, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W １ and W ６ Each of these independently represents either an oxygen atom or a sulfur atom, L ７ This indicates the seventh linker, L １０ This indicates the 10th linker, Z １ and Z ２ This indicates a functional substance, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, L １１ This indicates the 11th linker, L １４ This indicates the 14th linker, Z １ and Z ２ This indicates a functional substance, T １ and T ２ This represents a monovalent group, and the average modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%. [In the formula, Ig represents the immunoglobulin unit, W ７ , W ８ , W １１ , and W １２ Each of these independently represents either an oxygen atom or a sulfur atom, L １５ This indicates the 15th linker, L １８ This indicates the 18th linker, Z １ and Z ２ This indicates a functional substance, T ３ and T ４ This represents a monovalent group, and the modification percentage r per heavy chain in the immunoglobulin unit by the functional substance is 65-135%.

48. An affinity polypeptide or a salt thereof comprising an affinity moiety having affinity for the CH2-CH3 intercellular region of an antibody heavy chain, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are replaced with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence. SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC 49. A polynucleotide encoding an affinity polypeptide having affinity for the CH2-CH3 intercellular region of an antibody heavy chain, wherein the affinity polypeptide has an amino acid sequence in which two amino acid residues are replaced with amino acid residues having side-chain amino groups in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 13 or an amino acid sequence having 60% or more identity with said amino acid sequence. SEQ ID NO: 5: FNAQCQRRFYEALHDPNLNEEQRNARIRSIAEEC SEQ ID NO: 13: FNAQCQRRFYEALHDPNLNEEQRNARIRSIADDC

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