Compound containing antibody affinity polypeptide, modified antibody, antibody derivative, antibody-functional substance conjugate, and salts of these

Abstract

The present invention relates to technology for easily chemically modifying a heavy chain CH1 region in a constituent unit of an antibody. More specifically, the present invention relates to: a compound which contains (A) an affinity polypeptide containing an affinity moiety having an affinity for the heavy chain CH1 region of the antibody, and (B) a group that is reactive with the antibody; or a salt thereof.

Description

Compounds containing antibody-affinity polypeptides, modified antibodies, antibody derivatives, antibody-functional substance conjugates, and salts thereof

[0001] The present invention relates to compounds comprising antibody-affinity polypeptides, modified antibodies, antibody derivatives, antibody-functional substance conjugates, and salts thereof.

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

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

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

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

[0006] As an improvement over the C-CAP method, techniques have been reported that use a chemical synthesis technique using a specific compound containing an affinity peptide to prepare antibodies that do not contain a peptide moiety as a linker and that have a functional substance (e.g., a drug) regioselectively (Patent Documents 2 to 5). Avoiding the use of a linker containing a peptide moiety is desirable for clinical applications. These techniques propose several positions in an antibody that can be regioselectively modified with a drug, corresponding to various amino acid residues (e.g., lysine, tyrosine, serine, and threonine residues) in the CH2 and CH3 domains. However, it is not always easy to regioselectively modify an antibody with a functional substance and control the binding ratio between the antibody and the functional substance within a desired range.

[0007] Furthermore, Non-Patent Document 1 discloses a technique for labeling an antibody Fab fragment by photolabeling using a modified polypeptide of the C2 domain of Protein G.

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

[0009] Bioconjugate Chem. 2016, 27, 2095-2102

[0010] An object of the present invention is to develop a technique that allows for easy chemical modification of the heavy chain CH1 region in the building block of an antibody (in other words, an immunoglobulin unit comprising a heavy chain and a light chain).

[0011] A further object of the present invention is to develop a site-selectively modified antibody while easily chemically modifying the heavy chain CH1 region of the antibody building block.

[0012] As a result of extensive research, the present inventors have found that the heavy chain CH1 region of an antibody building block can be easily chemically modified by using (A) an affinity polypeptide containing an affinity portion having affinity for the CH1 region of the heavy chain of an antibody, and (B) a compound or its salt containing a group reactive to the antibody.

[0013] The compound of the present invention or a salt thereof can associate with the heavy chain of an antibody via an affinity polypeptide (A) containing an affinity moiety having affinity for the heavy chain CH1 region of the antibody, and then specifically reacts with the side chain of a specific amino acid residue in the heavy chain CH1 region of the antibody via the reactive group (R) to the antibody, thereby producing an affinity polypeptide-modified antibody or a salt thereof in which the CH1 region of the heavy chain of the antibody has been modified.

[0014] Patent Documents 1 to 5 disclose that an antibody can be regioselectively modified with a functional substance by using an affinity substance and a compound containing a group reactive to the antibody. However, they do not describe or suggest (1) the problem of developing a technique that can easily chemically modify the heavy chain CH1 region of an antibody, or (2) the use of a polypeptide containing an affinity moiety that has affinity for the heavy chain CH1 region of an antibody as an affinity polypeptide.

[0015] Non-Patent Document 1 discloses a method for site-specifically labeling an antibody Fab fragment using a modified polypeptide of the C2 domain of Protein G. However, this method relies on photolabeling, which requires light irradiation and is a complicated procedure. Furthermore, the modification efficiency is only about 50%, making it an inefficient method.

[0016] The present inventors have also succeeded in producing an antibody whose heavy chain CH1 region has been chemically modified by using the compound of the present invention or a salt thereof. Such an antibody is characterized in that it comprises (a) an immunoglobulin unit including a heavy chain and a light chain, and (b) a modifying unit (e.g., the above-mentioned affinity polypeptide, bioorthogonal functional group, or functional substance), and (c) the modifying unit is introduced into the heavy chain CH1 region of the above-mentioned immunoglobulin unit.

[0017] That is, the present invention is as follows: [1] A compound or a salt thereof comprising (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and (B) a group reactive with an antibody. [2] The compound or salt thereof according to [1], wherein the affinity polypeptide comprises an amino acid sequence including an amino acid residue having a side chain amino group, and the group reactive with an antibody is bound to the affinity polypeptide via a linker introduced to the side chain amino group of the amino acid residue having the side chain amino group. [3] The compound or salt thereof according to [2], wherein the affinity polypeptide has the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity thereto, in which an amino acid residue having a side chain amino group has been introduced at a specific amino acid residue. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE [4] The compound or salt thereof according to [3], wherein the specific amino acid residue is located near a target lysine present in the heavy chain CH1 region of the antibody in the three-dimensional configuration when the affinity polypeptide is coexistent with the antibody. [5] The compound or salt thereof according to [4], wherein the specific amino acid residue is located within 30 Å of a target lysine present in the heavy chain CH1 region of the antibody in the three-dimensional configuration when the affinity polypeptide is coexistent with the antibody. [6] The compound or salt thereof according to [4], wherein the target lysine present in the heavy chain CH1 region of the antibody is selected from lysine at positions 121, 147, 205, and 210 of the human IgG heavy chain according to EU numbering. [7] The compound or salt thereof according to [3], wherein the specific amino acid residue is selected from A at position 29, D at position 36, E at position 19, or E at position 15 of SEQ ID NO: 9. [8] The compound or salt thereof according to [1], wherein the CH1 region is a human CH1 region. [9] The compound or salt thereof according to [1], wherein the antibody is IgG.

[10] The compound or salt thereof according to [1], wherein the affinity polypeptide further comprises a tripeptide consisting of Gln-Glu-Thr (QET) or a dipeptide consisting of Gln-Glu (QE) at the N-terminus.

[11] The compound or salt thereof according to [1], wherein the compound is represented by the following formula (I): [In the formula, R represents the reactive group, L represents a linker, and A represents the affinity polypeptide.]

[12] The compound or salt thereof according to [2], wherein the compound or salt thereof further comprises (iii) a cleavable moiety between (i) the affinity polypeptide and (ii) the reactive group.

[13] The compound or salt thereof according to

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

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

[15] The compound or a salt thereof according to

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

[16] The compound or a salt thereof according to

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

[17] The compound or a salt thereof according to

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

[18] The compound or a salt thereof according to the following formula (Ib): [wherein R represents the reactive group; L ５ indicates the fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, and A represents the affinity polypeptide.

[19] The compound or a salt thereof according to

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

[20] The compound or a 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.

[0018]

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

[20] or a salt thereof.

[0019]

[22] An affinity polypeptide-modified antibody or a salt thereof, comprising, in the heavy chain CH1 region of the antibody, an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of the antibody.

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

[22] , comprising (a) immunoglobulin units comprising a heavy chain and a light chain, and (b) the affinity polypeptide, and (c) the affinity polypeptide has been introduced into the heavy chain CH1 region of the immunoglobulin unit.

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

[23] , wherein the affinity polypeptide has been introduced into the heavy chain CH1 region via modification of the amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region.

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

[24] , wherein one or more positions in the heavy chain CH1 region are selected from positions 121, 147, 205, and 210 of the human IgG heavy chain according to EU numbering.

[26] The affinity polypeptide according to

[22] , or a salt thereof, wherein the affinity polypeptide has an amino acid sequence in which an amino acid residue having a side chain amino group has been introduced into a specific amino acid residue in the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to the amino acid sequence, and the affinity polypeptide is modified with an amino acid group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region via a linker introduced to the side chain amino group of the amino acid residue having the side chain amino group. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE

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

[22] , wherein the affinity polypeptide has an amino acid sequence in which an amino acid residue having a side chain amino group has been introduced into a specific amino acid residue in the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to the amino acid sequence [In the formula, Ig represents an immunoglobulin unit comprising a heavy chain and a light chain, L represents a linker, A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin unit by the affinity polypeptide is 65 to 135%.]

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

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

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

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

[30] The affinity polypeptide-modified antibody or salt thereof according to the following formula (IIa): [wherein Ig represents the immunoglobulin unit; L １ indicates the first linker, L ２ represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin unit with the affinity polypeptide is 65 to 135%.

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

[29] , comprising a structural unit represented by the following formula (IIa-1): [wherein Ig represents the immunoglobulin unit; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ indicates a third linker, L ４represents a fourth linker, S represents a sulfur atom, A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin units by the affinity polypeptide is 65 to 135%.

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

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

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

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

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

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

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

[22] , wherein R represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, and A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin units by the affinity polypeptide is 65 to 135%.

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

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

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

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

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

[36] , wherein the additional affinity polypeptide is introduced into the heavy chain constant region via modification of amino groups in the side chains of lysine residues present at one or more positions in the heavy chain constant region.

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

[37] , wherein one or more positions in the heavy chain constant region are selected from positions 220, 226, 229, 246, 248, 274, 288, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering.

[0020]

[39] 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 including a heavy chain and a light chain to produce an affinity polypeptide-modified antibody or a salt thereof comprising the affinity polypeptide in the heavy chain CH1 region of the immunoglobulin unit.

[0021]

[40] An antibody derivative or salt thereof comprising a bioorthogonal functional group, the antibody derivative or salt thereof comprising (a) an immunoglobulin unit comprising a heavy chain and a light chain, and (b) a bioorthogonal functional group, and (c) the bioorthogonal functional group is introduced into the heavy chain CH1 region of the immunoglobulin unit.

[41] The antibody derivative or salt thereof according to

[40] , wherein the bioorthogonal functional group is introduced into the heavy chain CH1 region via modification of an amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region.

[42] The antibody derivative or salt thereof according to

[41] , wherein one or more positions in the heavy chain CH1 region are selected from positions 121, 147, 205, and 210 of the human IgG heavy chain according to EU numbering.

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

[44] The antibody derivative or salt thereof according to

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

[45] The antibody derivative or salt thereof according to

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

[46] The antibody derivative or salt thereof according to

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

[47] The antibody derivative or salt thereof according to

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

[48] The antibody derivative or salt thereof according to

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

[49] The antibody derivative or salt thereof according to

[48] , wherein the additional modifying moiety comprising an additional affinity polypeptide comprising a bioorthogonal functional group is introduced into the heavy chain constant region via modification of an amino group in the side chain of a lysine residue present at one or more positions in the heavy chain constant region.

[50] The antibody derivative or salt thereof according to

[49] , wherein one or more positions in the heavy chain constant region are selected from positions 220, 226, 229, 246, 248, 274, 288, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering.

[0022]

[51] A conjugate of an antibody and a functional substance, or a salt thereof, comprising (a) an immunoglobulin unit including a heavy chain and a light chain, and (b) a functional substance, and (c) the functional substance being introduced into the heavy chain CH1 region of the immunoglobulin unit.

[52] The conjugate or salt thereof according to

[51] , wherein the functional substance is introduced into the heavy chain CH1 region via modification of an amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region.

[53] The conjugate or salt thereof according to

[52] , wherein one or more positions in the heavy chain CH1 region are selected from positions 121, 147, 205, and 210 of the human IgG heavy chain according to EU numbering.

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

[55] The conjugate or a salt thereof according to

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

[56] The conjugate or a salt thereof according to

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

[57] The conjugate or a salt thereof according to

[51] , which contains a structural unit represented by the following formula (IVb-1): [wherein Ig represents the immunoglobulin unit; W１ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, Z represents a functional substance, and T ２ represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%.

[58] The conjugate or salt thereof according to

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

[59] The conjugate or salt thereof according to

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

[60] The conjugate or salt thereof according to

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

[61] The conjugate or salt thereof according to

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

[62] The conjugate or salt thereof according to

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

[63] The conjugate or salt thereof according to

[63] , wherein the one or more positions in the heavy chain constant region are selected from positions 220, 226, 229, 246, 248, 274, 288, 290, 317, 320, and 322 of a human IgG heavy chain according to EU numbering.

[0023]

[64] A method for producing an affinity polypeptide-free antibody or its salt, comprising: (A) an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody; and (B) an antibody; and (C) an affinity polypeptide-modified antibody or its salt further comprising a cleavable moiety between the affinity polypeptide (A) and the antibody (B), and cleaving the antibody or its salt with the cleavable moiety to produce an affinity polypeptide-free antibody or its salt.

[65] The method according to

[64] , wherein the affinity polypeptide has the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence containing one to two amino acid substitutions, deletions, additions, or insertions, in which an amino acid residue having a side chain amino group has been introduced at a specific amino acid residue.

[66] The method according to

[64] , wherein the cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the antibody upon cleavage, and the affinity polypeptide-free antibody or its salt is an antibody derivative or its salt comprising a bioorthogonal functional group.

[67] The method according to

[66] , wherein the antibody derivative or its salt comprising a bioorthogonal functional group is the antibody derivative or its salt according to

[43] or

[44] , or its salt.

[68] The method of

[64] , wherein the antibody or salt thereof not comprising an affinity polypeptide further comprises a bioorthogonal functional group between the antibody and the cleavable moiety, and the antibody or salt thereof not comprising an affinity polypeptide is an antibody derivative or salt thereof comprising a bioorthogonal functional group.

[69] The method of

[68] , wherein the antibody derivative or salt thereof comprising a bioorthogonal functional group is the antibody derivative or salt thereof according to

[45] or

[46] .

[0024]

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

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

[71] The method according to

[70] , wherein the conjugate or a salt thereof is the conjugate or a salt thereof according to any one of

[54] to

[55] .

[0025]

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

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

[73] The method according to

[72] , wherein the conjugate or a salt thereof is the conjugate or a salt thereof according to any one of

[56] to

[57] .

[0026]

[74] A method for producing a conjugate comprising an antibody and a functional substance, or a salt thereof, comprising reacting an antibody derivative comprising a bioorthogonal functional group, or a salt thereof, with a functional substance to produce a conjugate comprising an antibody and a functional substance, or a salt thereof, wherein the antibody derivative comprising a bioorthogonal functional group, or a salt thereof, is an antibody derivative comprising a bioorthogonal functional group, or a salt thereof, comprising (a) immunoglobulin units comprising a heavy chain and a light chain, and (b) the bioorthogonal functional group, and (c) the bioorthogonal functional group has been introduced into a heavy chain CH1 region in the immunoglobulin unit, and the conjugate comprising an antibody and a functional substance, or a salt thereof, is an antibody and functional substance conjugate, or a salt thereof, comprising (a) immunoglobulin units comprising a heavy chain and a light chain, and (b) the functional substance, and (c) the functional substance has been introduced into a heavy chain CH1 region in the immunoglobulin unit.

[75] The method according to

[74] , wherein the antibody derivative comprising a bioorthogonal functional group, or a salt thereof, is the antibody derivative according to any one of

[43] to

[46] , or a salt thereof.

[76] The method according to

[74] , wherein the conjugate or a salt thereof is the conjugate or a salt thereof according to any one of

[54] to

[57] .

[0027]

[77] An affinity polypeptide or a salt thereof, comprising an affinity portion having affinity for the heavy chain CH1 region of an antibody, wherein the affinity polypeptide has the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to the amino acid sequence, in which an amino acid residue having a side chain amino group has been introduced at a specific amino acid residue. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE

[0028]

[78] A polynucleotide encoding an affinity polypeptide comprising an affinity peptide having affinity for the heavy chain CH1 region of an antibody, wherein the affinity polypeptide has the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity thereto, in which an amino acid residue having a side chain amino group has been introduced at a specific amino acid residue. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE

[0029]

[79] An expression vector comprising the polynucleotide according to

[78] and a promoter operably linked thereto.

[80] A host cell comprising an expression unit comprising the polynucleotide according to

[78] and a promoter operably linked thereto.

[0030] According to the present invention, the heavy chain CH1 region of an antibody building block (an immunoglobulin unit comprising a heavy chain and a light chain) can be easily modified. Furthermore, according to the present invention, the heavy chain CH1 region of an antibody can be easily modified while providing a regioselectively modified antibody. By regioselectively modifying the heavy chain CH1 region, the modification can be performed at a site away from the binding sites involved in Fc binding and Fc receptor binding, such as FcRn and FcγR, making it possible to modify the antibody without losing its function. Furthermore, by regioselectively modifying the heavy chain CH1 region, it is possible to modify the Fab region protein and F(ab') ２ Modification of the domain protein is also possible.

[0031] Photograph showing the results of SDS-PAGE confirmation of the expression of affinity peptides #1 to #8. LC-MS / MS analysis results (MS spectrum) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-2-2). LC-MS / MS analysis results (HCD spectrum) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-2-2). LC-MS / MS analysis results (BioPharma Finder analysis) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-2-2). LC-MS / MS analysis results (MS spectrum) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-3-2). LC-MS / MS analysis results (HCD spectrum) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-3-2). LC-MS / MS analysis results (BioPharma Finder analysis) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-3-2). LC-MS / MS analysis results (MS spectrum) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-1-2). LC-MS / MS analysis results (HCD spectrum) of the modification site of trastuzumab into which two molecules of thiol groups have been introduced, obtained in Example (4-1-2). LC-MS / MS analysis of the modification site of trastuzumab into which two thiol groups have been introduced, obtained in Example (4-1-2) (analysis using BioPharma Finder). Figure (photograph) showing the results of confirming the expression of affinity peptides #9 to #12 by SDS-PAGE. Figure (photograph) showing the results of reacting affinity reagent #5-1 with trastuzumab in buffers of various pHs. Figure (photograph) showing the results of reacting affinity reagent #6-1 with trastuzumab in buffers of various pHs. Figure (photograph) showing the results of reacting affinity reagent #5-2 with trastuzumab in buffers of various pHs. Figure (photograph) showing the results of reacting affinity reagent #6-2 with trastuzumab in buffers of various pHs. FIG. 1 shows the results of reacting affinity reagent #8-2 with trastuzumab in buffer solutions of various pH values.

[0032] 1. Definitions of General Terms In this specification, terms and expressions used to describe a particular invention or item may also be used to describe other inventions or items. Therefore, the definitions, examples, and preferred examples of terms and expressions used to describe a particular invention or item may also be the same for other inventions or items described using such terms and expressions.

[0033] In the present invention, the term "antibody" is as follows. Furthermore, the term "immunoglobulin unit" corresponds to a monovalent or divalent monomer unit that is a constituent unit of such an antibody, and is an immunoglobulin unit comprising a heavy chain and a light chain. Therefore, the definitions, examples, and preferred examples of terms and expressions for the immunoglobulin unit, such as its origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, position of amino acid residues (e.g., lysine residues), and regioselectivity, are the same as those for antibodies described below, and are used interchangeably with the expression "antibody."

[0034] The origin of the antibody is not particularly limited, and may be derived from animals such as mammals and birds (e.g., chickens). Preferably, the immunoglobulin unit is derived from a mammal. Examples of such mammals include primates (e.g., humans, monkeys, chimpanzees), rodents (e.g., mice, rats, guinea pigs, hamsters, rabbits), pets (e.g., dogs, cats), livestock (e.g., cows, pigs, goats), and working animals (e.g., horses, sheep), preferably primates or rodents, more preferably humans.

[0035] The type of 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 tetravalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies with a specific sugar chain added (e.g., antibodies modified to have a sugar chain consensus sequence such as an N-glycan binding consensus sequence), bispecific antibodies, Fab region proteins, and F(ab') ２ Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. In the present invention, the antibody may be either a full-length antibody or an antibody fragment. The antibody fragment may be any antibody fragment containing a CH1 region, and antibody fragments containing both a variable region and a CH1 region are preferably used, but may also be antibody-like molecules containing only a CH1 region. The antibody may be a full-length antibody or a Fab region protein or F(ab') ２ The antibody is preferably a human IgG monoclonal antibody, more preferably a full-length human IgG monoclonal antibody.

[0036] Any antigen can be used as the antigen for the antibody. Examples of such antigens include proteins (including oligopeptides and polypeptides, and may also be proteins modified with biomolecules such as sugars (e.g., glycoproteins)), sugar chains, nucleic acids, and low-molecular-weight compounds. Preferably, the antibody may be an antibody whose antigen is a protein. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (e.g., extracellular matrix proteins), ligands, and soluble receptors.

[0037] More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following:

[0038] (1) Cancer area: PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2,, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesotheli n, CD138, c-Met, Ang2, VEGF- A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, グリピカン3, maccrophase inhibitory factor, CD74, Notch1, Notch 2. Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM- CSF, EphA3, B7-H3, CD123, gpA 33. Frizzled7 receptor, DLL4, VEG F, RSPO, LIV-1, SLITRK6, Nect in-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissu Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-カドヘリン, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73 , FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2. CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7 , EFNA4, FAP, DR5, CEA, Ly6E, CA6、CEACAM5、LAMP1、tissue factor, EPHA2, DR5, B7-H3, FGFR4 , FGFR2, α2-PI, A33, GDF15, CAIX, C D166, ROR1, GITR, BCMA, TBA, LAG- 3. EphA2, TIM-3, CD-200, EGFRvIII , CD16A, CD32B, PIGF, Axl, MICA / B , Thomsen-Friedenreich, CD39, CD 37、CD73、CLEC12A、Lgr3、トランスフェリンReceptor, TGFβ, IL-17, 5T4, RTK, Immune Supplier Protein, NaPi2b, ルイス blood type B antigen, A34, Lysil-Oxidase , DLK-1, TROP-2, α9インテグリン, TAG-72 (CA72-4), CD70,

[0039] (2) Autoimmune diseases / inflammatory diseases IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL-4αR, HAS , Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymic Stromal Lymphopoietin), LAMP (Alpha4 Beta 7 Integrin), IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74 , CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT

[0040] (3) Neurological diseases: CGRP, CD20, β-amyloid, β-amyloid protofibrin, calcitonin gene-related peptide receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, insulin receptor, tau protein, TDP-43, SOD1, TauC3, JC virus

[0041] (4) Infectious diseases: Clostridium Difficile toxin B, cytomegalovirus, respiratory syncytial virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. Aureus toxin, influenza A, alginate, Staphylococcus aureus, PD-L1, influenza B, Acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae

[0042] (5) Genetic and rare diseases: Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin

[0043] (6) Eye diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid

[0044] (7) Bone / orthopedics field Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15

[0045] (8) Blood diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI

[0046] (9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Edoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP

[0047] Specific examples of monoclonal antibodies include certain chimeric antibodies (e.g., rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, and ortatoxacimab), certain humanized antibodies (e.g., daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eculizumab (IgG2), mogamulizumab, pertuzumab, obinutuzumab, vedolizumab, pemprolizumab (IgG4), mepolizumab, elotuzumab, daratumumab, and ikesekizumab), and certain human antibodies (e.g., adalimumab (IgG1), panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, dupilumab (IgG4), secukinumab, evolocumab (IgG2), alirocumab, necitumumab, brodalumab (IgG2), olaratumab, pembrolizumab (IgG4)) (if no IgG subtype is mentioned, IgG1 is implied).

[0048] In the present invention, specific amino acid residues in the CH1 region of an antibody heavy chain can be regioselectively modified. Examples of such specific amino acid residues include lysine, tyrosine, serine, and threonine residues. For example, in human IgG, such as human IgG1, the following amino acid residues present in the heavy chain CH1 region can be exposed on the antibody surface, and these amino acid residues can be used to introduce specific cleavage sites (the positions of the amino acid residues are based on EU numbering; see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). (1) Exposed lysine residues: e.g., positions 121, 133, 147, 205, 210, 213, 214, 218, and 222. (2) Exposed tyrosine residues: positions 180 and 198. (3) Exposed serine residues: positions 120, 135, 139, 155, 164, 169, 187, 195, 197, and 209. (4) Exposed threonine residues: positions 116, 117, 119, 124, 131, 132, 134, 136, 157, 160, 165, 176, 177, 190, 191, 192, 207, and 219.

[0049] The positions of amino acid residues in an antibody and the positions of heavy chain constant regions (e.g., CH1 region) follow EU numbering (see http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). For example, when human IgG is the target, the lysine residue at position 121 corresponds to the fourth amino acid residue in the human IgG CH1 region, the lysine residue at position 147 corresponds to the 30th amino acid residue in the human IgG CH1 region, the lysine residue at position 205 corresponds to the 88th amino acid residue in the human IgG CH1 region, and the lysine residue at position 210 corresponds to the 93rd amino acid residue in the human IgG CH1 region.

[0050] Preferably, the specific amino acid residue in the heavy chain CH1 region to be regioselectively modified can be a lysine residue (e.g., a lysine residue at position 121, 147, 205, or 210). As used herein, "regioselective" or "regioselectivity" refers to a situation in which a specific structural unit capable of binding to a specific amino acid residue in an antibody is preferentially located in a specific region of the antibody, even though the specific amino acid residue is not preferentially located in a specific region of the antibody. Therefore, expressions related to regioselectivity, such as "regioselectively possessed," "regioselective binding," and "regioselective binding," mean that the retention or binding rate of a specific structural unit in a target region containing one or more specific amino acid residues is significantly higher than the retention or binding rate of the structural unit in a non-target region containing multiple amino acid residues that are homologous to the specific amino acid residues in the target region. Such regioselectivity may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or even 100%. According to the present invention, specific lysine residues in the heavy chain of an antibody can be regioselectively modified without using a peptide-containing linker. Peptide moieties have potential immunogenicity and are easily hydrolyzed in blood. Therefore, avoiding the use of linkers containing peptide moieties is desirable in clinical applications.

[0051] In the present invention, as long as a specific amino acid residue (e.g., a lysine residue at a specific position) in the heavy chain CH1 region is regioselectively modified, specific amino acid residues at other positions may also be regioselectively modified. For example, methods for regioselectively modifying specific amino acid residues at predetermined positions in an antibody are described in WO 2018 / 199337, WO 2019 / 240288, WO 2019 / 240287, and WO 2020 / 090979. Such specific amino acid residues may be amino acid residues (e.g., lysine residues, aspartic acid residues, glutamic acid residues, asparagine residues, glutamine residues, threonine residues, serine residues, tyrosine residues, cysteine ​​residues) having a side chain that is easily modified (e.g., amino group, carboxy group, amide group, hydroxy group, thiol group), but are preferably lysine residues having a side chain containing an amino group, tyrosine residues, serine residues, and threonine residues having a side chain containing a hydroxy group, or cysteine ​​residues having a side chain containing a thiol group, and more preferably lysine residues.

[0052] (Halogen Atom) Examples of halogen atoms include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0053] (Monovalent Group) Examples of the monovalent group include a monovalent hydrocarbon group and a monovalent heterocyclic group.

[0054] The monovalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 5, and particularly preferably 1 to 3) substituents described below.

[0055] (Monovalent Hydrocarbon Group and Related Terms) Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0056] A monovalent chain hydrocarbon group refers to a hydrocarbon group consisting only of a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of monovalent chain hydrocarbon groups include alkyl, alkenyl, and alkynyl. The alkyl, alkenyl, and alkynyl may be linear or branched.

[0057] The alkyl is preferably an alkyl having 1 to 12 carbon atoms, more preferably an alkyl having 1 to 6 carbon atoms, and even more preferably an alkyl having 1 to 4 carbon atoms. When the alkyl has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0058] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. When the alkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0059] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. When the alkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0060] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0061] The monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not have to be composed only of alicyclic hydrocarbons, and may contain a chain structure as part of it. Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0062] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 to 6 carbon atoms. When the cycloalkyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0063] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 or 6 carbon atoms. When the cycloalkenyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0064] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 or 6 carbon atoms. When the cycloalkynyl has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0065] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0066] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed solely of an aromatic ring, and it may contain a chain structure or an alicyclic hydrocarbon as part of it, and the aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, an aryl group having 6 to 12 carbon atoms is preferred, an aryl group having 6 to 10 carbon atoms is more preferred, and an aryl group having 6 carbon atoms is even more preferred. When the monovalent aromatic hydrocarbon group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms mentioned above. Examples of aryl groups having 6 to 12 carbon atoms include phenyl and naphthyl.

[0067] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0068] Among these, alkyl, cycloalkyl and aryl are preferred as the monovalent hydrocarbon group.

[0069] (Monovalent heterocyclic group and related terms) A monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group. The heteroatom constituting the heterocyclic group preferably contains one or more atoms selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom, and a silicon atom, and more preferably contains one or more atoms selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom.

[0070] The monovalent aromatic heterocyclic group is preferably an aromatic heterocyclic group having 1 to 15 carbon atoms, more preferably an aromatic heterocyclic group having 1 to 9 carbon atoms, and even more preferably an aromatic heterocyclic group having 1 to 6 carbon atoms. When the monovalent aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.

[0071] The monovalent non-aromatic heterocyclic group is preferably a non-aromatic heterocyclic group having 2 to 15 carbon atoms, more preferably a non-aromatic heterocyclic group having 2 to 9 carbon atoms, and even more preferably a non-aromatic heterocyclic group having 2 to 6 carbon atoms. When the monovalent non-aromatic heterocyclic group has a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0072] Among these, the monovalent heterocyclic group is preferably a 5- or 6-membered heterocyclic group.

[0073] (Divalent Group) The divalent group is a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, a divalent heterocyclic group, —C(═O)—, —C(═S)—, —NR1—, —C(═O)—NR １ -, -NR １ -C(=O)-, -C(=S)-NR １ -, -NR １ -C(=S)-, -O-, -S-, -(OR ２ )n-, and -(S-R ２ )m-, or a group having a main chain structure containing two or more of these groups (for example, 2 to 10, preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 5, and particularly preferably 2 or 3). １ represents a hydrogen atom or a substituent to be described later. ２ represents a divalent linear hydrocarbon group, a divalent cyclic hydrocarbon group, or a divalent heterocyclic group. n and m are each an integer of 1 to 10, preferably an integer of 1 to 8, more preferably an integer of 1 to 6, even more preferably an integer of 1 to 5, and particularly preferably an integer of 1 to 3.

[0074] The divalent linear hydrocarbon group is a linear alkylene, linear alkenylene, or linear alkynylene. The linear alkylene is a linear alkylene having 1 to 6 carbon atoms, preferably a linear alkylene having 1 to 4 carbon atoms. Examples of linear alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene. The linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, preferably a linear alkenylene having 2 to 4 carbon atoms. Examples of linear alkenylene include ethyleneylene, n-propynylene, n-butenylene, n-pentenylene, and n-hexenylene. The linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, preferably a linear alkynylene having 2 to 4 carbon atoms. Examples of the straight-chain alkynylene include ethynylene, n-propynylene, n-butynylene, n-pentynylene, and n-hexynylene. As the divalent straight-chain hydrocarbon group, a straight-chain alkylene is preferred.

[0075] The divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group. The arylene is preferably an arylene having 6 to 14 carbon atoms, more preferably an arylene having 6 to 10 carbon atoms, and particularly preferably an arylene having 6 carbon atoms. Examples of arylene include phenylene, naphthylene, and anthracenylene. The divalent non-aromatic cyclic hydrocarbon group is preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms, more preferably a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 4 to 10 carbon atoms, and particularly preferably a monocyclic divalent non-aromatic cyclic hydrocarbon group having 5 to 8 carbon atoms. Examples of divalent non-aromatic cyclic hydrocarbon groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. The divalent cyclic hydrocarbon group is preferably an arylene.

[0076] The divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group. The heteroatom constituting the heterocycle preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, phosphorus, boron, and silicon atoms, and more preferably contains one or more atoms selected from the group consisting of oxygen, sulfur, and nitrogen atoms. The divalent aromatic heterocyclic group is preferably a divalent aromatic heterocyclic group having 3 to 15 carbon atoms, more preferably a divalent aromatic heterocyclic group having 3 to 9 carbon atoms, and particularly preferably a divalent aromatic heterocyclic group having 3 to 6 carbon atoms. Examples of divalent aromatic heterocyclic groups include pyrrolediyl, furandiyl, thiophenediyl, pyridinediyl, pyridazinediyl, pyrimidinediyl, pyrazinediyl, triazinediyl, pyrazolediyl, imidazolediyl, thiazolediyl, isothiazolediyl, oxazolediyl, isoxazolediyl, triazolediyl, tetrazolediyl, indolediyl, purinediyl, anthraquinonediyl, carbazolediyl, fluorenediyl, quinolinediyl, isoquinolinediyl, quinazolinediyl, and phthalazinediyl. As the divalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferred, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferred, and a non-aromatic heterocyclic group having 3 to 6 carbon atoms is particularly preferred. Examples of divalent non-aromatic heterocyclic groups include pyrroledionediyl, pyrrolinedionediyl, oxiranediyl, aziridinediyl, azetidinediyl, oxetanediyl, thietanediyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxolanediyl, tetrahydrothiophenediyl, pyrrolinediyl, imidazolidinediyl, oxazolidinediyl, piperidinediyl, dihydropyrandiyl, tetrahydropyrandiyl, tetrahydrothiopyrandiyl, morpholinediyl, thiomorpholinediyl, piperazinediyl, dihydrooxazinediyl, tetrahydrooxazinediyl, dihydropyrimidinediyl, and tetrahydropyrimidinediyl. As the divalent heterocyclic group, a divalent aromatic heterocyclic group is preferred.

[0077] Preferably, the divalent group is alkylene, arylene, —C(═O)—, —NR １ -, -C(=O)-NR １ -, -NR １ -C(=O)-, -O-, and -(O-R ２ )n-, or alkylene, arylene, —C(═O)—, —NR １ -, -C(=O)-NR １ -, -NR １ -C(=O)-, -O-, and -(O-R ２ )n- is a divalent group having a main chain structure containing two or more groups selected from the group consisting of, １ is a hydrogen atom or alkyl, R ２ is alkylene or arylene, and n may be an integer of 1 to 5 (i.e., 1, 2, 3, 4, or 5). Alkylene, arylene, and alkyl are as defined above.

[0078] The main chain structure in the divalent group may be substituted with one or more (for example, 1 to 10, preferably 1 to 8, more preferably 1 to 6, still more preferably 1 to 5, and particularly preferably 1 to 3) substituents described below.

[0079] (Substituents) Examples of the substituents include: (i) a halogen atom; (ii) a monovalent hydrocarbon group; (iii) a monovalent heterocyclic group; (iv) an aralkyl; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or a monovalent hydrocarbon group; or (vi) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃare the same or different and represent a hydrogen atom or a monovalent hydrocarbon group; (vii) a nitro group, a sulfate group, a sulfonate group, a cyano group, and a carboxyl group.

[0080] The definitions, examples, and preferred examples of the halogen atom, monovalent hydrocarbon group, and monovalent heterocyclic group in the above substituents are the same as those described above.

[0081] Aralkyl refers to arylalkyl. The definitions, examples, and preferred examples of aryl and alkyl in arylalkyl are as described above. The aralkyl is preferably an aralkyl having 3 to 15 carbon atoms. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.

[0082] Preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 12 carbon atoms, phenyl, or naphthyl; (iii) an aralkyl having 3 to 15 carbon atoms; (iv) a 5- or 6-membered heterocycle; (v) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl having 1 to 12 carbon atoms; (vi) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and Rc are the same or different and each represents a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (vii) the same groups as those enumerated in (vii) above.

[0083] More preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 12 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, Rａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms; (iv) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 12 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0084] Even more preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 6 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(R ａ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; (iv) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ —C(═O)—O—, or Rb—C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 6 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0085] Particularly preferably, the substituents may be: (i) a halogen atom; (ii) an alkyl having 1 to 4 carbon atoms; (iii) R ａ -O-, R ａ -C(=O)-, R ａ -O-C(=O)- or R ａ -C(=O)-O-(Rａ represents a hydrogen atom or an alkyl having 1 to 4 carbon atoms; (iv) NR ｂ R ｃ -, NR ｂ R ｃ -C(=O)-,NR ｂ R ｃ -C(=O)-O- or R ｂ —C(═O)—NR ｃ - (R ｂ and R ｃ are the same or different and represent a hydrogen atom or alkyl having 1 to 4 carbon atoms; or (v) the same groups as those listed in (vii) above.

[0086] (Bio-orthogonal functional group) Bio-orthogonal functional groups are groups that do not react with biological components (e.g., amino acids, proteins, nucleic acids, lipids, sugars, phosphates), or react slowly with biological components, but selectively react with components other than biological components. Bio-orthogonal functional groups are well known in the art (e.g., Sharpless K.B. et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi C.R. et al., Science 291, 2357 (2001); Bertozzi C.R. et al., Nature Chemical Biology 1, 13 (2005)).

[0087] In the present invention, a bioorthogonal functional group for a protein is used as the bioorthogonal functional group. This is because the antibody to be derivatized with the reagent of the present invention is a protein. The bioorthogonal functional group for a protein is a group that does not react with the side chains of the 20 naturally occurring amino acid residues that make up proteins, or that reacts slowly with the side chains, but reacts with the desired functional group. The 20 naturally occurring amino acids that make up proteins are alanine (A), asparagine (N), cysteine ​​(C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (L). Among these 20 naturally occurring amino acids, glycine has no side chain (i.e., a hydrogen atom), and alanine, isoleucine, leucine, phenylalanine, and valine have hydrocarbon side chains (i.e., do not contain heteroatoms selected from the group consisting of sulfur, nitrogen, and oxygen atoms). These amino acids are inert to normal reactions. Therefore, the bioorthogonal functional group for proteins is a group that does not react or reacts slowly with the side chains of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, in addition to the side chains of these amino acids that have side chains that are inert to normal reactions, but reacts with the desired functional group.

[0088] Examples of such bioorthogonal functional groups include azide residues, aldehyde residues, thiol residues, alkene residues (in other words, it is sufficient that it has a vinylene (ethenylene) moiety, which is the smallest unit having a double bond between carbon atoms; the same applies below), alkyne residues (in other words, it is sufficient that it has an ethynylene moiety, which is the smallest unit having a triple bond between carbon atoms; the same applies below), halogen residues, tetrazine residues, nitrone residues, hydroxylamine residues, nitrile residues, hydrazine residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues (e.g., a carbonyl residue having a fluorine atom, chlorine atom, bromine atom, or iodine atom at the α-position; the same applies below), isonitrile residues, sydnone residues, and selenium residues.

[0089] The bioorthogonal functional group may be protected or unprotected. A bioorthogonal functional group refers to an unprotected bioorthogonal functional group or a protected bioorthogonal functional group. An unprotected bioorthogonal functional group corresponds to the bioorthogonal functional group described above. A protected bioorthogonal functional group is a group that generates a bioorthogonal functional group by cleavage of the protecting group. Cleavage of the protecting group can be carried out by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli selected from the group consisting of light, or (c) leaving the linker when using a cleavable linker containing a self-cleaving cleavable moiety. Such protecting groups and their cleavage conditions are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, J.M., Journal of American Chemical Society. Society. 132, 17928 (2010); Thompson, D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004)). Reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are as described below.

[0090] Protected bioorthogonal functional groups include, for example, disulfide residues, ester residues, acetal residues, ketal residues, imine residues, and vicinal diol residues.

[0091] Preferably, the bioorthogonal functional group is an unprotected bioorthogonal functional group.

[0092] More preferably, the bioorthogonal functional group may be a specific bioorthogonal functional group that exhibits excellent reactivity (e.g., reactivity level and / or reaction specificity) with other bioorthogonal functional groups. Examples of such bioorthogonal functional groups include azide residues, alkyne residues (preferably ring groups having a triple bond between carbon atoms, which may be substituted with the substituents described above), tetrazine residues, alkene residues, thiol residues, maleimide residues, thiol residues, furan residues, and halocarbonyl residues. Combinations of two bioorthogonal functional groups that can react with each other include, for example, combinations of azide residues and alkyne residues, combinations of tetrazine residues and alkene residues, combinations of tetrazine residues and alkyne residues, combinations of thiol residues and maleimide residues, combinations of furan residues and maleimide residues, combinations of thiol residues and halocarbonyl residues (substitution reaction in which the halogen is replaced by thiol), and combinations of thiol residues and other thiol residues (formation of a disulfide bond).

[0093] (Functional Substance) The functional substance is not particularly limited as long as it is a substance that imparts any function to the antibody, and examples thereof include drugs, labeling substances, affinity substances, transport substances, and stabilizers, but drugs, labeling substances, affinity substances, or transport substances are preferred. The functional substance may also be a single functional substance, or a substance in which two or more functional substances are linked together.

[0094] The drug may be a drug for any disease, such as cancer (e.g., lung cancer, stomach cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, brain tumor, melanoma), autoimmune diseases / inflammatory diseases (e.g., allergic diseases, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve diseases (e.g., cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), infectious diseases (e.g., bacterial infection, viral infection), genetic / rare diseases (e.g., hereditary spherocytosis, non-dystrophic myotonia), eye diseases (e.g., age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa), bone / orthopedic diseases (e.g., osteoarthritis), blood diseases (e.g., leukemia, purpura), and other diseases (e.g., diabetes, metabolic disorders such as hyperlipidemia, liver diseases, kidney diseases, lung diseases, circulatory system diseases, digestive system diseases). The drug may be a drug for preventing or treating a disease, or a drug for mitigating side effects.

[0095] More specifically, the drug may be an anticancer drug. Examples of anticancer drugs include chemotherapeutic agents, toxins, radioisotopes, and substances containing the same. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, and platinum compounds. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioactive isotopes of hydrogen atoms (e.g., 3H), radioactive isotopes of carbon atoms (e.g., 14C), radioactive isotopes of phosphorus atoms (e.g., 32P), radioactive isotopes of sulfur atoms (e.g., 35S), radioactive isotopes of yttrium (e.g., 90Y), radioactive isotopes of technetium (e.g., 99mTc), radioactive isotopes of indium (e.g., 111In), radioactive isotopes of iodine atoms (e.g., 123I, 125I, 129I, 131I), radioactive isotopes of samarium (e.g., 153Sm), radioactive isotopes of rhenium (e.g., 186Re), radioactive isotopes of astatine (e.g., 211At), and radioactive isotopes of bismuth (e.g., 212Bi). More specifically, the drugs include auristatins (MMAE, MMAF), maytansine (DM1, DM4), PBD (pyrrolobenzodiazepine), IGN, camptothecin analogs, calicheamicin, duocarmycin, eribulin, anthracyclines, dmDNA31, and tubulysin.

[0096] A labeling substance is a substance that enables detection of a target (e.g., tissue, cell, substance). Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin, digoxigenin, aptamers), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2′-bipyridyl)ruthenium, luminol), radioisotopes (e.g., those described above), and substances containing the same.

[0097] An affinity substance is a substance that has affinity for a target. Examples of affinity substances include affinity proteins or peptides such as antibodies, aptamers, lectins, and complementary chains to target nucleic acids. The affinity substance is preferably an affinity protein or affinity peptide, and more preferably an antibody. The species of animals from which antibodies used as functional substances are derived are the same as those described above.

[0098] The type of antibody used as the functional substance may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent or higher antibody (e.g., IgA antibody, IgM antibody). Preferably, the antibody is a monoclonal antibody. Examples of monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, antibodies with specific glycosylation (e.g., antibodies modified to have a glycosylation consensus sequence such as an N-glycosylated consensus sequence), bispecific antibodies, Fc region proteins, Fc fusion proteins, and disulfide-reduced antibodies. Examples of monoclonal antibody isotypes include IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. Examples of antibodies used as the functional substance include full-length antibodies and fragments thereof (fragment antibodies). The antibody fragment may be any fragment that maintains binding to a desired antigen, and may be, for example, Fab, Fab', F(ab') ２ , scFv, and VHH antibodies.

[0099] The antigenicity of the antibody used as the functional substance may be the same as or different from the antigenicity of the immunoglobulin unit in the antibody, antibody derivative, and conjugate of the present invention, preferably different. Furthermore, the origin of the antibody used as the functional substance may be the same as or different from the origin of the immunoglobulin unit, preferably different. Therefore, the antibody used as the functional substance may be a specific chimeric antibody, a specific humanized antibody, or a specific human antibody mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom. The antibody used as the functional substance may also be an IgG1, IgG2, IgG3, or IgG4 antibody mentioned in the specific examples of the monoclonal antibody above, or an antibody derived therefrom.

[0100] The transporter is a substance capable of transporting a compound. Preferred transporters are substances capable of encapsulating a compound in a protein shell (e.g., multimer) (e.g., ferritin, virus particles, virus-like particles).

[0101] Stabilizers are substances that enable antibody stabilization, and include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.

[0102] The functional substance may also be a peptide, a protein, a nucleic acid, an organic compound, an inorganic compound, a sugar chain, a lipid, a high molecular weight polymer, a metal (e.g., gold), or a chelator. Examples of peptides include cell membrane-permeable peptides, blood-brain barrier-permeable peptides, and peptide drugs. Examples of proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, antibodies, and ferritin. Examples of nucleic acids include DNA, RNA, and artificial nucleic acids. Examples of nucleic acids include RNA interference-inducing nucleic acids (e.g., siRNA), aptamers, and antisense. Examples of organic compounds include low-molecular-weight organic compounds such as proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds. Examples of inorganic compounds include silica, talc, and alumina.

[0103] (Salts) In the present invention, the term "salt" includes, for example, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, and salts with amino acids. Salts with inorganic acids include, for example, salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid. Salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Salts with inorganic bases include, for example, salts with alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., calcium, magnesium), and other metals such as zinc and aluminum, as well as ammonium. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine. Examples of salts with amino acids include salts with basic amino acids (e.g., arginine, histidine, lysine, ornithine) and acidic amino acids (e.g., aspartic acid, glutamic acid). The salt is preferably a salt with an inorganic acid (e.g., hydrogen chloride) or a salt with an organic acid (e.g., trifluoroacetic acid).

[0104] 2. Affinity Polypeptides or Salts Thereof, and Related Inventions 2-1. Affinity Polypeptides or Salts Thereof The present invention provides affinity polypeptides or salts thereof comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody.

[0105] The affinity polypeptide used in the present invention comprises an affinity moiety having affinity for the heavy chain CH1 region of an antibody. In one embodiment, the antibody is an immunoglobulin unit comprising a heavy chain and a light chain. Thus, the antibody's constituent unit is an immunoglobulin unit comprising two heavy chains and two light chains, or an immunoglobulin unit comprising one heavy chain and one light chain. Examples of antibodies comprising an immunoglobulin unit comprising two heavy chains and two light chains include bivalent antibodies (e.g., IgG, IgD, IgE), tetravalent or higher antibodies (e.g., IgA antibodies, IgM antibodies), chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and disulfide-reduced antibodies.

[0106] In one embodiment, the antibody is an antibody fragment containing the variable region and the CH1 region. The antibody fragment may be a Fab region protein or F(ab') ２ The Fab region protein is preferably contained in an immunoglobulin unit containing one heavy chain and one light chain, and is expressed as F(ab'). ２ The domain proteins are contained in an immunoglobulin unit which contains two heavy chains and two light chains.

[0107] Affinity moieties include, for example, polymeric substances of a predetermined structural unit (e.g., peptides (including oligopeptides, polypeptides, and proteins), nucleic acids (including oligonucleic acids and polysaccharides), and sugars (including oligosaccharides and polysaccharides)), as well as non-polymeric substances (e.g., low molecular weight compounds). Many substances, such as peptides, nucleic acids, sugars, and low-molecular-weight compounds, have been reported as usable affinity moieties having affinity for the constant region of an antibody heavy chain (see, for example, WO 2007 / 004748, WO 2008 / 054030, WO 2013 / 027796, WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979; Nomura Y et al., Nucleic Acids Res., 2010 Nov; 38(21):7822-9; Miyakawa S et al., RNA., 2008 Jun;14(6):1154-63, and see the scientific paper below).

[0108] Furthermore, substances that can be used as affinity moieties having affinity for the antibody heavy chain CH1 region can be obtained by any method. For example, such substances can be obtained by screening any substance library (e.g., a low molecular weight compound library, a peptide library, an aptamer library, a sugar library, a phage library, an mRNA library, a cDNA library) for substances having affinity for the constant region of the antibody heavy chain (e.g., high-throughput screening, phage display, SELEX, mRNA display, ribosome display, cDNA display, yeast display). Furthermore, when screening for substances having affinity for a specific region (e.g., the CH1 region) in the antibody constant region, partial peptides present in a specific region (e.g., the CH1 region) of various antibodies (e.g., IgG, IgA, IgM, IgD, IgE) can be used to efficiently obtain substances that can selectively bind to such a region.

[0109] The affinity portion may have affinity for the CH1 region of the antibody heavy chain. Here, "having affinity for the CH1 region" is not particularly limited as long as it has affinity for at least a portion of the CH1 region, and may have affinity for a partial region of the CH1 region, or for a region spanning the CH1 region and another CHX domain (e.g., an adjacent region). Therefore, it may have affinity only for a partial region of the CH1 region, or for a region spanning the CH1 region and the CH2 domain (e.g., an adjacent region of the CH1 region and the CH2 domain), and more preferably, it may have affinity only for a partial region of the CH1 region.

[0110] The CH1 region of the heavy chain of the antibody to which the affinity moiety has affinity may be derived from an animal (e.g., a mammal, a bird) as described above. The CH1 region of the heavy chain of the antibody may be preferably a mammalian CH1 region, more preferably a primate CH1 region or a rodent CH1 region, and even more preferably a human CH1 region.

[0111] The CH1 region of the heavy chain of the antibody to which the affinity moiety has affinity may be the CH1 region of a bivalent antibody (e.g., IgG, IgD, IgE) or a tetravalent or higher antibody (e.g., IgA antibody, IgM antibody). Such a CH1 region is preferably the CH1 region of a bivalent antibody (e.g., IgG, IgD, IgE), more preferably the CH1 region of an IgG.

[0112] In the present invention, the affinity moiety is an affinity peptide having affinity for the CH1 region of the antibody heavy chain. The affinity peptide can be obtained by the screening method described above (e.g., the method using the library described above or the display method described above).

[0113] The amino acid residues constituting the affinity peptide can be any of the 20 naturally occurring amino acids commonly constituting proteins, or unnatural amino acid residues, such as L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G) (hereinafter, the abbreviation "L" will be omitted).

[0114] For example, affinity peptides having affinity for the CH1 region of an antibody heavy chain and having no lysine residues can be used. More specifically, the following affinity peptides having affinity for the constant region of an antibody heavy chain and having no lysine residues may be used: (1) an affinity peptide comprising the amino acid sequence TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 9); (2) an affinity peptide comprising an amino acid sequence (having no lysine residues) having 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more identity to the amino acid sequence TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 9), and having affinity for the CH1 region of an antibody heavy chain; (3) An affinity peptide comprising an amino acid sequence in which one to several, preferably one or two, amino acid residues in the amino acid sequence of TTYRLVINGRTLRGETTTEAVDAETAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 9) are substituted with amino acid residues other than lysine residues, and having affinity for the heavy chain CH1 region of an antibody; (4) An affinity peptide comprising the amino acid sequence of TTYRLVINGRTLRGETTTEAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 10); (5) An affinity peptide comprising an amino acid sequence (containing no lysine residues) having 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more identity with the amino acid sequence of TTYRLVINGRTLRGETTTEAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 10), and having affinity for the heavy chain CH1 region of an antibody; (6) An affinity peptide comprising an amino acid sequence in which one to several, preferably one or two, amino acid residues in the amino acid sequence of TTYRLVINGRTLRGETTTEAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 10) are substituted with amino acid residues other than lysine residues, and having affinity for the heavy chain CH1 region of an antibody;

[0115] Substitutions of amino acid residues may be conservative substitutions. The term "conservative substitution" refers to replacing a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl group (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Amino acids with uncharged polar side chains and nonpolar side chains are sometimes collectively referred to as neutral amino acids. Preferably, conservative amino acid substitutions may be between aspartic acid and glutamic acid, between arginine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine, between leucine, isoleucine and alanine, and between glycine and alanine.

[0116] The affinity polypeptide or its salts may contain the above-mentioned natural amino acid residues or unnatural amino acid residues as the amino acid residues constituting it. When the affinity polypeptide contains only the above-mentioned natural amino acid residues, it can be produced, for example, by a polypeptide expression system using host cells, a cell-free synthesis system, or an organic synthesis system (e.g., solid-phase synthesis). When the affinity polypeptide contains unnatural 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, which enables large-scale production of the affinity polypeptide by a polypeptide expression system using host cells or a cell-free synthesis system.

[0117] The amino and carboxyl groups at the termini of the affinity polypeptide can be protected as appropriate. Examples of protecting groups for the N-terminal amino group include alkylcarbonyl groups (acyl groups) (e.g., butoxycarbonyl groups such as acetyl, propoxy, and tert-butoxycarbonyl), alkyloxycarbonyl groups (e.g., fluorenylmethoxycarbonyl), aryloxycarbonyl groups, and arylalkyl(aralkyl)oxycarbonyl groups (e.g., benzyloxycarbonyl). Preferably, the N-terminal amino group may be alkylated, formylated, or acetylated. Examples of protecting groups for the C-terminal 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.

[0118] Furthermore, when the N-terminal amino acid of the affinity polypeptide is glutamic acid (E) or glutamine (Q), the N-terminus can be protected using the side chain thereof. When the N-terminal amino acid is glutamic acid, the protected N-terminal glutamic acid can have a cyclic structure of pyroglutamic acid. When the N-terminal amino acid is glutamine, the protected N-terminal glutamine ... ２ ) can react with the amide group present in its side chain (pyroglutamylation) to form a pyroglutamic acid-type cyclic structure. Therefore, the N-terminal amino acid may preferably be glutamic acid or glutamine.

[0119] The affinity polypeptide may further contain a tripeptide consisting of Gln-Glu (QET) at the N-terminus. If the affinity peptide sequence already contains T at the N-terminus, it may further contain a dipeptide consisting of QE at the N-terminus. In this case, a polypeptide expression system using a host cell enables the protection of the N-terminal amino group by pyroglutamylation of Q, as well as the simple and large-scale secretion production of the affinity polypeptide (see WO 2013 / 062029 and WO 2020 / 090979). In this case, a signal peptide such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 31) can be added to the N-terminus of QET (see WO 2013 / 062029 and WO 2020 / 090979).

[0120] Furthermore, when the affinity peptide contains T at its N-terminus, it may further contain a dipeptide consisting of QE at the N-terminus, with a peptide linker sequence between QE and T. The peptide linker sequence may consist of 20 or more amino acid residues. The peptide linker sequence may consist of 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, 32 or more, 34 or more, 36 or more, 38 or more, or 40 or more amino acid residues. The peptide linker sequence may also consist of 60 or less, 58 or less, 56 or less, 54 or less, 52 or less, 50 or less, 48 ​​or less, or 46 or less amino acid residues. Suitable amino acid residues for the peptide linker sequence include, but are not limited to, alanine, proline, serine, and glycine. The peptide linkers disclosed in WO 2021 / 112249 and WO 2011 / 144756 may also be used.

[0121] The affinity polypeptide of the present invention or its salt can be used, for example, as a synthetic intermediate for the affinity polypeptide and the compound of the present invention or its salt comprising a reactive group for an antibody.Therefore, in addition to the affinity polypeptide, the affinity polypeptide of the present invention or its salt can be derivatized to contain only one specific reactive group that enables specific reaction with a partial compound comprising a reactive group for an antibody, in order to easily realize the homogeneous synthesis of the compound of the present invention or its salt comprising a reactive group for an antibody.When the affinity polypeptide contains only one specific reactive group, both the affinity polypeptide and the reactive group for an antibody can be specifically reacted through the specific reactive group in the affinity polypeptide, so that the compound of the present invention or its salt comprising a reactive group for an antibody can be easily produced as a homogeneous compound.

[0122] Examples of the specific reactive groups include the following: (1) an amino group (NH ２ , N.H.R. ３ , N.R. ３ R ４ .R ３ and R４ are each independently a monovalent group as described above, preferably a monovalent hydrocarbon group, more preferably an alkyl group, and even more preferably an alkyl group having 1 to 6 carbon atoms; (2) a residue capable of reacting with an amino group, such as an activated ester residue (e.g., an N-hydroxysuccinimide residue), a vinyl sulfone residue, a sulfonyl chloride residue, an isocyanate residue, an isothiocyanate residue, an aldehyde residue, a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residue, a 2-imino-2-methoxyethyl residue, or a diazonium terephthalate residue; (3) a carboxyl group (COOH); (4) a residue capable of reacting with a carboxyl group, such as an amino group as described above; (5) a hydroxyl group (OH) (including alcoholic and phenolic hydroxyl groups); and (6) a residue capable of reacting with a hydroxyl group, such as a diazonium residue, a diazodicarboxyl residue, or a 2,3-dihydro-1H-pyrazin-6-one residue. The specific reactive group may preferably be any of (1) to (4). More preferably, the specific reactive group may be any of (1) or (2), or any of (3) or (4). Alternatively, more preferably, the specific reactive group may be any of (1) or (3). The specific reactive group is even more preferably (1), and particularly preferably an amino group (NH ２ ) may also be used.

[0123] The affinity polypeptide or its salt may contain only one amino acid residue containing a specific reactive group. In such a case, the affinity polypeptide may contain (a) only one amino acid residue (e.g., lysine residue, aspartic acid residue, glutamic acid residue, tyrosine residue, threonine residue, or serine residue) having a specific reactive group (e.g., amino group, carboxyl group, or hydroxyl group) in its side chain. When the affinity polypeptide contains only one lysine residue having an amino group in its side chain, the N-terminus of the affinity polypeptide is preferably protected (the C-terminus may also be protected). When the affinity polypeptide contains only one amino acid residue (e.g., aspartic acid residue or glutamic acid residue) having a carboxyl group in its side chain, the C-terminus of the affinity polypeptide is preferably protected (the N-terminus may also be protected).

[0124] Alternatively, the affinity polypeptide may be a polypeptide that does not contain any amino acid residues having an amino group in the side chain (e.g., lysine residues) and has an amino group at the N-terminus, thereby containing only one amino group as a specific reactive group. The affinity polypeptide may also be a polypeptide that does not contain any amino acid residues having a carboxyl group in the side chain (e.g., aspartic acid residues, glutamic acid residues) and has a carboxyl group at the C-terminus, thereby containing only one carboxyl group as a specific reactive group.

[0125] Preferably, the affinity polypeptide containing only one specific reactive group may be a polypeptide containing an amino acid residue having an amino group in its side chain. When the affinity polypeptide is produced by an organic synthesis system (e.g., solid-phase synthesis), not only lysine residue, a natural amino acid constituting proteins, but also other amino acid residues having an amino group in their side chain (e.g., ornithine) can be used. More preferably, the affinity polypeptide containing only one specific reactive group contains an amino group (NH ２ ) in the side chain.

[0126] Preferably, the affinity peptide may further have the following characteristic (1) or (2), preferably characteristic (1):

[0127] (1) Affinity polypeptide or its salt containing only one amino group as a specific reactive group: The affinity polypeptide or its salt containing only one amino group as a specific reactive group may be an affinity polypeptide or its salt that does not contain a group that can react with an amino group (e.g., a carboxy group) in order to suppress undesired reactions (e.g., intramolecular reactions or intermolecular reactions). Therefore, in the above (1), the affinity peptide may not contain an amino acid residue having a carboxy group in its side chain.

[0128] (2) Affinity polypeptides or salts thereof containing only one carboxy group as a specific reactive group: The affinity polypeptides or salts thereof containing only one carboxy group as a specific reactive group may be affinity polypeptides or salts thereof that do not contain a group reactive with the carboxy group (e.g., an amino group) in order to suppress undesired reactions (e.g., intramolecular reactions or intermolecular reactions). Therefore, in the above (2), the affinity peptide may be one that does not contain an amino acid residue having an amino group in its side chain and / or one that has a protected N-terminus.

[0129] Preferably, the affinity peptide represented by the above formula (A') may further have the following characteristic (1') or (2'), preferably characteristic (1').

[0130] The affinity polypeptide containing only one specific reactive group can be an affinity peptide that has affinity for the heavy chain CH1 region of the above-mentioned antibody and does not contain a lysine residue (nor does it contain amino acid residues having side chain amino groups other than lysine), for example, peptides (1) to (4), in which one amino acid residue has been replaced with an amino acid residue having a side chain amino group (lysine residue, ornithine residue, diaminobutanoic acid residue, diaminopimelic acid residue, etc.).

[0131] In the amino acid sequence of the affinity polypeptide, the amino acid residue to be substituted with an amino acid residue having a side chain amino group is not particularly limited, but is preferably an amino acid residue located near a target lysine present in the heavy chain CH1 region of the antibody in the three-dimensional configuration when the affinity polypeptide is coexisted with the antibody. Examples of target lysines include lysine at position 121, lysine at position 147, lysine at position 205, and lysine at position 210. "Nearby" means, for example, within 30 Å.

[0132] When the affinity polypeptide has the amino acid sequence of SEQ ID NO: 9, the specific amino acid residue to be substituted with an amino acid residue having a side chain amino group is selected from, for example, A at position 29, D at position 36, E at position 19, and E at position 15 of SEQ ID NO: 9. When the affinity polypeptide has an amino acid sequence containing one to several amino acid substitutions, deletions, additions, or insertions in the amino acid sequence of SEQ ID NO: 9, the specific amino acid residue is, for example, a position corresponding to A at position 29, D at position 36, E at position 19, or E at position 15. When the affinity polypeptide has an amino acid sequence that is 60% or more identical to the amino acid sequence of SEQ ID NO: 9, the specific amino acid residue is, for example, a position corresponding to A at position 29, D at position 36, E at position 19, or E at position 15.

[0133] When the target lysine is the lysine at position 121, the specific amino acid residue substituted with the lysine residue in SEQ ID NO: 9 is preferably A at position 29; when the target lysine is the lysine at position 147, the specific amino acid residue substituted with the lysine residue is preferably D at position 36; when the target lysine is the lysine at position 205, the specific amino acid residue substituted with the lysine residue is preferably E at position 19; and when the target lysine is the lysine at position 210, the specific amino acid residue substituted with the lysine residue is preferably E at position 15.

[0134] When the affinity polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 10, the specific amino acid residue to be substituted with an amino acid residue having a side chain amino group is selected from, for example, V at position 29, D at position 36, E at position 19, or E at position 15 of SEQ ID NO: 10. When the affinity polypeptide is a polypeptide having an amino acid sequence containing one to several amino acid substitutions, deletions, additions, or insertions in the amino acid sequence of SEQ ID NO: 10, the specific amino acid residue is, for example, a position corresponding to V at position 29, D at position 36, E at position 19, or E at position 15. When the affinity polypeptide is a polypeptide having an amino acid sequence that is 60% or more identical to the amino acid sequence of SEQ ID NO: 10, the specific amino acid residue is, for example, a position corresponding to V at position 29, D at position 36, E at position 19, or E at position 15.

[0135] When the target lysine is the lysine at position 121, the specific amino acid residue substituted with a lysine residue in SEQ ID NO: 10 is preferably V at position 29; when the target lysine is the lysine at position 147, the specific amino acid residue substituted with a lysine residue is preferably D at position 36; when the target lysine is the lysine at position 205, the specific amino acid residue substituted with a lysine residue is preferably E at position 19; and when the target lysine is the lysine at position 210, the specific amino acid residue substituted with a lysine residue is preferably E at position 15.

[0136] 2-2. Related Inventions Regarding Affinity Polypeptides or Their Salts When the affinity polypeptides or their salts of the present invention are affinity polypeptides comprising an affinity peptide having affinity for the heavy chain CH1 region of an antibody, such affinity polypeptides can be prepared using a host cell comprising an expression unit comprising a polynucleotide encoding the affinity polypeptide and a promoter operably linked thereto, or using a cell-free system, etc. The present invention also provides such polynucleotides and host cells, as well as expression vectors that can be used to produce the host cells.

[0137] The polynucleotide of the present invention is a polynucleotide that encodes the affinity polypeptide of the present invention. The polynucleotide of the present invention may be DNA or RNA, but is preferably DNA.

[0138] The host cell of the present invention can be produced, for example, by a method using an expression vector containing a polynucleotide of the present invention (e.g., a competent cell method, an electroporation method), or by genome modification technology. When the expression vector is an integrative vector that undergoes homologous recombination with the genomic DNA of the host cell, the expression unit can be integrated into the genomic DNA of the host cell by transformation. On the other hand, when the expression vector is a non-integrative vector that does not undergo homologous recombination with the genomic DNA of the host cell, the expression unit is not integrated into the genomic DNA of the host cell by transformation, and can exist in the host cell as an expression vector, independent of the genomic DNA. Alternatively, genome editing technology (e.g., the CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)) can be used to integrate the expression unit into the genomic DNA of the host cell and modify the expression unit inherently contained in the host cell.

[0139] The present invention also provides an expression vector comprising a polynucleotide of the present invention and a promoter operably linked thereto. The expression vector of the present invention may further comprise elements that function in host cells, such as a terminator, a ribosome binding site, and a drug resistance gene. Examples of drug resistance genes include genes that are resistant to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0140] The expression vector may further comprise a region that enables homologous recombination with the genome of the host cell for homologous recombination with the genomic DNA of the host cell. For example, the expression vector may be designed so that the expression unit contained therein is located between a pair of homologous regions (e.g., homology arms homologous to a specific sequence in the genome of the host cell, or loxP, or FRT). The genomic region of the host cell into which the expression unit is to be introduced (the target of the homologous region) is not particularly limited, and may be the locus of a gene that is highly expressed in the host cell.

[0141] The expression vector may be a plasmid, a viral vector, a phage, or an artificial chromosome. The expression vector may also be an integrative vector or a non-integrative vector. An integrative vector may be a vector that is integrated in its entirety into the genome of a host cell. Alternatively, an integrative vector may be a vector that is integrated only in part (e.g., an expression unit) into the genome of a host cell. The expression vector may further be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and derivatives thereof.

[0142] Host cells for expressing the affinity polypeptide of the present invention include various prokaryotic cells such as Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (e.g., Corynebacterium glutamicum), and Bacillus bacteria (e.g., Bacillus subtilis), as well as Saccharomyces bacteria (e.g., Saccharomyces cerevisiae), Pichia bacteria (e.g., Pichia stipitis), and Aspergillus bacteria (e.g., Aspergillus oryzae). Various eukaryotic cells, including E. oryzae, can be used as hosts. Alternatively, insect cells, plant cells, and animal cells (e.g., mammalian cells such as Chinese hamster ovary (CHO) cells) can be used as hosts. A strain lacking a specific gene may also be used as a host. Examples of host cells include host cells that carry an expression vector in the cytoplasm and host cells into which a target gene has been introduced into the genome.

[0143] When the affinity polypeptide of the present invention contains a tripeptide consisting of Gln-Glu-Thr (QET) at the N-terminus, it is preferable to use an affinity polypeptide that can be prepared by a polypeptide secretion production method using coryneform bacteria as a host (WO 2013 / 062029). This method can add the N-terminal three residues of the Csp mature protein, Gln-Glu-Thr (QET), to the N-terminus of the target polypeptide, and can easily prepare large amounts of polypeptides containing a glutamine residue (Q) at the N-terminus, making it suitable for preparing affinity polypeptides. In this case, various signal peptides, such as a signal peptide (CspBss) consisting of the amino acid sequence MFNNRIRTAALAGAIAISTAASGVAIPAFA (SEQ ID NO: 31), can be added to the N-terminus of QET (see Examples, WO 2013 / 062029, WO 2020 / 090979). Examples of coryneform bacteria that can be used in this method include bacteria of the genus Corynebacterium (eg, Corynebacterium glutamicum, Corynebacterium stationis) and bacteria of the genus Brevibacterium.

[0144] The host cells of the present invention can be cultured in a medium having the composition described below using a predetermined culture device (e.g., test tube, flask, or jar fermenter). Culture conditions can be set appropriately. Specifically, the culture temperature may be 10°C to 37°C, the pH may be 6.5 to 7.5, and the culture time may be 1 hour to 100 hours. Culture may also be performed while controlling the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture medium may be used as a control index. Aeration and agitation conditions can be controlled so that the relative dissolved oxygen concentration (DO value) when the atmospheric oxygen concentration is 21% does not fall below, for example, 1% to 10%, preferably 3% to 8%. Culture may be performed by batch or fed-batch culture. In fed-batch culture, the culture can be continued by sequentially adding a solution serving as a sugar source or a solution containing phosphate to the culture medium, either continuously or discontinuously.

[0145] As a promoter for expressing the polynucleotide of the present invention, a promoter typically used for heterologous protein production in E. coli can be used, and examples thereof include strong promoters such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and T5 promoter, with PhoA, PhoC, and lac being preferred. Furthermore, examples of vectors that may be used include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and derivatives thereof.

[0146] Furthermore, a terminator, which is a transcription termination sequence, may be ligated downstream of the polynucleotide of the present invention. Examples of such terminators include the T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and Escherichia coli trpA gene terminator.

[0147] The medium may be a medium commonly used for culturing Escherichia coli, such as M9-casamino acid medium or LB medium. The medium may contain a predetermined carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, the medium may contain peptone, yeast extract, NaCl, glucose, MgSO4, etc. ４ ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc. may also be used. The culture conditions and production induction conditions are appropriately selected depending on the type of marker, promoter, host bacterium, etc. of the vector used.

[0148] The affinity polypeptide of the present invention can be recovered by the following methods. The affinity polypeptide of the present invention can be obtained as a disruptant or lysate by recovering the transformed cells of the present invention and then disrupting (e.g., sonication, homogenization) or lysing (e.g., lysozyme treatment) the cells. If the affinity polypeptide is secreted or leaked outside the cells, a sterilized solution containing the affinity polypeptide can be obtained from the culture medium by centrifugation or membrane filtration. The affinity polypeptide of the present invention can be obtained by subjecting such disruptant, lysate, or sterilized solution to techniques such as extraction, precipitation, filtration, and column chromatography.

[0149] 3. Compound or Salt Thereof The compound or salt thereof of the present invention comprises (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and (B) a group reactive with the antibody. The definitions, examples, and preferred examples of the affinity polypeptide and its constituent elements (e.g., affinity moieties such as affinity peptides) are as described above.

[0150] As the antibody-reactive group, a group reactive to an amino acid residue having a reactive side chain among the amino acid residues constituting an antibody (protein) can be used. Of the 20 naturally occurring amino acids constituting proteins as described above, glycine, which has no side chain, and alanine, isoleucine, leucine, phenylalanine, and valine, which have hydrocarbon side chains, are inactive in normal reactions. Therefore, the antibody-reactive group is a group capable of reacting with the side chains of one or more (e.g., two, three, or four) of the 14 amino acids consisting of asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Depending on conditions such as the amino acid composition of the antibody, one or more (e.g., two, three, or four) reactive groups may be contained in the compound of the present invention or a salt thereof. Preferably, the compound of the present invention or a salt thereof contains only one reactive group.

[0151] Preferably, the antibody-reactive group is a group capable of reacting with the side chain of any one of the 14 amino acids constituting proteins. The antibody-reactive group is more preferably a group specifically reactive with the side chain of any one of the amino acids lysine, tyrosine, tryptophan, or cysteine, even more preferably a group specifically reactive with the side chain of any one of the amino acids lysine, tyrosine, or tryptophan, and particularly preferably a group specifically reactive with the side chain of lysine or tyrosine, especially the side chain of lysine. For details of such reactive groups, see, for example, WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979.

[0152] The reactive group specific to the side chain of a lysine residue is the amino group (NH ２ ), and examples thereof include activated ester residues (e.g., N-hydroxysuccinimide residues), vinyl sulfone residues, sulfonyl chloride residues, isocyanate residues, isothiocyanate residues, aldehyde residues, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues, 2-imino-2-methoxyethyl residues, diazonium terephthalic acid residues, α-halogen-substituted acetamides, and α-halogen-substituted methyl ketones. ２ ) can produce, for example, an amide residue, a urea residue, a pyridine residue, a carbamate residue, or a sulfonamide residue as a linking moiety.

[0153] In certain embodiments, the compound of the present invention or a salt thereof has the following formula (I): [wherein R represents a group reactive with an antibody, L represents a linker, and A represents an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody.] The definitions, examples, and preferred examples of the group reactive with an antibody represented by R and the affinity polypeptide represented by A are as described above.

[0154] The linker is a divalent group. The divalent group may be substituted or unsubstituted. Examples of the divalent group include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0155] In certain embodiments, the compound of the present invention or a salt thereof may further comprise a cleavable moiety between the affinity polypeptide and the antibody-reactive group. In this case, the compound represented by formula (I) or a salt thereof may comprise a linker containing the cleavable moiety.

[0156] A cleavable moiety is a site that can be cleaved by a specific treatment under conditions (mild conditions) that do not cause denaturation or decomposition of a protein (e.g., cleavage of an amide bond). Therefore, a cleavable moiety can be said to be a site (a bond other than an amide bond) that can be cleaved by a specific cleavage treatment under mild conditions. Examples of such specific treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes, (b) treatment with physicochemical stimuli such as light, or (c) incubation when a cleavable linker containing a self-degrading cleavable moiety is used. Such cleavable linkers and the cleavage conditions thereof are common knowledge in the art (e.g., G. Leriche, L. Chisholm, A. Wagner, Bioorganic & Medicinal Chemistry. 20, 571 (2012); Feng P. et al., Journal of American Chemical Society. 132, 1500 (2010); Bessodes M. et al., Journal of Controlled Release, 99, 423 (2004); DeSimone, J.M., Journal of American Chemical Society. Society. 132, 17928 (2010); Thompson, D. H., Journal of Controlled Release, 91, 187 (2003); Schoenmarks, R. G., Journal of Controlled Release, 95, 291 (2004)). Reaction conditions for mild conditions (e.g., reaction temperature, reaction time, reaction solution) are as described below.Examples of the cleavable moiety include disulfide residues, acetal residues, ketal residues, ester residues, carbamoyl residues, alkoxyalkyl residues, imine residues, tertiary alkyloxycarbamate residues (e.g., tert-butyloxycarbamate residues), silane residues, hydrazone-containing residues (e.g., hydrazone residues, acylhydrazone residues, bisarylhydrazone residues), phosphoramidate residues, aconityl residues, trityl residues, azo residues, vicinal diol residues, selenium residues, aromatic ring-containing residues having an electron-withdrawing group, coumarin-containing residues, sulfone-containing residues, unsaturated bond-containing chain residues, and glycosyl residues.

[0157] The aromatic ring group having an electron-withdrawing group is preferably one having an aromatic ring group selected from the group consisting of aryl, aralkyl, aromatic heterocyclic group, and alkyl having an aromatic heterocyclic group, and more preferably aralkyl or alkyl having an aromatic heterocyclic group. The electron-withdrawing group is preferably bonded to the 2-position of the ring. Even more preferably, the aromatic ring-containing residue having an electron-withdrawing group is, for example, an aralkyl (e.g., benzyl) having an electron-withdrawing group at the 2-position. Examples of the electron-withdrawing group include halogen atoms, alkyl substituted with halogen atoms (e.g., trifluoromethyl), boronic acid residues, mesyl, tosyl, triflate, nitro, cyano, phenyl group, and keto group (e.g., acyl).

[0158] The definitions, examples, and preferred examples of groups such as alkyl, acyl (i.e., alkylcarbonyl), alkoxy (i.e., alkyloxy), aryl, aralkyl, etc. found as prefixes, suffixes, etc. in connection with the names of residues as cleavable moieties are the same as those described above.

[0159] Examples of the ester residue include ordinary ester residues composed of carbon atoms and oxygen atoms [e.g., alkyl esters (e.g., tertiary alkyloxycarbonyl such as tert-butyloxycarbonyl), aryl esters (e.g., phenacyl ester, 2-(diphenylphosphino)benzoate), glycosyl ester residues, and orthoester residues], ester residues containing a sulfur atom and an oxygen atom (e.g., thioester residues such as α-thiophenyl ester residues and alkylthioester residues), ester residues containing a phosphorus atom and an oxygen atom (e.g., phosphodiester residues, phosphotriester residues), and activated ester residues (e.g., N-hydroxysuccinimide residues).

[0160] Examples of sulfone-containing residues include sulfone residues and quinolinylbenzenesulfonate residues.

[0161] The silane residue is preferably a silane residue having a group selected from the group consisting of alkyl, aryl, aralkyl, and alkoxy. Examples of such silane residues include dialkyldialkoxysilane residues (e.g., dimethyldialkoxysilane, diethyldialkoxysilane) and diaryldialkoxysilane residues (e.g., diphenyldialkoxysilane).

[0162] Alkoxyalkyl (i.e., alkyloxyalkyl) residues are groups that combine alkyloxy and alkyl as described above, and include, but are not limited to, methoxymethyl, ethoxymethyl, methoxyethyl, and ethoxyethyl residues.

[0163] The unsaturated bond-containing chain residue is a residue containing an unsaturated bond moiety consisting of only carbon atoms (e.g., vinyl (ethenyl), the smallest unit having a double bond between carbon atoms, or acetylenyl (ethynyl), the smallest unit having a triple bond between carbon atoms), or a residue containing an unsaturated bond moiety (e.g., aldehyde, cyano) consisting of carbon atoms and heteroatoms (e.g., nitrogen atom, sulfur atom, oxygen atom). Examples of the unsaturated bond-containing chain residue include vinyl ether residue, cyanoethyl residue, ethylene residue, and malondialdehyde residue.

[0164] Examples of acidic substances (also referred to as electrophiles) include inorganic acidic substances such as hydrochloric acid, sulfuric acid, and nitric acid, and organic acidic substances such as formic acid, acetic acid, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 3-morpholinopropanesulfonic acid, sodium dihydrogenphosphate, citric acid, dodecylsulfuric acid, N-dodecanoylsarcosinic acid, and trifluoroacetic acid. Examples of sites that can be cleaved by acidic substances include alkyloxyarylalkyl residues, tertiary alkyloxycarbamate residues, acetal residues, silane residues, imine residues, vinyl ether residues, β-thiopropionate residues, trityl residues, hydrazone residues, aconityl residues, orthoester residues, carbamoyl residues, and 2-(diphenylphosphino)benzoate residues.

[0165] Examples of basic substances (also referred to as nucleophiles) include inorganic basic substances such as sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, ammonium acetate, etc., and organic basic substances such as hydroxylamine, triethylamine, N,N'-diisopropylamine, etc. Examples of sites that can be cleaved by basic substances include silane residues, cyanoethyl residues, sulfone residues, ethylene residues, glycosyl disuccinate residues, α-thiophenyl ester residues, unsaturated vinyl sulfide residues, malondialdehyde residues, acylhydrazone residues, and alkylthioester residues.

[0166] 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.

[0167] Examples of oxidizing agents include sodium periodate and oxidized glutathione. Examples of sites that can be cleaved by an oxidizing agent include vicinal diol residues and selenium residues.

[0168] Examples of the enzyme include trypsin, papain, TEV, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase, protease, matrix metalloprotease, lipase, endoglycosidase, and PN gauze F. Examples of the site cleavable by the enzyme include an ester residue, a phosphodiester residue, and a glycosyl residue.

[0169] Examples of the photocleavable moiety include a 2-nitrobenzyl residue, a phenacyl ester residue, an 8-quinolinebenzenesulfonate residue, a coumarin residue, a phosphotriester residue, a bisarylhydrazone residue, and a bimandithiopropionic acid residue.

[0170] Autolytic cleavable moieties include, for example, activated ester residues (eg, N-hydroxysuccinimide residues).

[0171] When the compound of the present invention or a salt thereof contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthogonal functional group on the reactive group side upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including conventional ester residues and other ester residues such as thioester residues), acetal residues (including conventional ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.

[0172] When the compound of the present invention or a salt thereof contains a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, it can be represented by the following formula (Ia): [wherein R represents a group reactive to an antibody, L １ indicates the first linker, L ２represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, and A represents an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody. The definitions, examples, and preferred examples of the antibody-reactive group represented by R, the affinity polypeptide represented by A, and the cleavable moiety represented by CLE(B) capable of generating a bioorthogonal functional group on the reactive group side upon cleavage are as described above.

[0173] L １ a first linker represented by ２ The second linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0174] In certain embodiments, the total number of atoms constituting the main chain of the first linker and the second linker may be 2 to 10. This total number of atoms may be 3 or more, or 4 or more. This total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, this total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0175] The number of atoms constituting the main chain of each of the first linker and the second linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0176] The main chains of the first linker and the second linker are composed of a chain structure, a cyclic structure, or a structure including a combination thereof. When the main chain is a chain structure that does not include a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure. On the other hand, when the main chain includes a cyclic structure, the number of atoms in the main chain can be determined by counting the number of atoms constituting the cyclic structure as the number of atoms in the main chain. Specifically, the number of atoms in the main chain in a cyclic structure can be determined by counting the number of atoms in the shortest path connecting two bonds in the cyclic structure (see, for example, the bolded paths in (a) to (d) below). When the main chain includes a combination of a chain structure and a cyclic structure, the number of atoms in the main chain can be determined by adding the number of atoms in the chain structure that does not include a cyclic structure to the number of atoms in the shortest path connecting two bonds in the cyclic structure. The method of counting the number of atoms in the main chain is similar for other linkers. - is a bond. In the case of (a), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 2. In the case of (b), the shortest path is the bold path, so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 3. In the case of (c), both paths are the shortest paths (equidistant), so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4. In the case of (d), the path of the condensation site is the shortest path, so the number of atoms in the divalent cyclic structure counted as the number of atoms in the main chain is 4.

[0177] In certain embodiments, the compound of the present invention or a salt thereof has the following formula (Ia-1): [wherein X represents a leaving group; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ indicates a third linker, L ４represents a fourth linker, S represents a sulfur atom, and A represents an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody. The definition, examples, and preferred examples of the affinity polypeptide represented by A are as described above.

[0178] The leaving group represented by X is a group that can be eliminated by a reaction between the carbon atom in C═W1 adjacent to X and an amino group. Those skilled in the art can appropriately select such a leaving group. Examples of such leaving groups include the following: (a) R Ａ -S (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom; (b) R Ａ -O (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom; (c) R Ａ - (R Ｂ -) N (where R Ａ and R Ｂ each independently represent a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom; or (d) a halogen atom.

[0179] Preferably, the leaving group represented by X may be: (a) R Ａ -S (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom; (b) R Ａ -O (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom; or (c) R Ａ - (R Ｂ -) N (where R Ａ and R Ｂeach independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom.

[0180] More preferably, the leaving group represented by X may be: (a) R Ａ -S (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom; or (b) R Ａ -O (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom.

[0181] Even more preferably, the leaving group represented by X may be: (a) R Ａ -S (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom.

[0182] Particularly preferably, the leaving group represented by X may be: (a') R Ａ -S (where R Ａ represents an optionally substituted monovalent aromatic hydrocarbon group (e.g., phenyl), and S represents a sulfur atom.

[0183] W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom. １ , W ２ and W ３ may be an oxygen atom.

[0184] L ３ a third linker represented by ４The fourth linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0185] In certain embodiments, the total number of atoms constituting the main chain of the third linker and the fourth linker may be 2 to 10. This total number of atoms may be 3 or more, or 4 or more. This total number of atoms may be 9 or less, 8 or less, or 7 or less. More specifically, this total number of atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0186] The number of atoms constituting the main chain of each of the third linker and the fourth linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0187] When the compound of the present invention or a salt thereof contains a cleavable moiety, it may further contain a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety. The bioorthogonal functional group is as described above. Preferably, the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with a substituent as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue.

[0188] When the compound of the present invention or a salt thereof further comprises a bioorthogonal functional group between the antibody-reactive group and the cleavable moiety, it can be represented by the following formula (Ib): [wherein R represents a group reactive to an antibody, L ５ indicates the fifth linker, L ６represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, and A represents an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody. The definitions, examples, and preferred examples of the antibody-reactive group represented by R, the affinity polypeptide represented by A, and the cleavable moiety represented by CLE are as described above.

[0189] L ５ a fifth linker represented by L ６ The sixth linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0190] In certain embodiments, the total number of atoms constituting the main chain in the fifth linker and the sixth linker may be 2 to 10. The total number of such atoms may be 3 or more, or 4 or more. The total number of such atoms may be 9 or less, 8 or less, or 7 or less. More specifically, the total number of such atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0191] The number of atoms constituting the main chain of each of the fifth linker and the sixth linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0192] The group containing a bioorthogonal functional group represented by B may be a group consisting of a bioorthogonal functional group, or may be a group containing a bioorthogonal functional group and another moiety. Examples of other moieties include the linking moiety between the bioorthogonal functional group and the linker. The linking moiety is, for example, a divalent group. The divalent group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the bioorthogonal functional group, the divalent group, and the substituent when the divalent group is substituted are as described above.

[0193] In certain embodiments, the compound of the present invention or a salt thereof has the following formula (Ib-1): [wherein X represents a leaving group; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ indicates the seventh linker, L ８ represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, and A represents an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody. The compound may be a compound represented by the formula (I) or a salt thereof. The definitions, examples, and preferred examples of the leaving group represented by X, the group containing a bioorthogonal functional group represented by B, and the affinity polypeptide represented by A are as described above.

[0194] W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom. １ , W ２ and W ３ may be an oxygen atom.

[0195] L ７ a seventh linker represented by L ８ The eighth linkers represented by may be the same or different divalent groups. The divalent groups may be substituted or unsubstituted. Examples of the divalent groups include those described above. When the divalent group is substituted, examples of the substituent include those described above.

[0196] In certain embodiments, the total number of atoms constituting the main chain of the seventh linker and the eighth linker may be 2 to 10. The total number of such atoms may be 3 or more, or 4 or more. The total number of such atoms may be 9 or less, 8 or less, or 7 or less. More specifically, the total number of such atoms may be 3 to 9, 4 to 8, or 4 to 7.

[0197] The number of atoms constituting the main chain of each of the seventh linker and the eighth linker may be 1 to 9. Such number of atoms may be 2 or more, or 3 or more. Such number of atoms may be 8 or less, 7 or less, or 6 or less. More specifically, such number of atoms may be 2 to 8, 3 to 7, or 3 to 6.

[0198] V represents an oxygen atom or a sulfur atom, and preferably, V may be a sulfur atom.

[0199] The above series of compounds or salts thereof can be prepared by reacting the affinity polypeptide of the present invention with a moiety containing a reactive group for an antibody. For example, such a reaction can be carried out in a suitable organic solvent (e.g., CH ２ Cl ２ The reaction can be carried out in an organic solvent containing an alkyl halide (e.g., methyl halide) such as methyl amine, and an amine such as triethylamine, at an appropriate temperature (e.g., about −10 to 30° C.). The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.

[0200] The production of the above-mentioned series of compounds or salts thereof can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), NMR, or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Such compounds or salts thereof can be appropriately purified by any method such as chromatography (e.g., the above-mentioned chromatography and affinity chromatography).

[0201] 4. Affinity Polypeptide-Modified Antibodies or Salts Thereof 4-1. Affinity Polypeptide-Modified Antibodies or Salts Thereof Comprising an Affinity Polypeptide (Including an Affinity Moiety) The present invention provides affinity polypeptide-modified antibodies or salts thereof, which comprise, in the CH1 region of the antibody heavy chain, an affinity polypeptide comprising an affinity moiety that has affinity for the CH1 region of the antibody heavy chain. Definitions, examples, and preferred examples of affinity polypeptides and antibodies, as well as components thereof (e.g., affinity moieties such as affinity peptides, and constant regions), are as described above.

[0202] Preferably, the affinity polypeptide-modified antibody or its salt comprises (a) an antibody structural unit (an immunoglobulin unit including a heavy chain and a light chain), and (b) an affinity polypeptide, and (c) the affinity polypeptide may be introduced into the heavy chain CH1 region of the immunoglobulin unit. The definitions, examples, and preferred examples of antibodies, immunoglobulin units, and affinity polypeptides, as well as their constituent elements (e.g., constant regions), are as described above.

[0203] The affinity polypeptide-modified antibody or its salt can contain an affinity polypeptide through modification of a functional group in the side chain of one or more (e.g., two, three, or four) of the 14 amino acid residues present in the CH1 region: asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. The affinity polypeptide-modified antibody or its salt can contain an affinity polypeptide through modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, tryptophan, or cysteine ​​present in the CH1 region, preferably through modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, or tryptophan, even more preferably through modification of a functional group in the side chain of lysine or tyrosine, and particularly preferably through modification of the amino group in the side chain of lysine. The positions of these amino acid residues in the CH1 region are as described above. The positions of the antibody or its salt modified with the affinity polypeptide can be confirmed by peptide mapping. The modification may be regioselective, as described above. Thus, in the formulae (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin units may have the corresponding modifying units regioselectively via functional groups in the side chains of the amino acid residues.

[0204] Preferably, the affinity polypeptide-modified antibody can contain the affinity polypeptide via modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the CH1 region of the heavy chain in the antibody building block (immunoglobulin unit including a heavy chain and a light chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be positions 121, 147, 205, or 210 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Thus, in the immunoglobulin units of formulas (II), (IIa), (IIa-1), (IIb), and (IIb-1) described below, the immunoglobulin unit may have the corresponding modifying unit regioselectively via the amino group in the side chain of the lysine residue.

[0205] The affinity polypeptide-modified antibody or its salt can be produced by reacting the compound of the present invention or its salt with an antibody (including an antibody fragment) or its salt comprising an immunoglobulin unit including a heavy chain and a light chain. The amount of the compound of the present invention or its salt relative to the antibody in the reaction (compound of the present invention or its salt / antibody) is not particularly limited, as it varies depending on factors such as the type of compound of the present invention or its salt and the antibody, but is, for example, 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 40, and particularly preferably 6 to 20.

[0206] Such a reaction can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, a reaction under such mild conditions can be carried out in an appropriate reaction system, such as a buffer solution, at room temperature (e.g., about 15 to 30°C). The pH of the buffer solution is, for example, 5 to 9, preferably 5.5 to 8.5, more preferably 7.0 to 8.5, and even more preferably 7.4 to 8.2. The buffer solution may contain a suitable catalyst. The reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours. For details of such a reaction, see, for example, G. J. L. Bernardes et al., Chem. Rev., 115, 2174 (2015); G. J. L. Bernardes et al., Chem. Asian. J., 4, 630 (2009); B. G. Davies et al., Nat. Commun., 5, 4740 (2014); A. Wagner et al., Bioconjugate. Chem., 25, 825 (2014).

[0207] In certain embodiments, the affinity polypeptide-modified antibody or salt thereof has the following formula (II): [wherein Ig represents an immunoglobulin unit comprising a heavy chain and a light chain, L represents a linker, A represents an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and the average modification percentage r of the immunoglobulin unit with the affinity polypeptide is 65 to 135%.] The definitions, examples, and preferred examples of the immunoglobulin unit represented by Ig, the linker represented by L, the affinity polypeptide represented by A, and the antibody are as described above (for example, with regard to the linker represented by L, see the linker represented by L in the compound of formula (I)).

[0208] The average modification percentage r of the immunoglobulin units with the affinity polypeptide is 65 to 135%. Here, the average modification percentage r refers to the average modification rate per modification site in the heavy chain CH1 region of the immunoglobulin units. For example, when the modification site is a specific lysine present in the heavy chain CH1 region, the average modification percentage r indicates the average proportion of immunoglobulin units in which an affinity peptide has been introduced at that lysine per total immunoglobulin units. 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 (which can be performed in conjunction with DARcalculator (Agilent software); see Examples).

[0209] The average modification percentage r may also be preferably 65 to 100%, more preferably 70 to 100%, even more preferably 75 to 100%, and particularly preferably 80 to 100%, 85 to 100%, 90 to 100%, or 95 to 100%. The upper limit of these average modification percentages may be a value equal to or less than the aforementioned percentages, such as 98% or less, or 96% or less. Alternatively, the average modification percentage r may be 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100%.

[0210] The above-mentioned degree of the average modification percentage r can be similarly applied to other average modification percentages r. That is, the above-mentioned degree of the average modification percentage r can be similarly applied not only to the below-described average modification percentage r by an affinity polypeptide, but also to the below-described average modification percentage r by any modification (e.g., a bioorthogonal functional group, a functional substance).

[0211] An antibody comprising a structural unit represented by formula (II) or a salt thereof can be produced by reacting a compound represented by formula (I) or a salt thereof with an antibody (including an antibody fragment) or a salt thereof comprising an immunoglobulin unit comprising a heavy chain and a light chain.

[0212] In certain embodiments, the affinity polypeptide-modified antibody or its salt may further comprise a cleavable moiety between the affinity polypeptide and the antibody (immunoglobulin unit). In this case, the antibody or its salt comprising the structural unit represented by formula (II) above may comprise a linker comprising a cleavable moiety. The definition, examples, and preferred examples of the cleavable moiety are as described above.

[0213] When the affinity polypeptide-modified antibody or its salt contains a cleavable moiety, the cleavable moiety may be capable of generating a bioorthogonal functional group on the antibody (immunoglobulin unit) upon cleavage. Examples of such cleavable moieties include disulfide residues, ester residues (including conventional ester residues and other ester residues such as thioester residues), acetal residues (including conventional ester residues and other acetal residues such as thioacetal residues), ketal residues, imine residues, and vicinal diol residues.

[0214] When the affinity polypeptide-modified antibody or a salt thereof contains a cleavable moiety capable of generating a bioorthogonal functional group on the antibody (immunoglobulin unit) side upon cleavage, the affinity polypeptide-modified antibody or a salt thereof may be represented by the following formula (IIa): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; １ indicates the first linker, L ２ represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, A represents an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and the average modification percentage r of the immunoglobulin unit by the affinity polypeptide is 65 to 135%. １ a first linker represented by L ２The definitions, examples, and preferred examples of the second linker, denoted by CLE(B), the cleavable moiety, denoted by A, the affinity polypeptide, and the average modification percentage, denoted by r, and the antibody are as described above.

[0215] An antibody comprising a structural unit represented by formula (IIa) or a salt thereof can be produced by reacting a compound represented by formula (Ia) or a salt thereof with an antibody (including an antibody fragment) or a salt thereof comprising an immunoglobulin unit comprising a heavy chain and a light chain.

[0216] In certain embodiments, the affinity polypeptide-modified antibody or salt thereof has the following formula (IIa-1): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; W represents an immunoglobulin unit containing a heavy chain and a light chain; １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ indicates a third linker, L ４ represents a fourth linker, S represents a sulfur atom, A represents an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and the average modification percentage r of the immunoglobulin unit by the affinity polypeptide is 65 to 135%. １ , W ２ and W ３ An atom represented by L ３ a third linker represented by L ４ The definitions, examples, and preferred examples of the fourth linker, denoted by A, the affinity polypeptide, denoted by A, and the average modification percentage, denoted by r, and the antibody are as described above.

[0217] An antibody comprising a structural unit represented by formula (IIa-1) or a salt thereof can be produced by reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody (including an antibody fragment) or a salt thereof comprising an immunoglobulin unit comprising a heavy chain and a light chain.

[0218] When the affinity polypeptide-modified antibody or its salt contains a cleavable moiety, it may further contain a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety. The bioorthogonal functional group is as described above. Preferably, the bioorthogonal functional group is an azide residue, an alkyne residue (preferably a ring group having a triple bond between carbon atoms, which may be substituted with a substituent as described above), a tetrazine residue, an alkene residue, a thiol residue, a maleimide residue, a thiol residue, a furan residue, or a halocarbonyl residue.

[0219] When the affinity polypeptide-modified antibody or a salt thereof further comprises a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety, it can be represented by the following formula (IIb): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; ５ represents the fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, A represents an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and the average modification percentage r of the immunoglobulin unit by the affinity polypeptide is 65 to 135%. ５ a fifth linker represented by L ６ The definitions, examples, and preferred examples of the sixth linker represented by (I), the group containing a bioorthogonal functional group represented by (B), the cleavage site represented by (CLE), the affinity polypeptide represented by (A), and the average modification percentage represented by (r), as well as the antibody, are as described above.

[0220] An antibody comprising a structural unit represented by formula (IIb) or a salt thereof can be produced by reacting a compound represented by formula (Ib) or a salt thereof with an antibody (including an antibody fragment) or a salt thereof comprising an immunoglobulin unit comprising a heavy chain and a light chain.

[0221] In certain embodiments, the affinity polypeptide-modified antibody or salt thereof has the following formula (IIb-1): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ indicates the seventh linker, L ８ represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, A represents an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody, and the average modification percentage r of the immunoglobulin unit by the affinity polypeptide is 65 to 135%. The immunoglobulin unit represented by Ig, W １ , W ２ and W ３ An atom represented by L ７ A seventh linker represented by L ８ The definitions, examples, and preferred examples of the eighth linker represented by (I), the group containing a bioorthogonal functional group represented by (B), the atom represented by (V), the affinity polypeptide represented by (A), and the average modification percentage represented by (r), as well as the antibody, are as described above.

[0222] An antibody comprising a structural unit represented by formula (IIb-1) or a salt thereof can be produced by reacting a compound represented by formula (Ib-1) or a salt thereof with an antibody (including an antibody fragment) or a salt thereof comprising an immunoglobulin unit comprising a heavy chain and a light chain.

[0223] The affinity polypeptide-modified antibody or its salt may further comprise an additional modifying moiety. Various methods are known for modifying antibodies. Thus, in the present invention, the affinity polypeptide-modified antibody or its salt may be modified to further comprise an additional modifying moiety. The additional modifying moiety may be introduced into the heavy chain or light chain of the antibody, preferably into the heavy chain of the antibody (particularly into the constant region of the heavy chain).

[0224] In certain embodiments, the additional modifying moiety may be an additional affinity polypeptide comprising an additional affinity moiety that has affinity for the constant region of the antibody heavy chain (e.g., an additional affinity peptide comprising an additional affinity moiety that has affinity for the constant region of the antibody heavy chain). The additional affinity moiety may be an additional affinity moiety that has affinity for the CH1 region, like the affinity moieties described above, or may be an additional affinity moiety that has affinity for a different region. That is, the additional affinity moiety may be an additional affinity polypeptide comprising an additional affinity moiety that has affinity for a region of the constant region of the antibody heavy chain other than the CH1 region, such as the Fc region, CH2 region, or CH3 region.

[0225] In certain embodiments, an additional modifying moiety having affinity for the heavy chain constant region of an antibody may be introduced into the heavy chain constant region via modification of the amino group in the side chain of a lysine residue present at one or more positions in the heavy chain constant region. An affinity polypeptide-modified antibody or a salt thereof can contain an additional modifying moiety via modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the heavy chain constant region (preferably the Fc region or CH2 domain) in the antibody structural unit (an immunoglobulin unit comprising a heavy chain and a light chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be selected from positions 246, 248, 274, 288, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, WO 2020 / 090979).

[0226] Many peptides have been reported as affinity peptides that have affinity for the constant region of an antibody heavy chain and contain one lysine residue (see, for example, WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, and WO 2020 / 090979). Therefore, such peptides can be used as additional affinity peptides in the present invention.

[0227] The additional modification moiety may also be introduced into the heavy chain constant region via a cysteine ​​generated by reducing a disulfide bond. Such a cysteine ​​may be at a position selected from positions 220, 226, and 229 of the human IgG heavy chain according to EU numbering. The additional modification moiety may also be introduced into the light chain constant region via a cysteine ​​generated by reducing a disulfide bond. Such a cysteine ​​may be at position 214 of the human IgG light chain according to EU numbering.

[0228] An affinity polypeptide-modified antibody can have an affinity polypeptide depending on the number of heavy chains. For example, when an antibody having one antibody heavy chain (e.g., a Fab antibody) is used, it is possible to obtain an antibody having a structural unit (or a plurality of structural units of a lower concept) represented by L-A or the like regioselectively in the target region of one antibody heavy chain. On the other hand, when an antibody having two antibody heavy chains (e.g., an IgG, IgD, IgE, F(ab') ２ By using an affinity polypeptide-modified antibody, it is possible to obtain an antibody having two structural units (or multiple types of structural units, which are a subordinate concept) represented by L-A, etc., regioselectively in the same target region of two antibody heavy chains. In other words, in an affinity polypeptide-modified antibody, the modification pattern with the affinity polypeptide can be made the same among multiple (e.g., two) heavy chains.

[0229] The production of the desired affinity polypeptide-modified antibody or salt thereof can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Regioselectivity can be confirmed by peptide mapping. Peptide mapping can be performed, for example, by protease treatment and mass spectrometry. Preferred proteases are endoproteases. Examples of such endoproteases include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N. The number of affinity polypeptides introduced can be confirmed by mass spectrometry (which can be used in combination with DAR Calculator (Agilent software)). The affinity polypeptide-modified antibody or salt thereof can be appropriately purified by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0230] 5. Antibodies or salts thereof not containing affinity polypeptides 5-1. Overview An antibody or salt thereof not containing an affinity polypeptide can be produced using an affinity polypeptide-modified antibody or salt thereof that contains (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and further contains (C) a cleavable moiety between (A) the affinity polypeptide and (B) the antibody (immunoglobulin unit).

[0231] More specifically, the method for producing an antibody or a salt thereof that does not contain an affinity polypeptide may be the following method 1-1 or 1-2.

[0232] (Method 1-1) A method for producing an affinity polypeptide-free antibody or its salt, which comprises (A) an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and (C) an affinity polypeptide-modified antibody or its salt further comprising a cleavable moiety between the affinity polypeptide (A) and the antibody (B) (immunoglobulin unit), by cleaving the cleavable moiety to produce an affinity polypeptide-free antibody or its salt.

[0233] (Method 1-2) A method for producing an affinity polypeptide-free antibody or its salt, comprising the following steps (1) and (2): (1) reacting (A) an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit), and (B) a compound or its salt comprising a reactive group with an antibody (immunoglobulin unit) and (C) further comprising a cleavable moiety between (A) the affinity polypeptide and (B) the reactive group, with an antibody or its salt comprising an immunoglobulin unit comprising a heavy chain and a light chain, to produce an affinity polypeptide-modified antibody or its salt comprising a cleavable moiety between the affinity polypeptide and the antibody; and (2) cleaving the affinity polypeptide-modified antibody or its salt comprising a cleavable moiety between the affinity polypeptide and the antibody with the cleavable moiety, to produce an affinity polypeptide-free antibody or its salt.

[0234] Examples of cleavage treatments include (a) treatment with one or more substances selected from the group consisting of acidic substances, basic substances, reducing agents, oxidizing agents, and enzymes as described above, (b) treatment with physicochemical stimuli such as light, or (c) incubation when using a cleavable linker containing a self-degrading cleavable moiety. For these cleavage treatments, see International Publication Nos. WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, and WO 2020 / 090979.

[0235] Such cleavage reactions can be appropriately carried out under conditions (mild conditions) that do not cause denaturation or decomposition of the protein (e.g., cleavage of amide bonds). For example, such mild conditions are as described above. Furthermore, when the cleavable site is an ester (e.g., a normal ester or other ester such as a thioester), the cleavage reaction can be carried out by incubating in a hydroxylamine hydrochloride solution (e.g., pH 4.0 to 8.0, 10 mM to 10 M) for an appropriate time (e.g., 1 hour) (e.g., Vance, N. et al., Bioconjugate Chem. 2019, 30, 148-160).

[0236] The production of an affinity polypeptide-free antibody or a salt thereof obtained by the cleavage reaction can be confirmed by, for example, electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, depending on the specific raw materials and the molecular weight of the product. Regioselectivity can be confirmed by peptide mapping as described above. The number of affinity polypeptides introduced can be confirmed by mass spectrometry (which can be performed in combination with DAR Calculator (Agilent software)). The affinity polypeptide-modified antibody or a salt thereof can be appropriately purified by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0237] Incidentally, when an affinity polypeptide-modified antibody or its salt containing a cleavable moiety between (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit) and (B) the antibody (immunoglobulin unit) (a) contains a cleavable moiety that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side upon cleavage as the cleavable moiety, or (b) contains a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety, an antibody derivative or its salt containing a bioorthogonal functional group can be produced as an antibody or its salt that does not contain an affinity polypeptide.

[0238] Furthermore, by reacting an antibody derivative or its salt containing a bioorthogonal functional group with a functional substance, a conjugate or its salt of an antibody and a functional substance can be produced as an antibody or its salt that does not contain an affinity polypeptide.

[0239] Hereinafter, as antibodies or salts thereof that do not contain affinity polypeptides, (1) antibody derivatives or salts thereof that contain bioorthogonal functional groups, and (2) conjugates or salts thereof of antibodies and functional substances will be described in detail.

[0240] 5-2. Antibody Derivatives or Salts Comprising at Least One Bioorthogonal Functional Group The present invention provides antibody derivatives or salts thereof comprising a bioorthogonal functional group, which comprise (a) an antibody building block (an immunoglobulin unit comprising a heavy chain and a light chain), and (b) a bioorthogonal functional group, and (c) the bioorthogonal functional group is introduced into the heavy chain CH1 region of the immunoglobulin unit. The definitions, examples, and preferred examples of antibodies, immunoglobulin units, and bioorthogonal functional groups, as well as their constituent elements (e.g., constant regions), are as described above.

[0241] The antibody derivative or salt thereof can contain a bioorthogonal functional group via modification of a functional group in the side chain of one or more (e.g., two, three, or four) of the 14 amino acid residues present in the CH1 region: asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. The antibody derivative or salt thereof can contain a bioorthogonal functional group via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, tryptophan, or cysteine ​​present in the CH1 region, preferably via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, or tryptophan, even more preferably via modification of a functional group in the side chain of lysine or tyrosine, and particularly preferably via modification of an amino group in the side chain of lysine. The positions of these amino acid residues in the CH1 region are as described above. The modification position of an antibody or salt thereof with a bioorthogonal functional group can be confirmed by peptide mapping. The modification may be site-selective, as described above. Therefore, in the formulae (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit may have the corresponding modifying unit site-selectively via a functional group in the side chain of the amino acid residue.

[0242] Preferably, the antibody derivative can contain a bioorthogonal functional group via modification of an amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the CH1 region of the heavy chain in the antibody building block (immunoglobulin unit including a heavy chain and a light chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be positions 121, 147, 205, or 210 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Thus, in formulas (IIIa), (IIIa-1), (IIIb), and (IIIb-1) described below, the immunoglobulin unit may have the corresponding modifying unit regioselectively via the amino group in the side chain of the lysine residue.

[0243] In certain embodiments, the antibody derivative or salt thereof has the following formula (IIIa): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; １ represents a first linker, B represents a group containing a bioorthogonal functional group, and the average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%. １ The definitions, examples, and preferred examples of the first linker represented by (B), the group containing a bioorthogonal functional group represented by (B), and the antibody are as described above. A particularly preferred bioorthogonal functional group is a thiol group. Here, the average modification percentage r refers to the average modification rate per modification site in the heavy chain CH1 region of the immunoglobulin unit. For example, if the modification site is a specific lysine present in the heavy chain CH1 region, it indicates the average percentage of immunoglobulin units in which a group containing a bioorthogonal functional group has been introduced into that lysine per total immunoglobulin unit.

[0244] In formula (IIIa), L １ The molecular weight of the partial structure represented by -B may be 700 or less. １ When the molecular weight of the partial structure represented by -B is 700 or less, the ratio of the molecular weight of the partial structure to the molecular weight of the entire antibody is very small, making purification based on differences in molecular weight relatively difficult for antibody derivatives or salts thereof having a bioorthogonal functional group. However, according to the present invention, which enables advanced control of DAR, it is possible to obtain highly purified antibody derivatives exhibiting the desired DAR without necessarily requiring purification based on differences in molecular weight. １ The molecular weight of the partial structure represented by -B is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0245] In another specific embodiment, the antibody derivative or salt thereof has the following formula (IIIa-1): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; W １ represents an oxygen atom or a sulfur atom, L ２ represents a third linker, SH represents a thiol group, and the average modification percentage r of the immunoglobulin unit with the bioorthogonal functional group is 65 to 135%. １ An atom represented by L ３ The definitions, examples, and preferred examples of the third linker, denoted by , and the average modification percentage, denoted by r, and the antibody are as described above.

[0246] In formula (IIIa-1), C(=W1)-L ３ The molecular weight of the partial structure represented by —SH may be 700 or less. C(═W1)-L ３ The molecular weight of the partial structure represented by —SH is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, 150 or less, or 100 or less.

[0247] In formula (IIIa-1), L ３ The third linker represented by (CH ２ ) ｎ１ n1 may be an integer of 1 to 10. Preferably, n1 may be an integer of 2 or greater. n1 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Particularly preferably, n1 is 2.

[0248] In yet another specific embodiment, the antibody derivative or salt thereof has the following formula (IIIb): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; ５ represents a fifth linker, B represents a group containing a bioorthogonal functional group, and T １represents a monovalent group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.]. The immunoglobulin unit represented by Ig, L ５ The definitions, examples, and preferred examples of the fifth linker represented by B, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the antibody, are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[0249] T1 is a monovalent group and can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of the monovalent group include those described above. When the monovalent group is substituted, examples of the substituent include those described above.

[0250] In certain embodiments, T １ The monovalent group represented by the formula (α) may be an optionally substituted hydroxyamino group. The optionally substituted hydroxyamino group can be represented by the following formula (α): NR ｉ -OR ｉｉ (α) [wherein, R ｉ , R ｉｉ are each independently a hydrogen atom or a monovalent hydrocarbon group.) Here, the monovalent hydrocarbon group may be substituted or unsubstituted. The definitions, examples, and preferred examples of the monovalent hydrocarbon group and the substituents when the monovalent hydrocarbon group is substituted are as described above. Preferably, the optionally substituted hydroxyamino group is NH-OR ｉｉ (where R ｉｉ represents an alkyl group.) More preferably, the optionally substituted hydroxyamino group is ｉｉ (where R ｉｉ represents an alkyl group having 1 to 6 carbon atoms.

[0251] In formula (IIIb), L ５ (-B)-T １ The molecular weight of the partial structure represented by may be 700 or less. ５ (-B)-T１ The molecular weight of the partial structure represented by the formula (I) is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0252] In yet another specific embodiment, the antibody derivative or salt thereof has the following formula (IIIb-1): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; W represents an immunoglobulin unit containing a heavy chain and a light chain; １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, B represents a group containing a bioorthogonal functional group, and T ２ represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.]. The immunoglobulin unit represented by Ig, W １ and W ２ An atom represented by L ７ The definitions, examples, and preferred examples of the seventh linker represented by B, the group containing the bioorthogonal functional group represented by B, and the average modification percentage represented by r, as well as the antibody, are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[0253] T ２ is a monovalent group that can be generated by cleavage of the cleavable moiety. The monovalent group may be substituted or unsubstituted. Examples of the monovalent group include those described above. When the monovalent group is substituted, examples of the substituent include those described above. T ２ The monovalent group represented by the formula may be an optionally substituted hydroxyamino group. Details of the optionally substituted hydroxyamino group are given in T １ It is similar to what was stated above.

[0254] In formula (IIIb-1), C(=W １ )-L ７ (-B)-C (=W ２ )-T ２The molecular weight of the partial structure represented by C(=W １ )-L ７ (-B)-C (=W ２ )-T ２ The molecular weight of the partial structure represented by the formula (I) is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0255] In formula (IIIb-1), L ７ The seventh linker represented by (CH ２ ) ｎ２ n2 may be an integer of 1 to 10. Preferably, n2 may be an integer of 2 or greater, or 3 or greater. n2 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less. Particularly preferably, n2 may be 3.

[0256] In formula (IIIb-1), the group containing a bioorthogonal functional group is NH—C(═O)—(CH ２ ) ｎ３ -N ３ n3 may be an integer of 1 to 10. Preferably, n3 may be an integer of 2 or more, 3 or more, or 4 or more. n3 may also be an integer of 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. Particularly preferably, n3 may be 4.

[0257] The antibody derivative or salt thereof containing a bioorthogonal functional group can be produced using an affinity polypeptide-modified antibody or salt thereof that contains (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit), and (B) an antibody (immunoglobulin unit), and further contains (C) a cleavable moiety between (A) the affinity polypeptide and (B) the antibody (immunoglobulin unit).

[0258] In certain embodiments, when the affinity polypeptide-modified antibody or its salt (a) contains a cleavable moiety that can be cleaved to generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side, an antibody derivative or its salt containing the bioorthogonal functional group can be produced.

[0259] More specifically, such a production method includes, for example, the following methods (2-1) to (2-6).

[0260] (Method 2-1) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising: (A) an affinity polypeptide containing an affinity portion having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit); and (B) an antibody (immunoglobulin unit); and (C) an affinity polypeptide-modified antibody or its salt further containing a cleavable portion (here, the cleavable portion is a cleavable portion that can generate a bioorthogonal functional group on the antibody (immunoglobulin unit) side upon cleavage) between the (A) affinity polypeptide and the (B) antibody (immunoglobulin unit), using the cleavable portion to produce an antibody derivative or its salt containing a bioorthogonal functional group.

[0261] (Method 2-2) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) reacting a compound or salt thereof containing (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit) and (B) a group reactive with the antibody (immunoglobulin unit), and further containing (C) a cleavable moiety between (A) the affinity polypeptide and (B) the reactive group (wherein the cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the antibody (immunoglobulin unit) reactive group side upon cleavage), with an antibody or salt thereof containing immunoglobulin units including a heavy chain and a light chain, to produce an affinity polypeptide-modified antibody or a salt thereof containing a cleavable moiety between the affinity polypeptide and the antibody (wherein the cleavable moiety is a cleavable moiety capable of generating a bioorthogonal functional group on the antibody side upon cleavage); and (2) An affinity polypeptide-modified antibody or its salt containing a cleavable moiety between the affinity polypeptide and the antibody is cleaved with the cleavable moiety to produce an antibody derivative or its salt containing a bioorthogonal functional group.

[0262] (Method 2-3) A method for producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIa) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIa) above.

[0263] (Method 2-4) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) reacting a compound represented by formula (Ia) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit including a heavy chain and a light chain to produce an antibody or a salt thereof containing a structural unit represented by formula (IIa); and (2) cleaving the antibody or salt thereof containing a structural unit represented by formula (IIa) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIa).

[0264] (Method 2-5) A method for producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIa-1) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIa-1) above.

[0265] (Method 2-6) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit including a heavy chain and a light chain to produce an antibody or a salt thereof containing a structural unit represented by formula (IIa-1); and (2) cleaving the antibody or salt thereof containing a structural unit represented by formula (IIa-1) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIa-1).

[0266] The above method 2-1 may be carried out by the above method 2-3 or 2-5. The above method 2-2 may be carried out by the above method 2-4 or 2-6. The above methods 2-2, 2-4, and 2-6 may further comprise reacting the affinity polypeptide of the present invention with a moiety containing a group reactive to an antibody to produce the compound of the present invention or a salt thereof.

[0267] In another specific embodiment, when the affinity polypeptide-modified antibody or its salt comprises (b) a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety, an antibody derivative or its salt comprising the bioorthogonal functional group can be produced.

[0268] More specifically, such production methods include, for example, the following (2-7) to (2-12).

[0269] (Method 2-7) A method for producing an antibody derivative or salt thereof containing a bioorthogonal functional group, comprising: (A) an affinity polypeptide containing an affinity portion having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit); and (B) an antibody (immunoglobulin unit); and (C) an affinity polypeptide-modified antibody or salt thereof further containing a cleavable portion between (A) the affinity polypeptide and (B) the antibody (immunoglobulin unit), and (D) a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable portion, using the cleavable portion to produce an antibody derivative or salt thereof containing a bioorthogonal functional group.

[0270] (Method 2-8) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) (A) an affinity polypeptide containing an affinity moiety having affinity for the heavy chain CH1 region of an antibody (immunoglobulin unit), and (B) a reactive group for the antibody (immunoglobulin unit), and (C) a cleavable moiety between (A) the affinity polypeptide and (B) the reactive group, and (D) a bioorthogonal functional group between the reactive group and the cleavable moiety. Reacting a compound or salt thereof with an antibody or salt thereof containing an immunoglobulin unit containing a heavy chain and a light chain to produce an affinity polypeptide-modified antibody or salt thereof containing a cleavable moiety between the affinity polypeptide and the antibody, and (D) a bioorthogonal functional group between the antibody (immunoglobulin unit) and the cleavable moiety; and (2) cleaving an affinity polypeptide-modified antibody or salt thereof containing a cleavable moiety between the affinity polypeptide and the antibody with the cleavable moiety to produce an antibody derivative or salt thereof containing a bioorthogonal functional group.

[0271] (Method 2-9) A method for producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIb) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb) above.

[0272] (Method 2-10) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following steps (1) and (2): (1) reacting a compound represented by formula (Ib) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit including a heavy chain and a light chain to produce an antibody or a salt thereof containing a structural unit represented by formula (IIb); and (2) cleaving the antibody or salt thereof containing a structural unit represented by formula (IIb) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIb).

[0273] (Method 2-11) A method for producing an antibody derivative or a salt thereof comprising a bioorthogonal functional group, comprising cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIb-1) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb-1) above.

[0274] (Method 2-12) A method for producing an antibody derivative or a salt thereof containing a bioorthogonal functional group, comprising the following (1) and (2): (1) reacting a compound represented by formula (Ib-1) or a salt thereof with an antibody or a salt thereof containing an immunoglobulin unit including a heavy chain and a light chain to produce an antibody or a salt thereof containing a structural unit represented by formula (IIb-1); and (2) cleaving the antibody or salt thereof containing a structural unit represented by formula (IIb-1) with a cleavable moiety to produce an antibody derivative or a salt thereof containing a structural unit represented by formula (IIIb-1).

[0275] The above method 2-7 may be carried out by the above method 2-9 or 2-11. The above method 2-8 may be carried out by the above method 2-10 or 2-11. The above methods 2-8, 2-10, and 2-12 may further comprise reacting the affinity 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.

[0276] The antibody derivative or its salt may further comprise an additional modifying moiety. Various methods are known for modifying antibodies. Thus, in the present invention, the antibody derivative or its salt may be modified to further comprise an additional modifying moiety. The additional modifying moiety may be introduced into the heavy chain or light chain of the antibody, preferably into the heavy chain of the antibody (particularly in the constant region of the heavy chain).

[0277] In certain embodiments, the additional modifying moiety may be an additional modifying moiety that includes a bioorthogonal functional group. The bioorthogonal functional group is the same as that described above. The bioorthogonal functional group contained in the additional modifying moiety may be the same as or different from the bioorthogonal functional group described in (b) above, but is preferably different.

[0278] In certain embodiments, the additional modifying moiety containing the bioorthogonal functional group may be introduced into the heavy chain constant region via modification of the amino group in the side chain of a lysine residue present at one or more positions in the heavy chain constant region. The antibody derivative or its salt can contain the additional modifying moiety via modification of the amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the heavy chain constant region (preferably the Fc region or CH2 domain) in the antibody structural unit (immunoglobulin unit containing a heavy chain and a light chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be selected from positions 246, 248, 274, 288, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, WO 2020 / 090979).

[0279] The antibody derivatives of the present invention having bioorthogonal functional groups can also be used in combination with other antibody derivatives having bioorthogonal functional groups depending on the number of heavy chains (i.e., L １Therefore, by using an antibody having a plurality of antibody heavy chains (for example, 1 to 8, preferably 1 to 4, more preferably 2) in the production method of the present invention, it is possible to obtain L-type antibodies in the same target region of the plurality of antibody heavy chains. １ It is possible to produce antibodies having a plurality of structural units represented by -B (or a plurality of structural units of a lower concept) in a site-selective manner.

[0280] For example, antibodies having two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab') ２ By using the antibody) in the production method of the present invention, it is possible to achieve L2 targeting in the same target region of two antibody heavy chains. １ It is possible to produce an antibody having two structural units represented by -B (or a plurality of structural units of a subordinate concept) regioselectively. That is, in an antibody having a bioorthogonal functional group, the modification pattern by the bioorthogonal functional group can be made the same among a plurality of (e.g., two) heavy chains. On the other hand, by using an antibody having one antibody heavy chain (e.g., a Fab antibody) in the production method of the present invention, it is possible to produce an antibody having two or more structural units represented by -B (or a plurality of structural units of a subordinate concept) regioselectively. １ It is possible to produce antibodies having one structural unit represented by -B (or a plurality of structural units of a lower concept) site-selectively.

[0281] The production of an antibody derivative or a salt thereof containing a bioorthogonal functional group can be confirmed, for example, by electrophoresis, chromatography (e.g., gel filtration chromatography, ion exchange chromatography, reverse-phase column chromatography, HPLC), or mass spectrometry, depending on the specific raw material and the molecular weight of the product. Regioselectivity can be confirmed by peptide mapping as described above. The number of bioorthogonal functional groups introduced can be confirmed by mass spectrometry (which can be performed in conjunction with DAR Calculator (Agilent software)). The antibody derivative or a salt thereof containing a bioorthogonal functional group can be appropriately purified by any method, such as chromatography (e.g., the chromatography described above and affinity chromatography).

[0282] 5-4. Conjugate of an antibody and at least one functional substance or a salt thereof The present invention provides a conjugate of an antibody and a functional substance or a salt thereof, which comprises (a) an antibody structural unit (an immunoglobulin unit including a heavy chain and a light chain) and (b) a functional substance, and (c) the functional substance is introduced into the heavy chain CH1 region of the immunoglobulin unit. Definitions, examples, and preferred examples of antibodies, immunoglobulin units, and conjugates of antibodies and functional substances, as well as their constituent elements (e.g., constant regions), are as described above.

[0283] The conjugate or its salt can contain a functional substance via modification of a functional group in the side chain of one or more (e.g., two, three, or four) of the 14 amino acid residues present in the CH1 region: asparagine, glutamine, methionine, proline, serine, threonine, tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. The conjugate or its salt can contain a functional substance via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, tryptophan, or cysteine ​​present in the CH1 region, preferably via modification of a functional group in the side chain of one of the amino acids lysine, tyrosine, or tryptophan, even more preferably via modification of a functional group in the side chain of lysine or tyrosine, and particularly preferably via modification of an amino group in the side chain of lysine. The positions of these amino acid residues in the CH1 region are as described above. The positions of the antibody or its salt modified with the functional substance can be confirmed by peptide mapping. The modification may be regioselective, as described above. Thus, in the formulae (IVa), (IVa-1), (IVb), and (IVb-1) described below, the immunoglobulin units may have the corresponding modifying units regioselectively via functional groups in the side chains of the amino acid residues.

[0284] Preferably, the conjugate can contain a functional substance via modification of an amino group in the side chain of one or more (preferably one or two, more preferably one) lysine residues in the CH1 region of the heavy chain in an antibody building block (an immunoglobulin unit including a heavy chain and a light chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be positions 121, 147, 205, or 210 of the human IgG heavy chain according to EU numbering. The modification may be regioselective, as described above. Thus, in formulas (IVa), (IVa-1), (IVb), and (IVb-1) described below, the immunoglobulin unit may have the corresponding modifying unit regioselectively via the amino group in the side chain of the lysine residue.

[0285] In certain embodiments, the conjugate or salt thereof has the following formula (IVa): [wherein Ig represents an immunoglobulin unit comprising a heavy chain and a light chain, L1 represents a first linker, Z represents a functional substance, and the average modification percentage r of the immunoglobulin unit with the functional substance is 65 to 135%.] The antibody or salt thereof may include an antibody comprising a structural unit represented by the formula: [wherein Ig represents an immunoglobulin unit comprising a heavy chain and a light chain, L1 represents a first linker, Z represents a functional substance, and the average modification percentage r of the immunoglobulin unit with the functional substance is 65 to 135%.] １ The definitions, examples, and preferred examples of the first linker represented by (I), the functional substance represented by (Z), and the antibody are as described above. A particularly preferred bioorthogonal functional group is a thiol group. Here, the average modification percentage r refers to the average modification rate per modification site in the heavy chain CH1 region of the immunoglobulin unit. For example, if the modification site is a specific lysine present in the heavy chain CH1 region, it represents the average percentage of immunoglobulin units in which a functional substance has been introduced into that lysine per total immunoglobulin unit.

[0286] In formula (IVa), L １ The molecular weight of the partial structure represented by -Z may be 700 or less. １When the molecular weight of the partial structure represented by -Z is 700 or less, the ratio of the molecular weight of the partial structure to the molecular weight of the entire antibody is very small, making purification of the conjugate or its salt based on the difference in molecular weight relatively difficult. However, according to the present invention, which enables a high degree of control of the DAR, a highly purified conjugate exhibiting a desired DAR can be obtained without necessarily requiring purification based on the difference in molecular weight. １ The molecular weight of the partial structure represented by -Z is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0287] In another particular embodiment, the conjugate or salt thereof has the following formula (IVa-1): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; W represents an immunoglobulin unit containing a heavy chain and a light chain; １ represents an oxygen atom or a sulfur atom, L ３ represents a third linker, Z represents a functional substance, and the average percentage modification r of the immunoglobulin unit with the functional substance is 65 to 135%. １ The atom represented by L3, the third linker represented by L3, the functional substance represented by Z, and the average modification percentage represented by r, as well as definitions, examples, and preferred examples of the antibody are as described above.

[0288] In formula (IVa-1), C (=W １ )-L ３ The molecular weight of the partial structure represented by -Z may be 700 or less. １ )-L ３ The molecular weight of the partial structure represented by -Z 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.

[0289] In formula (IVa-1), L ３ The partial structure represented by (CH ２) ｎ１ The definition, examples, and preferred examples of n1 are as described above.

[0290] In yet another particular embodiment, the conjugate or salt thereof has the following formula (IVb): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; ５ represents a fifth linker, Z represents a functional substance, and T １ represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%.]. The immunoglobulin unit represented by Ig, L ５ a fifth linker represented by １ The definitions, examples, and preferred examples of the monovalent group represented by , the average modification percentage represented by r, and the antibody are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[0291] In formula (IVb), L ５ (-Z)-T １ The molecular weight of the partial structure represented by may be 700 or less. ５ (-Z)-T １ The molecular weight of the partial structure represented by the formula (I) is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0292] In yet another particular embodiment, the conjugate or salt thereof has the following formula (IVb-1): where Ig represents an immunoglobulin unit containing a heavy chain and a light chain; W represents an immunoglobulin unit containing a heavy chain and a light chain; １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, Z represents a functional substance, and T ２represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%.]. The immunoglobulin unit represented by Ig, W １ and W ２ An atom represented by L ７ The definitions, examples, and preferred examples of the seventh linker represented by (I), the functional substance represented by (Z), the monovalent group represented by (T2), and the average modification percentage represented by (r), as well as the antibody, are as described above. A particularly preferred bioorthogonal functional group is an azide group.

[0293] In formula (IVb-1), C (=W １ )-L ７ (-Z)-C(=W ２ )-T ２ The molecular weight of the partial structure represented by C(=W １ )-L ７ (-Z)-C(=W ２ )-T ２ The molecular weight of the partial structure represented by the formula (I) is preferably 600 or less, more preferably 500 or less, even more preferably 400 or less, and particularly preferably 300 or less, 250 or less, 200 or less, or 100 or less.

[0294] In formula (IVb-1), L ７ The partial structure represented by (CH ２ ) ｎ２ The definition, examples, and preferred examples of n2 are as described above.

[0295] In formula (IVb-1), the group containing a bioorthogonal functional group is NH—C(═O)—(CH ２ ) ｎ３ -N ３ The definition, examples, and preferred examples of n3 are as described above.

[0296] The method for producing the conjugate or its salt may be carried out by reacting an antibody derivative or its salt containing a bioorthogonal functional group with a functional substance to produce a conjugate or its salt containing the antibody and the functional substance.

[0297] Alternatively, the method for producing the conjugate or its salt may be carried out by a method including the following (1) and (2): (1) producing an antibody derivative or its salt comprising a bioorthogonal functional group by the above-described method; and (2) reacting the antibody derivative or its salt comprising a bioorthogonal functional group with a functional substance to produce a conjugate or its salt comprising the antibody and the functional substance.

[0298] More specifically, methods for producing the conjugate or a salt thereof include, for example, the following methods (3-1) to (3-12).

[0299] (Method 3-1) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting an antibody derivative containing a structural unit represented by formula (IIIa) above or a salt thereof with a functional substance to produce a conjugate containing a structural unit represented by formula (IVa) above or a salt thereof.

[0300] (Method 3-2) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising the following steps (1) and (2): (1) cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIa) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIa) above; and (2) reacting the antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIa) above with a functional substance to produce a conjugate or a salt thereof comprising a structural unit represented by formula (IVa) above.

[0301] (Method 3-3) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, the method comprising the following steps (1) to (3): (1) reacting a compound represented by formula (Ia) or a salt thereof with an antibody or a salt thereof comprising an immunoglobulin unit including a heavy chain and a light chain, to produce an antibody or a salt thereof comprising the structural unit represented by formula (IIa); (2) cleaving the antibody or salt thereof comprising the structural unit represented by formula (IIa) with a cleavable moiety, to produce an antibody derivative or a salt thereof comprising the structural unit represented by formula (IIIa); and (3) reacting the antibody derivative or salt thereof comprising the structural unit represented by formula (IIIa) with a functional substance, to produce a conjugate or a salt thereof comprising the structural unit represented by formula (IVa).

[0302] (Method 3-4) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting an antibody derivative containing a structural unit represented by formula (IIIa-1) above or a salt thereof with a functional substance to produce a conjugate containing a structural unit represented by formula (IVa-1) above or a salt thereof.

[0303] (Method 3-5) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising the following steps (1) and (2): (1) cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIa-1) with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIa-1); and (2) reacting the antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIa-1) with a functional substance to produce a conjugate or a salt thereof comprising a structural unit represented by formula (IVa-1).

[0304] (Method 3-6) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising the following steps (1) to (3): (1) reacting a compound represented by formula (Ia-1) or a salt thereof with an antibody or a salt thereof comprising an immunoglobulin unit including a heavy chain and a light chain, to produce an antibody or a salt thereof comprising the structural unit represented by formula (IIa-1); (2) cleaving the antibody or salt thereof comprising the structural unit represented by formula (IIa-1) with a cleavable moiety, to produce an antibody derivative or a salt thereof comprising the structural unit represented by formula (IIIa-1); and (3) reacting the antibody derivative or salt thereof comprising the structural unit represented by formula (IIIa-1) with a functional substance, to produce a conjugate or a salt thereof comprising the structural unit represented by formula (IVa-1).

[0305] (Method 3-7) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting an antibody derivative containing a structural unit represented by formula (IIIb) above or a salt thereof with a functional substance to produce a conjugate containing a structural unit represented by formula (IVb) above or a salt thereof.

[0306] (Method 3-8) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising the following steps (1) and (2): (1) cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIb) above with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb) above; and (2) reacting an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb) above with a functional substance to produce a conjugate or a salt thereof comprising a structural unit represented by formula (IVb) above.

[0307] (Method 3-9) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, the method comprising the following steps (1) to (3): (1) reacting a compound represented by formula (Ib) or a salt thereof with an antibody or a salt thereof comprising an immunoglobulin unit including a heavy chain and a light chain, to produce an antibody or a salt thereof comprising a structural unit represented by formula (IIb); (2) cleaving the antibody or salt thereof comprising a structural unit represented by formula (IIb) with a cleavable moiety, to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb); and (3) reacting the antibody derivative or salt thereof comprising a structural unit represented by formula (IIIb) with a functional substance, to produce a conjugate or a salt thereof comprising a structural unit represented by formula (IVb).

[0308] (Method 3-10) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising reacting an antibody derivative containing a structural unit represented by formula (IIIb-1) above or a salt thereof with a functional substance to produce a conjugate containing a structural unit represented by formula (IVb-1) above or a salt thereof.

[0309] (Method 3-11) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising the following steps (1) and (2): (1) cleaving an antibody or a salt thereof comprising a structural unit represented by formula (IIb-1) with a cleavable moiety to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb-1); and (2) reacting an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb-1) with a functional substance to produce a conjugate or a salt thereof comprising a structural unit represented by formula (IVb-1).

[0310] (Method 3-12) A method for producing a conjugate of an antibody and a functional substance or a salt thereof, comprising the following steps (1) to (3): (1) reacting a compound represented by formula (IB-1) or a salt thereof with an antibody or a salt thereof comprising an immunoglobulin unit including a heavy chain and a light chain, to produce an antibody or a salt thereof comprising a structural unit represented by formula (IIb-1); (2) cleaving the antibody or salt thereof comprising a structural unit represented by formula (IIb-1) with a cleavable moiety, to produce an antibody derivative or a salt thereof comprising a structural unit represented by formula (IIIb-1); and (3) reacting the antibody derivative or salt thereof comprising a structural unit represented by formula (IIIb-1) with a functional substance, to produce a conjugate or a salt thereof comprising a structural unit represented by formula (IVb-1).

[0311] The above methods 3-1 to 3-12 may further comprise reacting the affinity polypeptide of the present invention with a moiety containing a reactive group to an antibody to produce the compound of the present invention or a salt thereof.

[0312] The conjugate or its salt may further comprise an additional modifying moiety. Various methods are known for modifying antibodies. Thus, in the present invention, the conjugate or its salt may be modified to further comprise an additional modifying moiety. The additional modifying moiety may be introduced into the heavy chain or light chain of the antibody, preferably into the heavy chain of the antibody (particularly into the constant region of the heavy chain).

[0313] In certain embodiments, the additional modifying moiety may be an additional modifying moiety containing a functional substance. The functional substance is the same as that described above. The functional substance contained in the additional modifying moiety may be the same as or different from the functional substance described in (b) above, but is preferably different.

[0314] In certain embodiments, the additional modifying moiety containing the functional substance may be introduced into the constant regions of the two heavy chains via modification of the amino groups in the side chains of lysine residues present at one or more positions in the constant regions of the heavy chains. The conjugate or a salt thereof can contain the additional modifying moiety via modification of the amino groups in the side chains of one or more (preferably one or two, more preferably one) lysine residues in the heavy chain constant region (preferably the Fc region or CH2 domain) in the antibody structural unit (immunoglobulin unit containing a heavy chain and a light chain). More specifically, the position of one or more (preferably one or two, more preferably one) lysine residues may be selected from positions 246, 248, 274, 288, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering (see, e.g., WO 2016 / 186206, WO 2018 / 199337, WO 2019 / 240287, WO 2019 / 240288, WO 2020 / 009165, and WO 2020 / 090979). The position at which an additional modifying moiety containing a functional substance is introduced is preferably different from the position at which the functional substance in (b) above is introduced.

[0315] The antibody having a functional substance (antibody and functional substance conjugate) produced by the production method of the present invention may also contain a functional substance (i.e., L １ This allows antibodies having functional substances to be synthesized by dividing them into L and Z depending on the number of heavy chains. １ This is because the antibody can be produced from an antibody having a bioorthogonal functional group that can have a structural unit represented by -B. Therefore, by using an antibody having multiple antibody heavy chains (for example, 1 to 8, preferably 1 to 4, more preferably 2), it is possible to produce L-type antibodies in the same target region of multiple antibody heavy chains. １ Antibodies having multiple structural units represented by -Z (or multiple structural units of a lower concept) site-selectively can be produced.

[0316] For example, antibodies having two antibody heavy chains (e.g., IgG, IgD, IgE, F(ab') ２By using the antibody) in the production method of the present invention, it is possible to achieve L2 targeting in the same target region of two antibody heavy chains. １ It is possible to produce an antibody having two structural units represented by -Z regioselectively. That is, in an antibody having a functional substance, the modification pattern with the functional substance can be made the same among a plurality of (e.g., two) heavy chains. On the other hand, by using an antibody having one antibody heavy chain (e.g., a Fab antibody) in the production method of the present invention, it is possible to produce an antibody having two structural units represented by -Z regioselectively. １ Antibodies can be produced that regioselectively have one structural unit represented by -Z.

[0317] 6. Uses The compound of the present invention or a salt thereof can easily modify the heavy chain CH1 region of an antibody building block (an immunoglobulin unit comprising a heavy chain and a light chain) (the average modification percentage r of an immunoglobulin unit is 65 to 135%). The compound of the present invention or a salt thereof can also site-selectively modify a specific amino acid residue (preferably a lysine residue) in the heavy chain of an immunoglobulin unit. Thus, the present invention provides a reagent for derivatizing an antibody, comprising the compound of the present invention or a salt thereof.

[0318] 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, a solution and a stabilizer (e.g., an antioxidant, a preservative). The solution is preferably an aqueous solution. Examples of aqueous solutions include water (e.g., distilled water, sterile distilled water, purified water, physiological saline), and buffer solutions (e.g., aqueous phosphate solution, Tris-hydrochloric acid buffer, carbonate-bicarbonate buffer, aqueous boric acid solution, glycine-sodium hydroxide buffer, citrate buffer, and acetate buffer), with buffer solutions being preferred. The pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5, more preferably 7.0 to 8.5, and even more preferably 7.4 to 8.2. The reagent of the present invention can be provided in liquid or powder form (e.g., lyophilized powder).

[0319] The affinity polypeptide-modified antibody, antibody derivative or salt thereof of the present invention is useful, for example, as an intermediate for preparing the conjugate of the present invention or a salt thereof.

[0320] The conjugate or salt thereof 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 thereof of the present invention, in which the CH1 region of the heavy chain in the antibody structural unit is specifically modified (the average modification percentage r of the immunoglobulin unit is 65 to 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 in an antibody-drug conjugate (ADC) changes the pharmacokinetics, drug release rate, and efficacy. For these reasons, next-generation ADCs are required to control the number and position of drugs to be conjugated. It is believed that maintaining a constant number and position will achieve the expected efficacy and resolve problems of variation in the conjugated drug and lot-to-lot differences, known as regulation. Therefore, the conjugate or salt thereof of the present invention can solve such regulation problems.

[0321] The conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition. Such a pharmaceutical composition may contain, in addition to the conjugate of the present invention or a salt thereof, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; flavorings such as citric acid, menthol, glycyrrhizin 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; dispersing agents such as surfactants; diluents such as water, saline, and orange juice; and base waxes such as cocoa butter, polyethylene glycol, and white kerosene. The conjugate of the present invention or a salt thereof may also have any modification that provides stability (eg, PEGylation).

[0322] Suitable formulations for oral administration include solutions in which an effective amount of the ligand is dissolved in a diluent such as water, physiological saline, or orange juice; capsules, sachets, or tablets containing an effective amount of the ligand as a solid or 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 and emulsified in a suitable dispersion medium.

[0323] The pharmaceutical composition is suitable for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration). Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0324] The dosage of the pharmaceutical composition varies depending on the type and activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug tolerance, body weight, age, etc., but can be set appropriately.

[0325] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0326] (Example 1) Design of Affinity Polypeptide and Secretory Expression of Affinity Polypeptide in C. glutamicum (1-1) Overview of Affinity Polypeptide Design In the present invention, in order to modify the Fab region of an immunoglobulin, it is preferable to bind a compound having a reactive group for the Fab region of an immunoglobulin to the affinity polypeptide. Furthermore, in order to remove the affinity polypeptide after modification of the Fab region of the immunoglobulin, it is preferable to include a cleavable moiety between the reactive group and the affinity polypeptide.

[0327] Furthermore, in order to achieve specific binding of a compound to a specific site in an affinity polypeptide, it is preferable to design the affinity polypeptide so that there is only one site capable of reacting with the compound. Therefore, when an affinity polypeptide is used as an affinity polypeptide and the affinity polypeptide is bound to a compound via the amino group in the side chain of a lysine residue (K) in the affinity polypeptide, it is preferable to design the affinity polypeptide so that there is only one lysine residue in the affinity polypeptide (note that an ornithine residue, a diaminobutanoic acid residue, or a diaminopimelic acid residue may be used instead of the lysine residue). Therefore, the affinity polypeptide was designed as follows: A) The polypeptide contains only one K residue; B) The N-terminal amino acid is changed to Q (glutamine), thereby pyroglutamylating and converting the N-terminal amino group to an amide.

[0328] According to the above rules A) and B), the following polypeptide affinity polypeptides (a) to (h) were designed.

[0329] (1-2) Preparation of Affinity Polypeptides The following affinity polypeptides were prepared: (a) Affinity peptide #1 QETTYRLVINGRTLRGETTTEAVDAATAERKFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 1)

[0330] (b) Affinity peptide #2 QETTYRLVINGRTLRGETTTEAVDAATAERVFRQYAWKNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 2)

[0331] (c) Affinity peptide #3 QETTYRLVINGRTLRGETTTKAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 3)

[0332] (d) Affinity peptide #4 QETTYRLVINGRTLRGKTTTEAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 4)

[0333] (e) Affinity peptide #5 QETTYRLVINGRTLRGETTTEAVDAETAAAKFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 5)

[0334] (f) Affinity peptide #6 QETTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANKNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 6)

[0335] (g) Affinity peptide #7 QETTYRLVINGRTLRGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 7)

[0336] (h) Affinity peptide #8 QETTYRLVINGRTLRGKTTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 8)

[0337] (1-3) Expression of Affinity Polypeptides Expression of these polypeptide affinity polypeptides was investigated using Corynex®. Expression using Corynex® utilized the CspB fusion method (WO 2013 / 062029), a technology that improves secretory production of a target polypeptide by inserting a base sequence encoding an amino acid sequence containing the N-terminal three residues Gln-Glu-Thr (QET) of the mature CspB protein between the base sequence encoding the signal peptide and the base sequence encoding the target polypeptide. Furthermore, because the N-terminus of the CspB tag is Q, there is the advantage that after cleavage of the signal sequence, the first Q residue can be pyroglutamylated to protect the N-terminal amino group. In other words, in addition to the above rules A), B), and C), consideration of the following rule D) is effective in improving expression of affinity polypeptides using Corynex®.

[0338] D) Addition of three QET residues to the N-terminus improves secretion efficiency with Corynex®

[0339] Below, an example of expression study using Corynex (registered trademark) is described.

[0340] (1-4) Construction of secretory expression plasmids for affinity peptides #1-8. Eight types of amino acid sequences for each of the affinity peptides were designed as affinity polypeptides, and the nucleotide sequences encoding these polypeptides were designed taking into account the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed to enable secretory expression by C. glutamicum.

[0341] Affinity peptide #1 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP1") of the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #1. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP1 are shown in SEQ ID NOs: 11 and 12, respectively.

[0342] Nucleotide sequence encoding CspBss-AP1: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactgaagctgtggatgcagctaccgcagaacgcaagtttcgccagtacgcatgggacaatggcgttaccggcgagtggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 11)

[0343] Amino acid sequence of CspBss-AP1: MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGETTTEAVDAATAERKFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 12)

[0344] Affinity peptide #2 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP2") of the 30 amino acid signal peptide of CspB derived from the C. glutamicum ATCC13869 strain, the N-terminal three amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #2. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP2 are shown in SEQ ID NOs: 13 and 14, respectively.

[0345] Nucleotide sequence encoding CspBss-AP2: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactgaagctgtggatgcagctaccgcagaacgcgtgtttcgccagtacgcatggaagaatggcgttaccggcgagtggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 13)

[0346] Amino acid sequence of CspBss-AP2 MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGETTTEAVDAATAERVFRQYAWKNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 14)

[0347] Affinity peptide #3 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP3") of the 30 amino acid signal peptide of CspB derived from the C. glutamicum ATCC13869 strain, the N-terminal three amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #3. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP3 are shown in SEQ ID NOs: 15 and 16, respectively.

[0348] Nucleotide sequence encoding CspBss-AP3: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactaaggctgtggatgcagctaccgcagaacgcgtgtttcgccagtacgcatgggacaatggcgttaccggcgagtggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 15)

[0349] Amino acid sequence of CspBss-AP3: MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGETTTKAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 16)

[0350] Affinity peptide #4 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP4") of the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #4. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP4 are shown in SEQ ID NOs: 17 and 18, respectively.

[0351] Nucleotide sequence encoding CspBss-AP4: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtaagaccaccactgaagctgtggatgcagctaccgcagaacgcgtgtttcgccagtacgcatgggacaatggcgttaccggcgagtggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 17)

[0352] Amino acid sequence of CspBss-AP4 MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGKTTTEAVDAATAERVFRQYAWDNGVTGEWTYDDATRTFTVTE (SEQ ID NO: 18)

[0353] Affinity peptide #5 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP5") of the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal three amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #5. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP5 are shown in SEQ ID NOs: 19 and 20, respectively.

[0354] Nucleotide sequence encoding CspBss-AP5: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactgaagctgtggatgcagagaccgcagcagctaagtttgcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 19)

[0355] Amino acid sequence of CspBss-AP5: MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGETTTEAVDAETAAAKFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 20)

[0356] Affinity peptide #6 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP6") of the 30 amino acid signal peptide of CspB derived from the C. glutamicum ATCC13869 strain, the N-terminal three amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #6. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP6 are shown in SEQ ID NOs: 21 and 22, respectively.

[0357] Nucleotide sequence encoding CspBss-AP6: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactgaagctgtggatgcagagaccgcagcagctgcgtttgcgcagtacgcaaacaagaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 21)

[0358] Amino acid sequence of CspBss-AP6: MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANKNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 22)

[0359] Affinity peptide #7 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP7") of the 30 amino acid residue signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #7. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP7 are shown in SEQ ID NOs: 23 and 24, respectively.

[0360] Nucleotide sequence encoding CspBss-AP7: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactaaggctgtggatgcagagaccgcagcagctgcgtttgcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 23)

[0361] Amino acid sequence of CspBss-AP7: MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGETTTKAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 24)

[0362] Affinity peptide #8 was secreted and expressed as a fusion protein (hereinafter referred to as "CspBss-AP8") of the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain, the N-terminal 3 amino acid residues QET of the mature CspB protein derived from the same strain, and affinity peptide #8. The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP8 are shown in SEQ ID NOs: 25 and 26, respectively.

[0363] Nucleotide sequence encoding CspBss-AP8: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagaccacctatcgcctcgtgatcaacggacgtactctgcgtggtaagaccaccactgaagctgtggatgcagagaccgcagcagctgcgtttgcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 25)

[0364] Amino acid sequence of CspBss-AP8: MFNNRIRTAALAGAIAISTAASGVAIPAFAQETTYRLVINGRTLRGKTTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 26)

[0365] CspBss-AP1, CspBss-AP2, CspBss-AP3, CspBss-AP4, CspBss-AP5, CspBss-AP6, CspBss-AP7, and CspBss-AP8 are upstream of the base sequence described in C. glutamicum ATCC13869 strain. The promoter of the cspB gene was linked, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. Expression cassettes for eight types of affinity polypeptides were designed and totally synthesized. The fully synthesized DNA fragment (affinity polypeptide expression cassette) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774, to construct the affinity polypeptide secretion expression plasmids pPK4_CspBss-AP1, pPK4_CspBss-AP2, pPK4_CspBss-AP3, pPK4_CspBss-AP4, pPK4_CspBss-AP5, pPK4_CspBss-AP6, pPK4_CspBss-AP7, and pPK4_CspBss-AP8. The nucleotide sequences of the inserted fragments confirmed that the expression cassettes for the affinity polypeptides were constructed as designed. The base sequence was determined using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3500xL Genetic Analyzer (Applied Biosystems).

[0366] (1-5) C. Secretory expression of affinity polypeptide in glutamicum Using the pPK4_CspBss-AP1, pPK4_CspBss-AP2, pPK4_CspBss-AP3, pPK4_CspBss-AP4, pPK4_CspBss-AP5, pPK4_CspBss-AP6, pPK4_CspBss-AP7, and pPK4_CspBss-AP8 constructed above, C. The C. glutamicum YDK0107 strain was transformed with this vector to obtain strains YDK0107 / pPK4_CspBss-AP1, YDK0107 / pPK4_CspBss-AP2, YDK0107 / pPK4_CspBss-AP3, YDK0107 / pPK4_CspBss-AP4, YDK0107 / pPK4_CspBss-AP5, YDK0107 / pPK4_CspBss-AP6, YDK0107 / pPK4_CspBss-AP7, and YDK0107 / pPK4_CspBss-AP8.

[0367] Each of the obtained transformants was cultured at 30°C for 72 hours in an MMTG liquid medium containing 25 mg / L of kanamycin (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloric acid hydrolyzate (total nitrogen content 0.2 g), calcium carbonate 50 g, and water to make 1 L, adjusted to pH 7.0).

[0368] After the culture was completed, each culture medium was centrifuged, and 6.5 μL of the resulting culture supernatant was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tris Gel (Thermo Fisher Scientific), followed by staining with Quick-CBB (Wako). As a result, a polypeptide band estimated to be affinity peptide #1 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP1 strain (Fig. 1, lane 3), a polypeptide band estimated to be affinity peptide #2 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP2 strain (Fig. 1, lane 4), a polypeptide band estimated to be affinity peptide #3 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP3 strain (Fig. 1, lane 5), and a polypeptide band estimated to be affinity peptide #4 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP4 strain (Fig. 1, lane 6). A polypeptide band estimated to be affinity peptide #5 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP5 strain (Fig. 1, lane 7), a polypeptide band estimated to be affinity peptide #6 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP6 strain (Fig. 1, lane 8), a polypeptide band estimated to be affinity peptide #7 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP7 strain (Fig. 1, lane 9), and a polypeptide band estimated to be affinity peptide #8 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP8 strain (Fig. 1, lane 10). 10).

[0369] (Example 2) Purification of Affinity Polypeptides (2-1) Purification of Affinity Peptide #6 The culture supernatant of #6 prepared in Example 1 was filtered through a 0.22 μm filter. 360 ml of eluent A (50 mM Tris-HCl, pH 8.0) was added to 40 ml of the filtrate to prepare a load sample. Purification using an AKTA pure 150 (manufactured by Cytiva) was carried out under the following conditions. Column: HiTrap Q FF (manufactured by Cytiva) Eluent A: 50 mM Tris-HCl, pH 8.0 Eluent B: 50 mM Tris-HCl, 1 M NaCl, pH 8.0 Flow rate: 5.0 ml / min Gradient: 0-100% B / 20 CV Detector: Detection was performed at a wavelength of 280 nm.

[0370] (2-2) Purification of Affinity Peptides #5, 7, and 8 The culture supernatants of #5, #7, and #8 were left standing overnight at 37°C and then filtered through a 0.22 μm filter. 360 ml of eluent A (50 mM Tris-HCl, pH 8.0) was added to 40 ml of each filtrate to prepare a load sample. Purification using an AKTA pure 150 (Cytiva) was performed under the following conditions. Column: HiPrep Q FF 16 / 10 (manufactured by Cytiva) Eluent A: 50 mM Tris-HCl, pH 8.0 Eluent B: 50 mM Tris-HCl, 1 M NaCl, pH 8.0 Flow rate: 5.0 ml / min Gradient: 0-50% B / 20 CV Detector: Detection was performed at a wavelength of 280 nm.

[0371] 40-60 ml of the eluted fraction containing the target substance was collected and concentrated to 4 ml using an Amicon Ultra-3K (Merck). 56 ml of water for injection was added to the concentrate, and it was again concentrated to 4 ml. This process was repeated once more. Water for injection was added to the concentrate to make the final volume 20 ml. The obtained concentrate was subjected to HPLC, and the peak of the target substance was confirmed.

[0372] Example 3 Preparation of Compounds Having Affinity Polypeptides for Antibodies, Cleavable Moieties, and Reactive Groups Compounds having cleavable moieties and reactive groups were conjugated to the affinity peptides #5 to #8 purified in Example 2.

[0373] (3-1) Synthesis of Affinity Reagent #5-1 According to a previous report (WO2019 / 0240287), affinity reagent #5-1 was synthesized by amidation using compound (L-1) having a cleavable moiety and a reactive group and affinity peptide #5 purified in Example (2-2). MS (ESI) m / z: z=5,1334.900 [M+5H] ５＋

[0374] (3-2) Synthesis of Affinity Reagent #5-2 According to a previous report (WO2019 / 0240287), affinity reagent #5-2 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #5 purified in Example (2-2). MS (ESI) m / z: z=5,1337.35 [M+5H] ５＋

[0375] (3-3) Synthesis of Affinity Reagent #6-1 According to a previous report (WO2019 / 0240287), affinity reagent #6-1 was synthesized by amidation using compound (L-1) having a cleavable moiety and a reactive group and affinity peptide #6 purified in Example (2-1). MS (ESI) m / z: z=5,1326.05 [M+5H] ５＋

[0376] (3-4) Synthesis of Affinity Reagent #6-2 According to a previous report (WO2019 / 0240287), affinity reagent #6-2 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #6 purified in Example (2-1). MS (ESI) m / z: z=5,1328.90 [M+5H] ５＋

[0377] (3-5) Synthesis of Affinity Reagent #7-1 According to a previous report (WO2019 / 0240287), affinity reagent #7-1 was synthesized by amidation using compound (L-1) having a cleavable moiety and a reactive group and affinity peptide #7 purified in Example (2-2). MS (ESI) m / z: z=5,1323.35 [M+5H] ５＋

[0378] (3-6) Synthesis of Affinity Reagent #7-2 According to a previous report (WO2019 / 0240287), affinity reagent #7-2 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #7 purified in Example (2-2). MS (ESI) m / z: z=5,1326.15 [M+5H] ５＋

[0379] (3-7) Synthesis of Affinity Reagent #8-1 According to a previous report (WO2019 / 0240287), affinity reagent #8-1 was synthesized by amidation using compound (L-1) having a cleavable moiety and a reactive group and affinity peptide #8 purified in Example (2-2). MS (ESI) m / z: z=5,1323.30 [M+5H] ５＋

[0380] (3-8) Synthesis of Affinity Reagent #8-2 According to a previous report (WO2019 / 0240287), affinity reagent #8-2 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #8 purified in Example (2-2). MS (ESI) m / z: z=5,1326.05 [M+5H] ５＋

[0381] (Example 4) Modification of antibody using affinity reagent (4-1) Modification of antibody using affinity reagent #8-2 (4-1-1) Synthesis of trastuzumab bound to two molecules of affinity reagent #8-2

[0382] A DMF solution of the affinity reagent #8-2 synthesized in Example (3-8) (10 equivalents relative to the antibody) was added to a solution of the anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) in borate buffer (20 mM borate, pH 8.2), and the mixture was shaken for 1 hour. The reaction solution was purified using Nap25 (Cytiva) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161251, where two molecules of affinity reagent were bound to trastuzumab.

[0383] (4-1-2) Synthesis of Trastzumab Having Two Thiol Groups

[0384] The antibody obtained in Example (4-1) was subjected to a thioester group cleavage reaction (treatment with hydroxylamine) according to a previously reported method (WO 2019 / 240287), thereby obtaining a thiol group-introduced antibody (T-S-8) of the above structural formula, in which two thiol groups had been introduced. When the mass was measured by ESI-TOFMS, a peak was confirmed at 148,417, indicating that the cleavage reaction had progressed.

[0385] (4-1-3) Synthesis of ADC with DAR = 2

[0386] To a solution of the antibody (T-S-8) having two thiol groups obtained in Example (4-1-2) in PBSE buffer (10 mM Phosphate Buffered Saline (PBS), 10 mM EDTA, pH 7.4), a solution of the known compound MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) in dimethylacetamide (5 equivalents, dimethylacetamide (DMA) (8% v / v) solution) was added, and the mixture was shaken at room temperature for 1 hour. The reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva), and an ADC (T-M-5) with a DAR of 2 was obtained. The analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and ESI-TOFMS analysis was performed, and a peak was observed at 151066.

[0387] (4-2) Modification of antibody using affinity reagent #5-1 (4-2-1) Synthesis of trastuzumab with two molecules of affinity reagent #5-1 bound According to Example (4-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #5-1 synthesized in Example (3-1) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161342, where two molecules of affinity reagent were bound to trastuzumab.

[0388] (4-2-2) Synthesis of Trastzumab Having Two Thiol Groups According to Example (4-1-2), a thiol-group-introduced antibody having two thiol groups introduced thereinto was obtained from the antibody obtained in Example (4-2-1). HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced thereinto.

[0389] (4-2-3) Synthesis of ADC with DAR = 2 According to Example (4-1-3), the antibody (T-S-5) having two thiol groups obtained in Example (4-2-2) was reacted with the known compound MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) to obtain an ADC (T-M-5) with DAR = 2. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that two molecules of MC-Val-Cit-PABA-MMAE had been introduced.

[0390] (4-3) Modification of antibody using affinity reagent #5-2 (4-3-1) Synthesis of trastuzumab with two molecules of affinity reagent #5-2 bound According to Example (4-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #5-2 synthesized in Example (3-2) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161367, where two molecules of affinity reagent were bound to trastuzumab.

[0391] (4-3-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced thereinto was obtained from the antibody obtained in Example (4-3-1). HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced thereinto.

[0392] (4-3-3) Synthesis of ADC with DAR = 2 According to Example (4-1-3), the antibody (T-S-5) having two thiol groups obtained in Example (4-3-2) was reacted with the known compound MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) to obtain an ADC (T-M-5) with DAR = 2. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that two molecules of MC-Val-Cit-PABA-MMAE had been introduced.

[0393] (4-4) Modification of antibody using affinity reagent #6-1 (4-4-1) Synthesis of trastuzumab with two molecules of affinity reagent #6-1 bound According to Example (4-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #6-1 synthesized in Example (3-3) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161253, where two molecules of affinity reagent were bound to trastuzumab.

[0394] (4-4-2) Synthesis of Trastzumab Having Two Thiol Groups According to Example (4-1-2), a thiol-group-introduced antibody having two thiol groups introduced thereinto was obtained from the antibody obtained in Example (4-4-1). HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced thereinto.

[0395] (4-4-3) Synthesis of ADC with DAR = 2 According to Example (4-1-3), the antibody (T-S-6) having two thiol groups obtained in Example (4-4-2) was reacted with the known compound MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) to obtain an ADC (T-M-6) with DAR = 2. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that two molecules of MC-Val-Cit-PABA-MMAE had been introduced.

[0396] (4-5) Modification of antibody using affinity reagent #6-2 (4-5-1) Synthesis of trastuzumab with two molecules of affinity reagent #6-2 bound According to Example (4-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #6-2 synthesized in Example (3-4) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161280, where two molecules of affinity reagent were bound to trastuzumab.

[0397] (4-5-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced thereinto was obtained from the antibody obtained in Example (4-5-1). HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced thereinto.

[0398] (4-5-3) Synthesis of ADC with DAR = 2 According to Example (4-1-3), the antibody (T-S-6) having two thiol groups obtained in Example (4-5-2) was reacted with the known compound MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) to obtain an ADC (T-M-6) with DAR = 2. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that two molecules of MC-Val-Cit-PABA-MMAE had been introduced.

[0399] (4-6) Modification of antibody using affinity reagent #8-1 (4-6-1) Synthesis of trastuzumab with two molecules of affinity reagent #8-1 bound According to Example (4-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #8-1 synthesized in Example (3-7) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161224, where two molecules of affinity reagent were bound to trastuzumab.

[0400] (4-6-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (4-6-1) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0401] (4-6-3) Synthesis of ADC with DAR = 2 According to Example (4-1-3), the antibody (T-S-6) having two thiol groups obtained in Example (4-6-2) was reacted with the known compound MC-Val-Cit-PABA-MMAE (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) to obtain an ADC (T-M-6) with DAR = 2. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), HIC-HPLC analysis was performed to confirm that two molecules of MC-Val-Cit-PABA-MMAE had been introduced.

[0402] (Example 5) Reaction with human IgG antibody using affinity reagent (5-1) Modification of Cetuximab using affinity reagent (5-1-1) Modification of Cetuximab using affinity reagent #5-2 According to Example (4-1-1), anti-human EGFR monoclonal antibody Cetuximab (Merck Biopharmaceuticals) was reacted with a DMF solution of the affinity reagent #5-2 synthesized in Example (3-2) to obtain cetuximab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 165807, where two molecules of affinity reagent bound to cetuximab.

[0403] (5-1-2) Synthesis of Cetuximab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-1-1) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0404] (5-1-3) Modification of Cetuximab using Affinity Reagent #8-2 According to Example (4-1-1), the anti-human EGFR monoclonal antibody Cetuximab (Merck Biopharmaceuticals) and the DMF solution of affinity reagent #8-2 synthesized in Example (3-8) were reacted to obtain Cetuximab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 165710 where two molecules of affinity reagent bound to Cetuximab.

[0405] (5-1-4) Synthesis of Cetuximab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-1-3) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0406] (5-2) Modification of Rituximab using affinity reagent (5-2-1) Modification of Rituximab using affinity reagent #5-2 According to Example (4-1-1), the anti-CD20 monoclonal antibody Rituximab (Zenyaku Kogyo) was reacted with a DMF solution of the affinity reagent #5-2 synthesized in Example (3-2), to obtain Rituximab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 160392, where two molecules of affinity reagent bound to Rituximab.

[0407] (5-2-2) Synthesis of Rituximab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-2-1) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0408] (5-2-3) Modification of Rituximab using affinity reagent #8-2 According to Example (4-1-1), the anti-CD20 monoclonal antibody Rituximab (Zenyaku Kogyo) was reacted with a DMF solution of the affinity reagent #8-2 synthesized in Example (3-8) to obtain Rituximab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 160443, where two molecules of affinity reagent bound to Rituximab.

[0409] (5-2-4) Synthesis of Rituximab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-2-3) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0410] (5-3) Modification of Infliximab Using Affinity Reagent (5-3-1) Modification of Infliximab Using Affinity Reagent #5-2 According to Example (4-1-1), anti-human TNF-α monoclonal antibody infliximab (Tanabe Mitsubishi Pharma) and Example (3-2) by reacting the DMF solution of affinity reagent #5-2 synthesized in the affinity reagent was obtained. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161986 where two molecules of affinity reagent were bound to infliximab.

[0411] (5-3-2) Synthesis of infliximab having two thiol groups

[0112] According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-3-1) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0412] (5-3-3) Modification of infliximab using affinity reagent #8-2 According to Example (4-1-1), anti-human TNF-α monoclonal antibody infliximab (Zenyaku Kogyo) and the DMF solution of affinity reagent #8-2 synthesized in Example (3-8) were reacted to obtain infliximab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 161870 where two molecules of affinity reagent bound to infliximab.

[0413] (5-3-4) Synthesis of infliximab having two thiol groups

[0112] According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-3-3) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0414] (5-4) Modification of Pembrolizumab Using Affinity Reagent (5-4-1) Modification of Pembrolizumab Using Affinity Reagent #5-2 According to Example (4-1-1), the anti-human PD-1 monoclonal antibody Pembrolizumab (MSD) and the affinity reagent #5-2 synthesized in Example (3-2) were reacted with a DMF solution to obtain infliximab bound to two molecules of affinity reagent. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 162041 where two molecules of affinity reagent were bound to Pembrolizumab.

[0415] (5-4-2) Synthesis of Pembrolizumab Having Two Thiol Groups According to Example (4-1-2), a thiol group-introduced antibody having two thiol groups introduced thereinto was obtained from the antibody obtained in Example (5-4-1). HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced thereinto.

[0416] (5-4-3) Modification of Pembrolizumab Using Affinity Reagent #8-2 According to Example (4-1-1), the anti-human PD-1 monoclonal antibody Pembrolizumab (MSD) was reacted with a DMF solution of the affinity reagent #8-2 synthesized in Example (3-8), to obtain Pembrolizumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 162082, where two molecules of affinity reagent bound to Pembrolizumab.

[0417] (5-4-4) Synthesis of Pembrolizumab Having Two Thiol Groups According to Example (4-1-2), a thiol-group-introduced antibody having two thiol groups introduced from the antibody obtained in Example (5-4-3) was obtained. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0418] Example 6: F(ab) using affinity molecules ２ and modification of Fab molecules (6-1) Trastuzumab F(ab) ２ and Preparation of Fab Molecules (6-1-1) Trastuzumab F(ab) ２ Synthesis of

[0419] Immobilized Pepsin (Thermo Fisher) was added to a 10 mg / mL solution of anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) in acetate buffer (50 mM sodium acetate, pH 4.5), and the mixture was shaken at 37°C for 4 hours. The reaction solution was purified using a NAP-25 desalting column (Cytiva), and then trastuzumab F(ab) was purified using AKTA pure25 (Cytiva). ２ The molecule was obtained. Fab was analyzed by ESI-TOFMS according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was observed at 97290.

[0420] Purification using AKTA pure 25 (Cytiva) was carried out under the following conditions: Column: RESOURCE S (Cytiva) Eluent A: 50 mM sodium acetate, 0.1% Tween 20 (pH 5.0) Eluent B: 50 mM sodium acetate, 1 M NaCl, 0.1% Tween 20 (pH 5.0) Flow rate: 5.0 ml / min Detector: Detection was performed at wavelengths of 215 and 280 nm.

[0421] (6-1-2) Synthesis of Trastuzumab Fab

[0422] F(ab) obtained in Example (6-1-1) ２A 2-mercaptoethylamine PBSE buffer solution (10 mM) was added to a PBSE buffer solution (10 mM Phosphate Buffered Saline (PBS), 10 mM EDTA, pH 7.4) and shaken at 37 ° C for 1 hour. The reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva) to obtain Fab. Fab analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak at 48646 was observed by ESI-TOFMS analysis.

[0423] (6-2) F(ab) using affinity reagent 5-2 ２ (6-2-1) Modification of Trastuzumab F(ab) using affinity reagent 5-2 ２ Modification of

[0424] Trastuzumab F(ab) synthesized in Example (6-1-1) according to Example (4-1-1) ２ By reacting the affinity reagent #5-2 synthesized in Example (3-4) with a DMF solution, two molecules of the affinity reagent bound to F(ab) ２ According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed to confirm F(ab) ２ A peak was confirmed at 110437 where two molecules of the affinity reagent bound to the target.

[0425] (6-2-2) Trastuzumab F(ab) having two thiol groups ２ Synthesis of

[0426] According to Example (4-1-2), F(ab) obtained in Example (6-2-1) ２ A thiol group-introduced antibody (D-S-5) of the above structural formula, in which two thiol groups had been introduced, was obtained from the above. HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that two thiol groups had been introduced.

[0427] (6-3) Modification of Fab with Affinity Reagent 5-2 (6-3-1) Modification of Trastuzumab Fab with Affinity Reagent 5-2

[0428] According to Example (4-1-1), the Fab of trastuzumab synthesized in Example (6-1-2) was reacted with a DMF solution of the affinity reagent #5-2 synthesized in Example (3-4) to obtain Fab bound to one molecule of the affinity reagent. ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and F(ab) ２ A peak was confirmed at 55219, where one molecule of the affinity reagent bound to the target.

[0429] (6-3-2) Synthesis of Trastuzumab Fab Having One Thiol Group

[0430] According to Example (4-1-2), a thiol group-introduced antibody (F-S-5) of the following structural formula was obtained, in which two thiol group molecules were introduced from the Fab obtained in Example (6-3-1). HIC-HPLC analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and it was confirmed that one thiol group molecule had been introduced.

[0431] (Example 7) Peptide Mapping (7-1) Peptide Mapping by Trypsin Treatment Peptide mapping was carried out on the trastuzumab having two thiol groups obtained in Examples (4-1-2), (4-2-2) and (4-3-2) by the following steps.

[0432] (7-1-1) Trypsin treatment of trastuzumab containing two thiol groups. 10 μL of sample solution, 75.5 μL of 250 mM Tris-HCl buffer (pH 7.5) containing 7.5 M guanidine hydrochloride, and 1.5 μL of 500 mM dithiothreitol aqueous solution were added to a 1.5 mL low-absorption microtest tube. After 30 minutes of incubation at room temperature, 3.5 μL of 500 mM iodoacetamide aqueous solution was added and reacted for 30 minutes at room temperature in the dark. After the reaction, the buffer was replaced with 100 mM Tris-HCl buffer (pH 7.5) using a desalting column. 10 μL of an aqueous solution containing 3 μg of trypsin and 1 μg of Lys-C was added, and the mixture was subjected to enzymatic digestion at 37°C for 1 hour. After digestion, 1 μL of formic acid and 2 μL of acetonitrile were added to stop the reaction. The sample solution was then subjected to LC-MS / MS analysis.

[0433] (7-1-2) LC-MS / MS Measurement of Trastuzumab Having Two Thiol Groups (Analytical Instrument) HPLC: Vanquish Duo (Thermo Fisher Scientific) Mass Spectrometer: Tribrid Mass Spectrometer Orbitrap Fusion (Thermo Fisher Scientific)

[0434] (HPLC analysis conditions) Analytical column: Acquity UPLC CSH C18 (130 Å, 1.7 μm, 2.1 × 150 mm (Waters)) Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Flow rate: 0.2 mL / min Sample injection volume: 5 μL Gradient conditions (B%): 2% (0-2 min), 2% → 37% (2-72 min), 37% → 80% (72-72.5 min), 80% (72.5-77.5 min)

[0435] (Mass spectrometer analysis conditions) Ionization method: ESI, positive mode Scan type: data dependent acquisition Activation type: higher-energy collisional dissociation (HCD) Data acquisition was performed using the accompanying software Xcalibur 4.3 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 3.3 (Thermo Fisher Scientific).

[0436] (7-1-3) Analysis of Modification Sites in Trastuzumab Having Two Thiol Groups Modification site analysis of the LC-MS / MS measurement results was performed using BioPharma Finder 5.1 (Thermo Fisher Scientific). Analysis using BioPharma Finder was performed with the S / N Threshold set to 16 for the thiol-introduced products obtained in (4-2-2) and (4-1-2) and 98 for the thiol-introduced product obtained in (4-3-2), and the MS Noise Level set to 2000 for all analyses. The digestive enzyme was set to trypsin, and the specificity was set to strict. Static modifications included carbamidomethyl (+57.021 Da) modification of cysteine ​​residues with iodoacetamide. Dynamic modifications included oxidation of methionine and tryptophan residues (+15.995 Da), deamidation of glutamic acid and aspartic acid residues (+0.984 Da), and modification of lysine residues (thiol-introduced carbamidomethylated with iodoacetamide (+145.019 Da)). In addition, pyroglutamylation of glutamic acid residues (-18.011 Da) was set as a dynamic modification to the N-terminus of the protein, and addition of lysine (+128.095 Da) was set as a dynamic modification to the C-terminus of the protein. Furthermore, a filter was set to include only those with a confidence score of 80 or higher, a mass accuracy of 5 ppm or less at the time of peptide identification, and MS / MS observation. Regarding the residue numbers of lysine residues, those on the heavy chain VH domain and light chain were represented by the number in the sequence (i.e., the N-terminal amino acid was numbered first; the same applies below), and those on the heavy chain CH1, CH2, and CH3 domains were represented using EU numbering.

[0437] (7-1-4) Amino acid sequences used in the analysis The following amino acid sequences (1) and (2) shown in SEQ ID NOs: 29 and 30 were used as data for the amino acid sequences to be searched for modification sites. (1) Amino acid sequence of the heavy chain of trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 29) (2) Amino acid sequence of the light chain of trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30)

[0438] (7-2) Analysis of modification sites of trastuzumab with two thiol groups by LC-MS / MS

[0439] (7-2-1) Analysis Results of Modification Sites of Trastuzumab Obtained in Example (4-2-2) Introduced with Two Thiol Groups by LC-MS / MS As a result of the analysis using LC-MS / MS, the MS spectrum (measured value: m / z 1366.30822, theoretical value: 1366.30859, trivalent) of the peptide fragment of WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK (SEQ ID NO: 27), a peptide consisting of 38 amino acid residues containing the modification site on the lysine residue by trypsin digestion of trastuzumab (thiol-introduced product (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) was observed ( FIG. 2 ), and the HCD spectrum indicated that the heavy chain EU A product ion of m / z 1560.80 (theoretical value: 1560.81), corresponding to monovalent y14, was confirmed, indicating modification of the lysine residue at position 121 in the EU numbering (Figure 3). Furthermore, analysis using BioPharma Finder demonstrated that modification of the lysine residue at position 121 occurred highly selectively (Figure 4). These results demonstrate that in the trastuzumab into which two thiol groups obtained in (4-2-2) above had been introduced, regioselective conjugation had occurred to Lys121 in the EU numbering on the heavy chain of the antibody.

[0440] (7-2-2) Analysis Results of Modification Sites of Trastuzumab Obtained in Example (4-3-2) Introduced with Two Thiol Groups by LC-MS / MS As a result of the analysis using LC-MS / MS, the MS spectrum (measured value: m / z 1366.30781, theoretical value: 1366.30859, trivalent) of the peptide fragment of WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK (SEQ ID NO: 27), a peptide consisting of 38 amino acid residues containing the modification site on the lysine residue by trypsin digestion of trastuzumab (thiol-introduced product (+145.019 Da) subjected to carbamidomethylation with iodoacetamide) was observed ( FIG. 5 ), and the HCD spectrum indicated that the heavy chain EU A product ion of m / z 1560.80 (theoretical value: 1560.81), corresponding to monovalent y14, was confirmed, indicating modification of the lysine residue at position 121 in the EU numbering (Figure 6). Furthermore, analysis using BioPharma Finder demonstrated that modification of the lysine residue at position 121 occurred highly selectively (Figure 7). These results demonstrate that in the trastuzumab into which two thiol groups obtained in (7-2-2) above had been introduced, regioselective conjugation had occurred to Lys121 in the EU numbering on the heavy chain of the antibody.

[0441] (7-2-3) Analysis of the Modification Site of Trastuzumab Obtained in Example (4-1-2) with Two Thiol Groups Introduced by LC-MS / MS As a result of the analysis using LC-MS / MS, the MS spectrum (measured value: m / z 1372.47258, theoretical value: 1372.47321, pentavalent) of the peptide fragment DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK (SEQ ID NO: 28), which is a peptide consisting of 38 amino acid residues including the modification site on the lysine residue by trypsin digestion of trastuzumab (thiol-introduced product (+145.019 Da) that has been carbamidomethylated with iodoacetamide), was observed ( FIG. 8 ). The HCD spectrum indicated that the heavy chain EU A product ion of m / z 1290.10 (theoretical value: 1289.61), corresponding to divalent y22, was confirmed (FIG. 9), indicating modification of the lysine residue at position 210 in the EU numbering. Furthermore, analysis using BioPharma Finder demonstrated that modification of the lysine residue at position 210 occurred highly selectively (FIG. 10). These results demonstrate that in the trastuzumab into which two thiol groups obtained in (4-1-2) above had been introduced, regioselective conjugation had occurred to Lys210 in the EU numbering on the heavy chain of the antibody.

[0442] (Example 8) Design of affinity polypeptides and secretory expression of affinity polypeptides #9, 10, 11, and 12 in C. glutamicum (8-1) Preparation of affinity polypeptides The following affinity polypeptides were prepared: (i) Affinity peptide #9 QEPAPAAPAPAAPAPAPAPAPAPAPAPAGGTTYRLVINGRTLRGETTTEAVDAETAAAKFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 32)

[0443] (j) Affinity peptide #10 QEPAPAAPAPAAPAAPAPAAPAAPAPAPAAPAPAPAPAPAPAPAPAPAPAPAAPAPAGGTTYRLVINGRTLRGETTTEAVDAETAAAKFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 33)

[0444] (k) Affinity peptide #11 QEPAPAAPAPAAPAPAPAPAAPAPAPAGGTTYRLVINGRTLRGKTTTEAVDAETAAAAFAQQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 34)

[0445] (l) Affinity peptide #12 QEPAPAAPAPAAPAAPAPAAPAAPAPAPAAPAPAPAPAPAPAPAPAPAPAPAAPAPAGGTTYRLVINGRTLRGKTTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 35)

[0446] (8-2) Construction of secretory expression plasmids for affinity peptides #9-12 Four types of amino acid sequences for each of the affinity peptides described above were designed as affinity polypeptides, and the nucleotide sequences encoding these polypeptides were designed taking into account the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed to enable secretory expression by C. glutamicum.

[0447] Affinity peptide #9 was secreted and expressed as a fusion protein of affinity peptide #9 and the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain (hereinafter referred to as "CspBss-AP9"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP9 are shown in SEQ ID NOs: 36 and 37, respectively.

[0448] Nucleotide sequence encoding CspBss-AP9: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatccccagcattcgctcaggagccagcaccagctgccccggctccggctgcgcctgctgcacctgcgccagctgcacctgcacctgcgccagctgcacctgcagcaccggcacctgggaggcac cacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactgaagctgtggatgcagagaccgcagcagctaagttt gcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 36)

[0449] Amino acid sequence of CspBss-AP9: MFNNRIRTAALAGAIAISTAASGVAIPAFAQEPAPAAPAPAAAPAPAPAAPAAPAPAGGTTYRLVINGRTLRGETTTEAVDAETAAAKFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 37)

[0450] Affinity peptide #10 was secreted and expressed as a fusion protein of affinity peptide #10 and the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain (hereinafter referred to as "CspBss-AP10"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP10 are shown in SEQ ID NOs: 38 and 39, respectively.

[0451] Base sequence encoding CspBss-AP10 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagccagcaccag ctgccccggctccggctgcgcctgctgcacctgcgccagctgcacctgcagcaccggcacctgctgctccagcagcacccgctcctgcagctcccgctgccccagc tccagcggcgcctgcggcgccagcacctggaggcaccacctatcgcctcgtgatcaacggacgtactctgcgtggtgaaaccaccactgaagctgtggatgcagag accgcagcagctaagtttgcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 38)

[0452] Amino acid sequence of CspBss-AP10 MFNNRIRTAALAGAIAISTAASGVAIPAFAQEPAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAPAAPAAPAPAAPAPAAPAPAGGTTYRLVINGRTLRGETTTEAVDAETAAAKFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 39)

[0453] Affinity peptide #11 was secreted and expressed as a fusion protein of the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain and affinity peptide #11 (hereinafter referred to as "CspBss-AP11"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP11 are shown in SEQ ID NOs: 40 and 41, respectively.

[0454] Nucleotide sequence encoding CspBss-AP11: atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatccccagcattcgctcaggagccagcaccagctgccccggctccggctgcgcctgctgcacctgcgccagctgcacctgcgccagctgcacctgcagcaccggcacctggaggcac cacctatcgcctcgtgatcaacggacgtactctgcgtggtaagaccaccactgaagctgtggatgcagagaccgcagcagctgcgtt gcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 40)

[0455] Amino acid sequence of CspBss-AP11 MFNNRIRTAALAGAIAISTAASGVAIPAFAQEPAPAAPAPAAPAAPAPAAPAAPAPAAPAGGTTYRLVINGRTLRGKTTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 41)

[0456] Affinity peptide #12 was secreted and expressed as a fusion protein of affinity peptide #12 and the 30 amino acid signal peptide of CspB derived from C. glutamicum ATCC13869 strain (hereinafter referred to as "CspBss-AP12"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AP12 are shown in SEQ ID NOs: 42 and 43, respectively.

[0457] Base sequence encoding CspBss-AP12 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctcaggagccagcaccag ctgccccggctccggctgcgcctgctgcacctgcgccagctgcacctgcagcaccggcacctgctgctccagcagcacccgctcctgcagctcccgctgccccagc tccagcggcgcctgcggcgccagcacctggaggcaccacctatcgcctcgtgatcaacggacgtactctgcgtggtaagaccaccactgaagctgtggatgcagag accgcagcagctgcgtttgcgcagtacgcaaacgacaatggcgttgatggcgtttggacctacgacgatgccactcgcaccttcaccgtcactgagtaa (SEQ ID NO: 42)

[0458] Amino acid sequence of CspBss-AP12 MFNNRIRTAALAGAIAISTAASGVAIPAFAQEPAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAPAAPAAPAPAPAAPAAPAPAAPAPAGGTTYRLVINGRTLRGKTTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE (SEQ ID NO: 43)

[0459] CspBss-AP9, CspBss-AP10, CspBss-AP11, and CspBss-AP12 are upstream of the base sequence described in C. glutamicum ATCC13869 strain-derived cspB gene promoter was linked, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. Expression cassettes for four types of affinity polypeptides were designed and totally synthesized. The totally synthesized DNA fragment (affinity polypeptide expression cassette) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774, and the affinity polypeptide secretion expression plasmids pPK4_CspBss-AP9, pPK4_CspBss-AP10, pPK4_CspBss-AP11, and pPK4_CspBss-AP12 were constructed, respectively. The nucleotide sequences of the inserts were determined using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3500xL Genetic Analyzer (Applied Biosystems), confirming that the expression cassettes for the affinity polypeptides were constructed as designed.

[0460] (8-3) C. Secretory expression of affinity polypeptide in glutamicum Using the pPK4_CspBss-AP9, pPK4_CspBss-AP10, pPK4_CspBss-AP11, and pPK4_CspBss-AP12 constructed above, C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain YDK0107 / pPK4_CspBss-AP9 strain, YDK0107 / pPK4_CspBss-AP10 strain, YDK0107 / pPK4_CspBss-AP11 strain, and YDK0107 / pPK4_CspBss-AP12 strain.

[0461] Each of the obtained transformants was cultured at 30°C for 72 hours in an MMTG liquid medium containing 25 mg / L of kanamycin (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloric acid hydrolyzate (total nitrogen content 0.2 g), calcium carbonate 50 g, and water to make 1 L, adjusted to pH 7.0).

[0462] After the culture was completed, each culture medium was centrifuged, and 6.5 μL of the resulting culture supernatant was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tris Gel (Thermo Fisher Scientific), followed by staining with Quick-CBB (Wako). As a result, a polypeptide band presumed to be affinity peptide #9 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP9 strain (Fig. 11, Lanes 3-5), a polypeptide band presumed to be affinity peptide #10 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP10 strain (Fig. 11, Lanes 9-11), a polypeptide band presumed to be affinity peptide #11 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP11 strain (Fig. 11, Lanes 6-8), and a polypeptide band presumed to be affinity peptide #12 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AP12 strain (Fig. 11, Lanes 10-11). A polypeptide band presumed to be #12 was detected (FIG. 11, Lanes 12-14).

[0463] Example 9: Purification of affinity polypeptides #9, 10, 11, and 12 Culture supernatants of affinity polypeptides #9, 10, 11, and 12 were incubated at 37°C for two days. 5-7 ml of each filtrate was added with 9 volumes of eluent A (20 mM Tris-HCl, pH 8.0) to prepare the load sample. Purification using the AKTA pure 150 (Cytiva) was performed under the following conditions: Column: HiPrep Q FF 16 / 10 (Cytiva); Eluent A: 20 mM Tris-HCl, pH 8.0; Eluent B: 20 ​​mM Tris-HCl, 1 M NaCl, pH 8.0; Flow rate: 5.0 mL / min; Gradient: 0-50% B / 20 CV; Detector: Detection at a wavelength of 280 nm.

[0464] In #9 and #10, 60 mL of the elution fraction containing the target substance was collected and concentrated to 4 mL using an Amicon Ultra-3K (Merck). 56 mL of water for injection was added to the concentrate, and the concentrate was again concentrated to 4 mL. 56 mL of water for injection was added to the concentrate, and the concentrate was again concentrated to 2.4 mL. In #11 and #12, 80 mL of the elution fraction containing the target substance was collected and concentrated to 6 mL using an Amicon Ultra-3K (Merck). 84 mL of water for injection was added to the concentrate, and the concentrate was again concentrated to 6 mL. 84 mL of water for injection was added to the concentrate, and the concentrate was again concentrated to 4.9 mL. The obtained concentrate was subjected to HPLC, and the peak of the target substance was confirmed.

[0465] (Example 10) Preparation of affinity polypeptides for antibodies, compounds having a cleavable moiety and a reactive group Compounds having a cleavable moiety and a reactive group were conjugated to affinity peptides #5 to #12 purified in Examples 2 and Y.

[0466] (10-1) Synthesis of Affinity Reagent #5-3 According to a previous report (WO2022 / 191283), affinity reagent #5-3 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #5 purified in Example (2-2). MS (ESI) m / z: z=1385.72 [M+5H] ５＋

[0467] (10-2) Synthesis of Affinity Reagent #6-3 According to a previous report (WO2022 / 191283), affinity reagent #6-3 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #6 purified in Example (2-1). MS (ESI) m / z: z=1376.92 [M+5H] ５＋

[0468] (10-3) Synthesis of Affinity Reagent #8-3 According to a previous report (WO2022 / 191283), affinity reagent #8-3 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #8 purified in Example (2-2). MS (ESI) m / z: z=1370.52 [M+5H] ５＋

[0469] (10-4) Synthesis of Affinity Reagent #9-1 According to a previous report (WO2019 / 0240287), affinity reagent #9-1 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #9 purified in Example 8. MS (ESI) m / z: z=1753.80 [M+5H] ５＋

[0470] (10-5) Synthesis of Affinity Reagent #9-2 According to a previous report (WO2022 / 191283), affinity reagent #9-2 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #9 purified in Example Y. MS (ESI) m / z: z=1798.52 [M+5H] ５＋

[0471] (10-6) Synthesis of Affinity Reagent #10-1 According to a previous report (WO2019 / 0240287), affinity reagent #10-1 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #10 purified in Example 8. MS (ESI) m / z: z=2140.80 [M+5H] ５＋

[0472] (10-7) Synthesis of Affinity Reagent #10-2 According to a previous report (WO2022 / 191283), affinity reagent #10-2 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #10 purified in Example 8. MS(ESI) / z:z=2185.52[M+5H] ５＋

[0473] (10-8) Synthesis of Affinity Reagent #11-1 According to a previous report (WO2019 / 0240287), affinity reagent #11-1 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and Affinity Peptide #11 purified in Example 8. MS (ESI) m / z: z=1741.80 [M+5H] ５＋

[0474] (10-9) Synthesis of Affinity Reagent #11-2 According to a previous report (WO2022 / 191283), affinity reagent #11-2 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #11 purified in Example 8. MS (ESI) m / z: z=1786.52 [M+5H] ５＋

[0475] (10-10) Synthesis of Affinity Reagent #12-1 According to a previous report (WO2019 / 0240287), affinity reagent #12-1 was synthesized by amidation using compound (L-2) having a cleavable moiety and a reactive group and affinity peptide #12 purified in Example 8. MS (ESI) m / z: z=2129.80 [M+5H] ５＋

[0476] (10-11) Synthesis of Affinity Reagent #12-2 According to a previous report (WO2022 / 191283), affinity reagent #12-2 was synthesized by amidation using compound (L-3) having a cleavable moiety and a reactive group and affinity peptide #12 purified in Example 8. MS (ESI) m / z: z=2174.52 [M+5H] ５＋

[0477] (Example 11) Modification of antibody using affinity reagent (11-1) Modification of antibody using affinity reagent #9-1 (11-1-1) Synthesis of trastuzumab bound to two molecules of affinity reagent #9-1

[0478] A DMSO solution of the affinity reagent #9-1 synthesized in Example (10-4) (7 equivalents relative to the antibody) was added to a solution of anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) in borate buffer (20 mM borate, pH 8.2), and the mixture was shaken for 1 hour. The reaction solution was purified using Nap25 (Cytiva) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 165534, where two molecules of affinity reagent were bound to trastuzumab.

[0479] (11-1-2) Synthesis of Trastzumab Having Two Thiol Groups

[0480] The antibody obtained in Example (11-1-1) was subjected to a thioester group cleavage reaction (treatment with hydroxylamine) according to a previously reported method (WO 2019 / 240287), yielding a thiol group-introduced antibody (T-S-9) with the following structural formula, in which two thiol groups had been introduced. When the mass was measured by ESI-TOFMS, a peak was confirmed at 148,406, indicating that the cleavage reaction had progressed.

[0481] (11-1-3) Synthesis of ADC with DAR = 2

[0482] A dimethylacetamide solution (5 equivalents, dimethylacetamide (DMA) (8% v / v) solution) of the payload linker (P1) described in WO2023 / 054714 was added to a PBSE buffer solution (10 mM Phosphate Buffered Saline (PBS), 10 mM EDTA, pH 7.4) of the antibody (T-S-9) having two thiol groups obtained in Example (11-1-2), and the mixture was shaken at room temperature for 1 hour. The reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva), and an ADC (T-M-9) with a DAR of 2 was obtained. The analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and ESI-TOFMS analysis was performed, and a peak was observed at 151418.

[0483] (11-2) Modification of antibody using affinity reagent 10-1 (11-2-1) Synthesis of trastuzumab with two molecules of affinity reagent 10-1 bound According to Example (11-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #10-1 synthesized in Example (10-6) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 169297, where two molecules of affinity reagent were bound to trastuzumab.

[0484] (11-2-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (11-1-2), a thiol group-introduced antibody (T-S-10) of the following structural formula was obtained, in which two thiol groups were introduced from the antibody obtained in Example (11-2-1). ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was confirmed at 148399, indicating that two thiol groups were bound to trastuzumab.

[0485] (11-2-3) Synthesis of ADC with DAR = 2 According to Example (11-1-3), the antibody (T-S-10) having two thiol groups obtained in Example (11-2-2) was reacted with the payload linker (P1) described in WO2023 / 054714 to obtain an ADC with DAR = 2 (T-M-5). According to a previous report (Anal.Chem., 2019,91,20,12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 151410, where two molecules of P1 were bound to trastuzumab.

[0486] (11-3) Modification of antibody using affinity reagent 11-1 (11-3-1) Synthesis of trastuzumab with two molecules of affinity reagent 11-1 bound According to Example (11-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #11-1 synthesized in Example (10-8), thereby obtaining trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 165419, where two molecules of affinity reagent were bound to trastuzumab.

[0487] (11-3-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (11-1-2), a thiol group-introduced antibody (T-S-11) of the following structural formula was obtained, in which two thiol groups were introduced from the antibody obtained in Example (11-3-1). ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was confirmed at 148562, indicating that two thiol groups were bound to trastuzumab.

[0488] (11-3-3) Synthesis of ADC with DAR = 2 According to Example (11-1-3), the antibody (T-S-11) having two thiol groups obtained in Example (11-3-2) was reacted with the payload linker (P1) described in WO2023 / 054714 to obtain an ADC with DAR = 2 (T-M-5). According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 151418, where two molecules of P1 were bound to trastuzumab.

[0489] (11-4) Modification of antibody using affinity reagent 12-1 (11-4-1) Synthesis of trastuzumab with two molecules of affinity reagent 12-1 bound According to Example (11-1-1), anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) was reacted with a DMF solution of the affinity reagent #12-1 synthesized in Example (10-10), thereby obtaining trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 169461, where two molecules of affinity reagent were bound to trastuzumab.

[0490] (11-4-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (11-1-2), a thiol group-introduced antibody (T-S-12) of the following structural formula was obtained, in which two thiol groups were introduced from the antibody obtained in Example (11-4-1). ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was confirmed at 148401, indicating that two thiol groups were bound to trastuzumab.

[0491] (11-4-3) Synthesis of ADC with DAR = 2 According to Example (11-1-3), the antibody (T-S-12) having two thiol groups obtained in Example (11-4-2) was reacted with the payload linker (P1) described in WO2023 / 054714 to obtain an ADC with DAR = 2 (T-M-5). According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 151408, where two molecules of P1 were bound to trastuzumab.

[0492] (11-5) Modification of antibody using affinity reagent #11-2 (11-5-1) Synthesis of trastuzumab bound to two molecules of affinity reagent #11-2

[0493] A DMSO solution of the affinity reagent #11-2 synthesized in Example (10-9) (7 equivalents relative to the antibody) was added to a solution of the anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) in borate buffer (20 mM borate, pH 8.2), and the mixture was shaken for 1 hour. The reaction solution was purified using Nap25 (Cytiva) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 170096, where two molecules of affinity reagent were bound to trastuzumab.

[0494] (11-5-2) Synthesis of Trastzumab Having Two Azide Groups

[0495] The antibody obtained in Example (11-5-1) was subjected to a thioester group cleavage reaction (treatment with methoxyamine) according to a previously reported method (WO 2022 / 191283), yielding an azide-introduced antibody (Ta-a-11) having the following structural formula, in which two azide groups had been introduced. When the mass was measured by ESI-TOFMS, a peak was confirmed at 148922, indicating that the cleavage reaction had progressed.

[0496] (11-5-3) Synthesis of ADC with DAR = 2

[0497] To a solution of the antibody (T-a-11) having two azide groups obtained in Example (11-5-2) in acetate buffer (20 mM sodium acetate, pH 5.5), a solution of the known compound DBCO-DM1 (Organic & Biomolecular Chemistry, 2016, 14, 9501-9518) in dimethylacetamide (5 equivalents, dimethylacetamide (DMA) (8% v / v) solution) was added, and the mixture was shaken at room temperature for 12 hours. The reaction solution was purified using a NAP-25 desalting column (manufactured by Cytiva) to obtain an ADC (T-D-1) with a DAR of 2. The analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and ESI-TOFMS analysis was performed, and a peak was observed at 150863.

[0498] (11-6) Modification of antibody using affinity reagent #7-2 (11-6-1) Synthesis of trastuzumab bound to two molecules of affinity reagent #7-2

[0499] A DMSO solution of the affinity reagent #9-1 synthesized in Example (3-6) (7 equivalents relative to the antibody) was added to a solution of the anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) in borate buffer (20 mM borate, pH 8.2), and the mixture was shaken for 1 hour. The reaction solution was purified using Nap25 (Cytiva) to obtain trastuzumab with two molecules of affinity reagent bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 165534, where two molecules of affinity reagent were bound to trastuzumab.

[0500] (11-6-2) Synthesis of Trastuzumab Having Two Thiol Groups According to Example (11-1-2), a thiol group-introduced antibody of the following structural formula, in which two thiol groups were introduced from the antibody obtained in Example (11-6-1), was obtained. ESI-TOFMS analysis was performed according to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), and a peak was confirmed at 148562, indicating that two thiol groups were bound to trastuzumab.

[0501] (11-6-3) Synthesis of ADC with DAR = 2 According to Example (11-1-3), an ADC with DAR = 2 was obtained by reacting the antibody having two thiol groups obtained in Example (11-6-2) with the payload linker (P1) described in WO2023 / 054714. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 151418, where two molecules of P1 were bound to trastuzumab.

[0502] (Example 12) Modification of antibody using two types of affinity reagents (12-1) Preparation of antibody complex of DAR4 using affinity reagent #5-2 (12-1-1) Synthesis of trastuzumab bound to four molecules of affinity reagent

[0503] A solution of anti-human HER2 monoclonal antibody trastuzumab (Chugai Pharmaceutical) in borate buffer (20 mM borate, pH 8.2) was added to a DMSO solution of affinity reagent #5-2 (7 equivalents relative to the antibody) synthesized in Example (3-2) and a DMSO solution of affinity reagent Z (WO2022 / 154116) (6 equivalents relative to the antibody), and the mixture was shaken for 1 hour. The reaction solution was purified using Nap25 (manufactured by Cytiva) to obtain trastuzumab in which two molecules of two affinity reagents were bound. According to a previous report (Anal. Chem., 2019, 91, 20, 12724-12732), ESI-TOFMS analysis was performed, and a peak was confirmed at 170300, where four molecules of affinity reagent were bound to trastuzumab.

[0504] (12-1-2) Synthesis of Trastzumab Havi...

Claims

A compound or a salt thereof comprising: (A) an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of an antibody; and (B) a group reactive to the antibody. The compound or salt thereof according to claim 1, wherein the affinity polypeptide comprises an amino acid sequence including an amino acid residue having a side chain amino group, and a group reactive to the antibody is bound to the affinity polypeptide via a linker introduced into the side chain amino group of the amino acid residue having the side chain amino group. The affinity polypeptide is a compound or salt thereof according to claim 2, which has an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to said amino acid sequence, in which an amino acid residue having a side chain amino group has been introduced into a specific amino acid residue. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE The compound or salt thereof according to claim 3, wherein the specific amino acid residue is located near the target lysine present in the heavy chain CH1 region of the antibody in the three-dimensional configuration when the affinity polypeptide is coexistent with the antibody. The compound or salt thereof according to claim 4, wherein the specific amino acid residue is located within 30 Å of the target lysine present in the heavy chain CH1 region of the antibody in the three-dimensional configuration when the affinity polypeptide is coexistent with the antibody.

5. The compound or salt thereof according to claim 4, wherein the target lysine present in the heavy chain CH1 region of the antibody is selected from lysine at positions 121, 147, 205, and 210 of a human IgG heavy chain according to EU numbering. The compound or salt thereof according to claim 3, wherein the specific amino acid residue is selected from A at position 29, D at position 36, E at position 19, or E at position 15 of SEQ ID NO:

9.

2. The compound or salt thereof according to claim 1, wherein the CH1 region is a human CH1 region. The compound or salt thereof according to claim 1, wherein the antibody is an IgG. The compound or salt thereof according to claim 1, wherein the affinity polypeptide further comprises a tripeptide consisting of Gln-Glu-Thr (QET) or a dipeptide consisting of Gln-Glu (QE) at the N-terminus. The compound has the following formula (I):

2. The compound according to claim 1, or a salt thereof, represented by the formula: wherein R represents the reactive group, L represents a linker, and A represents the affinity polypeptide.

2. The compound or salt thereof according to claim 1, wherein the compound or salt thereof further comprises (iii) a cleavable moiety between (i) the affinity polypeptide and (ii) the reactive group. The compound or salt thereof according to claim 12, wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the reactive group side upon cleavage. The compound has the following formula (Ia): [wherein R represents the reactive group; L １ indicates the first linker, L ２ represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the reactive group side upon cleavage, and A represents the affinity polypeptide. The compound has the following formula (Ia-1): [wherein X represents a leaving group, W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ indicates a third linker, L ４ represents a fourth linker, S represents a sulfur atom, and A represents the affinity polypeptide.

16. The compound of claim 15, or a salt thereof, wherein the leaving group is selected from the following: (a) R Ａ -S (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and S represents a sulfur atom; (b) R Ａ -O (where R Ａ represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and O represents an oxygen atom; (c) R Ａ - (R Ｂ -) N (where R Ａ and R Ｂ each independently represents a hydrogen atom, an optionally substituted monovalent hydrocarbon group, or an optionally substituted monovalent heterocyclic group, and N represents a nitrogen atom; or (d) a halogen atom.

13. The compound or salt thereof of claim 12, wherein the compound or salt thereof further comprises (iv) a bioorthogonal functional group between (ii) the reactive group and (iii) the cleavable moiety. The compound has the following formula (Ib): [wherein R represents the reactive group; L ５ indicates the fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, and A represents the affinity polypeptide. The compound is represented by the following formula (Ib-1): [wherein X represents a leaving group, W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ indicates the seventh linker, L ８ represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, and A represents the affinity polypeptide. 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. A reagent for antibody derivatization, comprising the compound according to any one of claims 1 to 20 or a salt thereof. An affinity polypeptide-modified antibody or a salt thereof, which comprises, in the heavy chain CH1 region of an antibody, an affinity polypeptide comprising an affinity moiety having affinity for the heavy chain CH1 region of the antibody. The affinity polypeptide-modified antibody or salt thereof according to claim 22, comprising (a) an immunoglobulin unit comprising a heavy chain and a light chain, and (b) the affinity polypeptide, and (c) the affinity polypeptide is introduced into the heavy chain CH1 region of the immunoglobulin unit. The affinity polypeptide-modified antibody or salt thereof according to claim 23, wherein the affinity polypeptide is introduced into the heavy chain CH1 region via modification of the amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region. The affinity polypeptide-modified antibody or salt thereof according to claim 24, wherein one or more positions in the heavy chain CH1 region are selected from positions 121, 147, 205, and 210 of the human IgG heavy chain according to EU numbering. The affinity polypeptide is an affinity polypeptide-modified antibody or salt thereof according to claim 22, which has an amino acid sequence in which an amino acid having a side chain amino group has been introduced at a specific amino acid residue in the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to said amino acid sequence, and which is modified at the amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region via a linker introduced to the side chain amino group of the amino acid having the side chain amino group. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE The affinity polypeptide-modified antibody has the following formula (II): The affinity polypeptide-modified antibody or salt thereof according to claim 22, comprising a structural unit represented by the formula: (wherein Ig represents an immunoglobulin unit comprising a heavy chain and a light chain, L represents a linker, A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin unit with the affinity polypeptide is 65 to 135%). The affinity polypeptide-modified antibody or salt thereof according to claim 22, wherein the antibody or salt thereof further comprises (iii') a cleavable moiety between (i') the affinity polypeptide and (ii') the antibody. The affinity polypeptide-modified antibody or its salt according to claim 28, wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the immunoglobulin unit side upon cleavage. The affinity polypeptide-modified antibody has the following formula (IIa): [wherein Ig represents the immunoglobulin unit; L １ indicates the first linker, L ２ represents a second linker, CLE(B) represents a cleavable moiety capable of generating a bioorthogonal functional group on the immunoglobulin unit side upon cleavage, A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin unit with the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody has the following formula (IIa-1): [wherein Ig represents the immunoglobulin unit; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ３ indicates a third linker, L ４ represents a fourth linker, S represents a sulfur atom, A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin units with the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody or salt thereof according to claim 28, wherein the antibody or salt thereof further comprises (iv') a bioorthogonal functional group between (ii') the antibody and (iii') the cleavable moiety. 　 The affinity polypeptide-modified antibody has the following formula (IIb): [wherein Ig represents the immunoglobulin unit; L ５ indicates the fifth linker, L ６ represents a sixth linker, B represents a group containing a bioorthogonal functional group, CLE represents a cleavable moiety, and A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin units by the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody has the following formula (IIb-1): [wherein Ig represents the immunoglobulin unit; W １ , W ２ and W ３ each independently represents an oxygen atom or a sulfur atom, L ７ indicates the seventh linker, L ８ represents an eighth linker, B represents a group containing a bioorthogonal functional group, V represents an oxygen atom or a sulfur atom, and A represents the affinity polypeptide, and the average modification percentage r of the immunoglobulin units with the affinity polypeptide is 65 to 135%. The affinity polypeptide-modified antibody or salt thereof according to claim 22, wherein the affinity polypeptide-modified antibody further comprises an additional modifying moiety. The affinity polypeptide-modified antibody or salt thereof according to claim 35, wherein the additional modifying portion is an additional affinity polypeptide comprising an affinity portion having affinity for the heavy chain constant region of the antibody, and the additional affinity polypeptide is contained in the heavy chain constant region of the antibody. The affinity polypeptide-modified antibody or salt thereof according to claim 36, wherein the additional affinity polypeptide is introduced into the heavy chain constant region via modification of the amino group in the side chain of a lysine residue present at one or more positions in the heavy chain constant region. The affinity polypeptide-modified antibody or salt thereof according to claim 37, wherein one or more positions in the heavy chain constant region are selected from positions 220, 226, 229, 246, 248, 274, 288, 290, 317, 320, and 322 of the human IgG heavy chain according to EU numbering. A method for producing an affinity polypeptide-modified antibody or its salt, comprising reacting the antibody derivatization reagent described in claim 21 with an antibody comprising an immunoglobulin unit including a heavy chain and a light chain to produce an affinity polypeptide-modified antibody or its salt comprising the affinity polypeptide in the heavy chain CH1 region of the immunoglobulin unit. An antibody derivative or salt thereof comprising a bioorthogonal functional group, comprising (a) an immunoglobulin unit comprising a heavy chain and a light chain, and (b) a bioorthogonal functional group, and (c) the bioorthogonal functional group is introduced into the heavy chain CH1 region of the immunoglobulin unit. The antibody derivative or salt thereof according to claim 40, wherein the bioorthogonal functional group is introduced into the heavy chain CH1 region via modification of an amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region.

42. The antibody derivative or salt thereof according to claim 41, wherein the one or more positions in the heavy chain CH1 region are selected from positions 121, 147, 205, and 210 of the human IgG heavy chain according to EU numbering. The antibody derivative or salt thereof containing a bioorthogonal functional group has the following formula (IIIa): [wherein Ig represents the immunoglobulin unit; L １ represents a first linker, B represents a group containing a bioorthogonal functional group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%. The antibody derivative or salt thereof containing a bioorthogonal functional group is represented by the following formula (IIIa-1): [wherein Ig represents the immunoglobulin unit; W １ represents an oxygen atom or a sulfur atom, and L ３ represents a third linker, SH represents a thiol group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%. The antibody derivative or salt thereof containing a bioorthogonal functional group is represented by the following formula (IIIb): [wherein Ig represents the immunoglobulin unit; L ５ represents a fifth linker, B represents a group containing a bioorthogonal functional group, and T １ represents a monovalent group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%. The antibody derivative or salt thereof containing a bioorthogonal functional group is represented by the following formula (IIIb-1): [wherein Ig represents the immunoglobulin unit; W １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, B represents a group containing a bioorthogonal functional group, and T ２ represents a monovalent group, and the average modification percentage r of the immunoglobulin units with the bioorthogonal functional group is 65 to 135%.

41. The antibody derivative or salt thereof of claim 40, wherein the antibody derivative further comprises an additional modifying moiety.

48. The antibody derivative or salt thereof according to claim 47, wherein the additional modifying moiety comprises an additional affinity polypeptide comprising a bioorthogonal functional group, and the bioorthogonal functional group is contained in the heavy chain constant region of the antibody.

49. The antibody derivative or salt thereof according to claim 48, wherein an additional modifying moiety comprising an additional affinity polypeptide comprising the bioorthogonal functional group is introduced into the heavy chain constant region via modification of an amino group in the side chain of a lysine residue present at one or more positions in the heavy chain constant region.

50. The antibody derivative or salt thereof according to claim 49, wherein the one or more positions in the heavy chain constant region are selected from positions 220, 226, 229, 246, 248, 274, 288, 290, 317, 320, and 322 of a human IgG heavy chain according to EU numbering. A conjugate of an antibody and a functional substance, or a salt thereof, comprising (a) an immunoglobulin unit including a heavy chain and a light chain, and (b) a functional substance, and (c) the functional substance is introduced into the heavy chain CH1 region of the immunoglobulin unit.

52. The conjugate or salt thereof according to claim 51, wherein the functional substance is introduced into the heavy chain CH1 region via modification of an amino group in the side chain of a lysine residue present at one or more positions in the heavy chain CH1 region.

53. The conjugate or salt thereof according to claim 52, wherein the one or more positions in the heavy chain CH1 region are selected from positions 121, 147, 205, and 210 of a human IgG heavy chain according to EU numbering. The conjugate or salt thereof has the following formula (IVa): [wherein Ig represents the immunoglobulin unit; L １ represents a first linker, Z represents a functional substance, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%. The conjugate or a salt thereof is represented by the following formula (IVa-1): [wherein Ig represents the immunoglobulin unit; W １ represents an oxygen atom or a sulfur atom, and L ３ represents a third linker, Z represents a functional substance, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%. The conjugate or salt thereof is represented by the following formula (IVb): [wherein Ig represents the immunoglobulin unit; L ５ represents a fifth linker, Z represents a functional substance, and T １ represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%. The conjugate or a salt thereof is represented by the following formula (IVb-1): [wherein Ig represents the immunoglobulin unit; W １ , and W ２ each independently represents an oxygen atom or a sulfur atom, L ７ represents a seventh linker, Z represents a functional substance, and T ２ represents a monovalent group, and the average percentage modification r of the immunoglobulin units with the functional substance is 65 to 135%.

52. The conjugate or salt thereof according to claim 51, wherein the functional substance is a drug, a labeling substance, an affinity polypeptide, a transport substance, or a stabilizer.

52. The conjugate or salt thereof according to claim 51, wherein the affinity polypeptide is a full-length antibody or a fragment thereof.

52. The conjugate of claim 51, or a salt thereof, wherein the conjugate further comprises an additional modifying moiety. The conjugate or salt thereof according to claim 60, wherein the additional modifying moiety comprises a functional substance, and the additional modifying moiety comprising the functional substance is contained in a heavy chain constant region of an antibody. The conjugate or salt thereof according to claim 60, wherein the additional modifying moiety comprising the functional substance is introduced into the heavy chain constant region via modification of an amino group in a side chain of a lysine residue present at one or more positions in the heavy chain constant region.

63. The conjugate or salt thereof according to claim 62, wherein the one or more positions in the heavy chain constant region are selected from positions 220, 226, 229, 246, 248, 274, 288, 290, 317, 320, and 322 of a human IgG heavy chain according to EU numbering. A method for producing an affinity polypeptide-free antibody or its salt, which comprises (A) an affinity polypeptide comprising an affinity portion having affinity for the heavy chain CH1 region of an antibody, and (B) an antibody, and (C) an affinity polypeptide-modified antibody or its salt further comprising a cleavable portion between the affinity polypeptide (A) and the antibody (B), and cleaving the cleavable portion to produce an affinity polypeptide-free antibody or its salt. The method for producing the affinity polypeptide described in claim 64, wherein the affinity polypeptide has an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence containing the amino acid sequence with one to two amino acid substitutions, deletions, additions or insertions, in which an amino acid having a side chain amino group has been introduced into a specific amino acid residue. The method of claim 64, wherein the cleavable moiety is a cleavable moiety that can generate a bioorthogonal functional group on the antibody upon cleavage, and the antibody or salt thereof that does not contain an affinity polypeptide is an antibody derivative or salt thereof that contains a bioorthogonal functional group. The method of claim 66, wherein the antibody derivative or salt thereof comprising a bioorthogonal functional group is the antibody derivative or salt thereof according to claim 43 or 44.

65. The method of claim 64, wherein the antibody or salt thereof not comprising the affinity polypeptide further comprises a bioorthogonal functional group between the antibody and the cleavable moiety, and the antibody or salt thereof not comprising the affinity polypeptide is an antibody derivative or salt thereof comprising the bioorthogonal functional group. The method of claim 68, wherein the antibody derivative or salt thereof comprising a bioorthogonal functional group is the antibody derivative or salt thereof according to claim 45 or 46. A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) Producing an antibody derivative or a salt thereof containing a bioorthogonal functional group by the method of claim 66; and (2) Reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing the antibody and the functional substance. The method according to claim 70, wherein the conjugate or a salt thereof is the conjugate or a salt thereof according to any one of claims 54 to 55. A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, comprising the following (1) and (2): (1) Producing an antibody derivative or a salt thereof containing a bioorthogonal functional group by the method of claim 68; and (2) Reacting an antibody derivative or a salt thereof containing a bioorthogonal functional group with a functional substance to produce a conjugate or a salt thereof containing the antibody and the functional substance. The method according to claim 72, wherein the conjugate or a salt thereof is the conjugate or a salt thereof according to any one of claims 56 to 57. A method for producing a conjugate comprising an antibody and a functional substance or a salt thereof, comprising: reacting an antibody derivative or salt thereof comprising a bioorthogonal functional group with a functional substance to produce a conjugate or salt thereof comprising the antibody and the functional substance; The antibody derivative or salt thereof comprising a bioorthogonal functional group comprises (a) an immunoglobulin unit comprising a heavy chain and a light chain, and (b) a bioorthogonal functional group, and (c) the bioorthogonal functional group is introduced into a heavy chain CH1 region in the immunoglobulin unit; A method for producing a conjugate or a salt thereof comprising an antibody and a functional substance, the conjugate or a salt thereof comprising (a) an immunoglobulin unit comprising a heavy chain and a light chain, and (b) a functional substance, and (c) the functional substance is introduced into the heavy chain CH1 region of the immunoglobulin unit. The method according to claim 74, wherein the antibody derivative or its salt comprising a bioorthogonal functional group is the antibody derivative or its salt according to any one of claims 43 to 46. The method according to claim 74, wherein the conjugate or salt thereof is the conjugate or salt thereof according to any one of claims 54 to 57. An affinity polypeptide or a salt thereof comprising an affinity portion having affinity for the heavy chain CH1 region of an antibody, wherein the affinity polypeptide has an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to the amino acid sequence, in which an amino acid having a side chain amino group has been introduced at a specific amino acid residue. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE A polynucleotide encoding an affinity polypeptide comprising an affinity peptide having affinity for the heavy chain CH1 region of an antibody, wherein the affinity polypeptide has an amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more identity to said amino acid sequence, in which an amino acid residue having a side chain amino group has been introduced into a specific amino acid residue. SEQ ID NO: 9: TTYRLVINGRTLRGETTTEAVDAETAAAAFAQYANDNGVDGVWTYDDATRTFTVTE 79. An expression vector comprising the polynucleotide of claim 78 and a promoter operably linked thereto.

79. A host cell comprising an expression unit comprising the polynucleotide of claim 78 and a promoter operably linked thereto.

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