Peptide crosslinking agent and crosslinked peptide crosslinked with said crosslinking agent

Abstract

A protein or peptide crosslinking agent which is represented by formula (I). [In the formula, A is a hydrogen atom, a C1-6 alkyl group which may be substituted with a phenyl group or a halogen atom, or a phenyl group.]

Description

[Technical Field]

[0001] The present invention relates to a method for producing a crosslinked peptide. [Background technology]

[0002] Protein and peptide drugs are among the biopharmaceuticals currently attracting the most attention. In particular, antibody drugs, primarily IgG antibodies, and peptide drugs have recently come into use in the pharmaceutical field, and are becoming increasingly important in industrial and pharmaceutical applications.

[0003] The present inventors have previously reported that IgG can be purified using a column immobilized with a peptide ligand containing a specific sequence in which a 17-residue IgG peptide is cyclized by disulfide bonds, in order to construct an alternative system to Protein A columns used for purifying antibody pharmaceuticals (Patent Document 1). However, IgG peptide columns have the drawback of being unable to be reused due to the low alkaline resistance of the disulfide bonds present in the peptide. To address this issue, the present inventors have conducted extensive research into methods for increasing alkaline resistance, and have discovered an IgG-binding peptide that dramatically improves resistance to alkaline washing by crosslinking the thiol groups of cysteine ​​residues in the peptide or protein using 1,3-dichloroacetone (Patent Document 2). However, although this crosslinking provides strong alkaline resistance, it also presents a new problem: its affinity for IgG (Kd = 4.9 μM) is low, resulting in impaired functionality. Therefore, we attempted to improve the crosslinked structure by redesigning it so that it could maintain alkali resistance without significantly reducing functionality, as occurs with dichloroacetone crosslinking. As a result, we found that by changing one of the thiols in the disulfide to a methyl group and converting the S-S bond to a CH-S bond, the affinity, although still slightly low, increased to a Kd value of 340 nM, approaching a practical crosslinked structure with alkali resistance (Patent Document 3). However, this method had the problem of low production yield, because homoserine in the peptide was first chlorinated before reacting with the thiol group of cysteine. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 027796 [Patent Document 2] International Publication No. 2018 / 092867 [Patent Document 3] International Publication No. 2020 / 075670 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the present invention aims to provide a method for producing a crosslinked structure that has high alkali resistance and maintains functionality in high yield without significantly reducing affinity by further redesigning the crosslinked structure. [Means for solving the problem]

[0006] The present inventors further improved the structure of the crosslinker, and as a result, succeeded in producing an IgG-binding peptide that retains alkali resistance and high affinity for IgG (Kd = 45 nM) by using 1,1-dichloroacetone and its derivatives as crosslinkers, which are usually highly reactive and not used in aqueous solutions. This invention has succeeded in providing a crosslinking technology that maintains the function of proteins and peptides while maintaining alkali resistance and high yields that can be used practically as pharmaceuticals.

[0007] A crosslinking agent for proteins or peptides according to a first aspect of the present invention is represented by the following formula (I): Contains compounds .

[0008] [ka] [In the formula, Hal represents a halogen atom, and the two halogen atoms may be the same or different. A is a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, or a phenyl group.

[0009] A method for producing a crosslinked protein or peptide according to a second aspect of the present invention is a method for producing a crosslinked protein or peptide in which at least two thiol groups carried by a single or separate protein or peptide represented by the following formula are bound to each other: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.] The protein or peptide is reacted with the crosslinking agent according to the first aspect of the present invention to convert the two thiol groups to the following groups: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] This includes bonding the components to each other via a

[0010] The protein or peptide may be a protein or peptide in which all or some of the at least two thiol groups form a disulfide bond, and the method may include generating two thiol groups by reducing the disulfide bond.

[0011] A method for improving the alkaline tolerance of a protein or peptide according to a third aspect of the present invention comprises crosslinking thiol groups in the protein or peptide by the method according to the second aspect of the present invention to obtain a crosslinked protein or peptide.

[0012] A method for producing a protein or peptide according to a fourth aspect of the present invention is a method for producing a protein or peptide having a reactive functional group represented by the following formula bound thereto, [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] Obtaining a crosslinked protein or peptide by the method according to the second aspect of the present invention; The method comprises reacting the resulting cross-linked protein or peptide with NH2-LZ (L represents a linker, and Z represents a reactive functional group) to introduce a reactive functional group into the cross-linked portion of the cross-linked protein or peptide.

[0013] A method for producing a protein or peptide according to a fifth aspect of the present invention is a method for producing a protein or peptide having a reactive functional group represented by the following formula bound thereto, [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] A cross-linked protein or peptide represented by the formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.] with NH2-LZ (where L represents a linker and Z represents a reactive functional group), thereby introducing a reactive functional group into the crosslinked portion of the crosslinked protein or peptide.

[0014] A method for producing a conjugate of a protein or peptide and a drug according to a sixth aspect of the present invention is a method for producing a conjugate of a protein or peptide and a drug represented by the following formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.] obtaining a protein or peptide having a reactive functional group bound thereto by the method according to the fourth or fifth aspect of the present invention; The method comprises reacting the resulting protein or peptide having the reactive functional group bound thereto with a drug having a functional group capable of reacting with the reactive functional group, thereby binding the drug to the crosslinked portion of the crosslinked protein or peptide.

[0015] A method for producing a conjugate of a protein or peptide and a drug according to a seventh aspect of the present invention is a method for producing a conjugate of a protein or peptide and a drug represented by the following formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.] A protein or peptide having a reactive functional group represented by the following formula attached thereto: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] and reacting the reactive functional group with a drug having a functional group capable of reacting with the reactive functional group, thereby binding the drug to the crosslinked portion of the crosslinked protein or peptide.

[0016] In the methods according to the fourth to seventh aspects of the present invention, the linker may include a moiety that can be cleaved by a protease.

[0017] In the methods according to the second to seventh aspects of the present invention, the protein or peptide may be a peptide of 5 to 50 amino acids.

[0018] In the methods according to the second to seventh aspects of the present invention, the thiol groups to be crosslinked may be thiol groups present in a single protein or peptide.

[0019] In the methods according to the second to seventh aspects of the present invention, the protein or peptide may be an Fc-binding peptide.

[0020] In the methods according to the second to seventh aspects of the present invention, the thiol groups to be crosslinked may be thiol groups present in an isolated protein or peptide.

[0021] The cross-linked protein or peptide according to an eighth aspect of the present invention is represented by the following formula:

[0022] [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.]

[0023] A protein or peptide having a reactive functional group bound thereto according to a ninth aspect of the present invention is represented by the following formula:

[0024] [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.]

[0025] A conjugate of a protein or peptide and a drug according to a tenth aspect of the present invention is represented by the following formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.]

[0026] The present invention No. a protein or peptide according to aspect 9; andIn the conjugate according to the tenth aspect of the present invention, the linker may comprise a moiety that is cleavable by a protease.

[0027] In the protein or peptide according to the eighth and ninth aspects of the present invention and the complex according to the tenth aspect of the present invention, the protein or peptide may be a peptide of 5 to 50 amino acids.

[0028] In the proteins or peptides according to the eighth and ninth aspects of the present invention, and the conjugate according to the tenth aspect of the present invention, the thiol groups to be crosslinked may be thiol groups present within a single protein or peptide.

[0029] In the protein or peptide according to the eighth and ninth aspects of the present invention and the complex according to the tenth aspect of the present invention, the protein or peptide may be an Fc-binding peptide.

[0030] In the Fc-binding peptide, the amino group of a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, a diaminopropionic acid, an arginine residue, or the amino acid at position 1 may be modified with DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DMA (dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate dihydrochloride), DMS (dimethyl suberimidate dihydrochloride), DTBP (dimethyl 3,3'-dithiobispropionimidate dihydrochloride), or DSP (dithiobis(succinimidyl propionic acid)).

[0031] In the proteins or peptides according to the eighth and ninth aspects of the present invention, and the conjugate according to the tenth aspect of the present invention, the thiol groups to be crosslinked may be thiol groups present in the separated proteins or peptides.

[0032] A method for producing a molecule having the Fc region of IgG bound to a cross-linked Fc-binding peptide according to an eleventh aspect of the present invention comprises contacting a molecule having the Fc region of IgG with a protein or peptide according to the eighth or ninth aspect of the present invention, or a complex according to the tenth aspect of the present invention.

[0033] A method for producing a molecule having an IgG Fc region bound to a cross-linked Fc-binding peptide according to a twelfth aspect of the present invention comprises the steps of: An Fc-binding peptide bound to a molecule having the Fc region of IgG is reacted with a crosslinking agent according to the first aspect of the present invention to convert two thiol groups in the Fc-binding peptide to the following groups: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] This includes bonding the components to each other via a

[0034] An Fc-binding peptide according to a thirteenth aspect of the present invention, to which a reactive functional group Z represented by the following formula is attached: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, L represents a linker, and Z represents a reactive functional group] A method for producing a molecule having an IgG Fc region bound to A cross-linked Fc-binding peptide represented by the formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] and reacting a molecule having an IgG Fc region bound to the Fc-binding peptide with NH2-LZ (L represents a linker, and Z represents a reactive functional group) to introduce a reactive functional group into the cross-linking portion of the cross-linked Fc-binding peptide.

[0035] An Fc-binding peptide conjugated to drug D represented by the following formula according to a fourteenth aspect of the present invention: [ka] A method for producing a molecule having an IgG Fc region bound to Fc-binding peptide having a reactive functional group Z represented by the following formula [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, L represents a linker, and Z represents a reactive functional group] The method includes reacting a molecule having an IgG Fc region to which the reactive functional group Z is bound with a drug having a functional group that can react with the reactive functional group Z, thereby binding the drug to the crosslinked portion of the crosslinked protein or peptide.

[0036] The methods according to the eleventh to fourteenth aspects of the present invention may further comprise covalently binding the Fc-binding peptide to a molecule having the Fc region of IgG.

[0037] A molecule comprising an IgG Fc region according to the fifteenth aspect of the present invention is bound to a protein or peptide according to the eighth or ninth aspect of the present invention, or a complex according to the tenth aspect of the present invention.

[0038] In the molecule according to the fifteenth aspect of the present invention, the Fc-binding peptide may be covalently bound to the molecule having the Fc region of IgG.

[0039] A carrier according to a sixteenth aspect of the present invention has a peptide according to the eighth or ninth aspect of the present invention bound thereto.

[0040] A method for purifying a molecule having an IgG Fc region according to a seventeenth aspect of the present invention comprises: contacting a liquid containing a molecule having the Fc region of IgG with the carrier according to the sixteenth aspect of the present invention; Washing to remove components that do not bind to the carrier; and and recovering the molecules having the Fc region of the IgG by eluting the components bound to the carrier.

[0041] A method for producing a crosslinked protein or peptide according to an eighteenth aspect of the present invention is a method for producing a crosslinked protein or peptide in which at least two thiol groups carried by a protein or peptide represented by the following formula are bound to each other, [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, and Protein / Peptide represents a protein or a peptide] synthesizing the protein or peptide by the Fmoc method; and In said synthesis, in place of at least one cysteine ​​residue, [ka] The present invention includes using a compound represented by the formula:

[0042] A method for producing a protein or peptide according to a nineteenth aspect of the present invention is a method for producing a protein or peptide having a reactive functional group represented by the following formula bound thereto, [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; L represents a linker; Z represents a reactive functional group; and D represents a drug.] synthesizing the protein or peptide by the Fmoc method; and In said synthesis, in place of at least one cysteine ​​residue, [ka] The present invention includes using a compound represented by the formula: [Effects of the Invention]

[0043] The SH bonds in the amino acids obtained by the method of the present invention can form crosslinked structures with high yield. Furthermore, due to their excellent alkali resistance, the bonds are not cleaved even when exposed to alkaline conditions. Therefore, highly alkali-resistant proteins and peptides can be provided, and they can be used for proteins and peptides that are used or washed under alkaline conditions. Furthermore, because the crosslinked structure maintains the structure of the original protein or peptide, and its function is not impaired, they can be used to stabilize various peptides and proteins. [Brief explanation of the drawings]

[0044] [Figure 1] 1 is a schematic diagram of an intramolecular SS bond. A shows an SS bond in a natural protein or peptide. B shows a cross-linking bond in the prior art. C shows a cross-linking bond of the present invention. [Figure 2] This graph shows the results of DBC evaluation using a 1,1-dichloroacetone cross-linked peptide derivative immobilized column. The vertical axis represents absorbance at 280 nm, and the horizontal axis represents elution time. The dashed line indicates the 10% breakthrough point. The solid line represents the results after 1 to 5 alkaline washes (darker lines represent more washes), and the dotted line represents the results after 10 to 30 alkaline washes (darker lines represent more washes). [Figure 3] 1 is a graph showing the change in DBC of a peptide-immobilized column due to alkaline washing, where the vertical axis represents the rate of change (%) and the horizontal axis represents the number of washings. [Figure 4] FIG. 1 is a schematic diagram of a cross-linked peptide of the present invention. [Figure 5] 1 shows the results of liquid chromatography mass spectrometry (LC-MS) analysis of oxytocin before and after the crosslinking reaction in Example 11. A and B show elution chromatograms of the SS-oxidized form of oxytocin before the crosslinking reaction and the reaction product after the crosslinking reaction, respectively. [Figure 6] 1 shows the results of LC-MS analysis of vasopressin before and after the cross-linking reaction in Example 12. A and B show elution chromatograms of the SS-oxidized form of vasopressin before the cross-linking reaction and the reaction product after the cross-linking reaction, respectively. [Figure 7] FIG. 1 shows the results of reverse-phase high performance liquid chromatography (HPLC) analysis of the SS-oxidized form of oxytocin exposed to α-chymotrypsin in Example 13. [Figure 8] FIG. 1 shows the results of reverse-phase HPLC analysis of SH-reduced oxytocin exposed to α-chymotrypsin in Example 13. [Figure 9] FIG. 1 shows the results of reverse-phase HPLC analysis of the 2,2-dichloroacetophenone-bridged reaction product exposed to α-chymotrypsin in Example 13. [Figure 10] FIG. 12 shows the change in each molecular species after the addition of α-chymotrypsin in Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0045] (Crosslinking agent) In one embodiment, the cross-linking agent for cross-linking proteins or peptides is represented by the following formula (I):

[0046] [ka] [In the formula, Hal represents a halogen atom, and the two halogen atoms may be the same or different; and A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, or a phenyl group]

[0047] As used herein, the term "alkyl group" refers to a linear, branched, or cyclic saturated hydrocarbon group. In this specification, C represents the number of carbon atoms, and a C1-6 alkyl group refers to an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, a sec-butyl group, a t-butyl group, an isobutyl group, a pentyl group, an isopentyl group, a 2,3-dimethylpropyl group, a hexyl group, and a cyclohexyl group.

[0048] The number of substituents on the C1-6 alkyl group in group A can be 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 3, 2, and / or 1, and an example thereof is trifluoromethane.

[0049] The term "halogen atom" refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom, a chlorine atom, or a bromine atom, more preferably a fluorine atom or a chlorine atom.

[0050] Preferably, the two Hal are the same halogen atom, and may be, for example, both fluorine atoms, both chlorine atoms, or both bromine atoms.

[0051] Examples of the crosslinking agent include 1,1-dichloroacetone, 1,1-dichloropinacoline, and 2,2-dichloroacetophenone.

[0052] [ka]

[0053] (Method for synthesizing crosslinking agents) Dichlorination of A-alkyl chains can be performed according to the method described by Gallucci et al. (Gallucci, R.R. and Going, R., Journal of Organic Chemistry, 1981, vol. 46, #12, pp. 2532-2538). On the other hand, dichlorination of A-phenyl chains can be performed according to the method described by Zhang et al. (Shao-Lin Zhang, Zheng Yang, Xiaohui Hu, Kin Yip Tam, Bioorganic & Medicinal Chemistry Letters, vol. 28, Issue 21, 15 November 2018, pp. 3441-3445).

[0054] (Method for producing cross-linked proteins or peptides) In one embodiment, the method for producing a cross-linked protein or peptide comprises binding at least two thiol groups of a single or separate protein or peptide represented by the formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.] The protein or peptide is reacted with the crosslinking agent according to the present embodiment to convert the two thiol groups into the following groups: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] This includes bonding the components to each other via a

[0055] The method for producing a crosslinked protein can be represented, for example, by the following reaction scheme.

[0056] [ka]

[0057] The crosslinking reaction between the crosslinker and the thiol group can be carried out by adding a solution of a protein or peptide dissolved in a buffer such as PBS and stirring at room temperature for 30 minutes to 1 day to cleave disulfide bonds and generate SH groups. The crosslinker dissolved in acetonitrile is then added to the solution and stirred at room temperature for 30 minutes to 1 day. The reaction solution can be purified by HPLC or other methods, if necessary. The starting protein or peptide may be protected with Fmoc if necessary. In this case, a base such as piperidine is added to deprotect the Fmoc protecting group of the protein or peptide after the reaction. The reaction solution can be further purified by HPLC (C18 reverse-phase column) if necessary. The presence or absence of a crosslinked protein or peptide can be confirmed, for example, by confirming the molecular weight by LC-MS analysis.

[0058] The crosslinking agent of this embodiment may crosslink SH groups present in two separate proteins or peptides, in which case the two proteins or peptides are reacted with the crosslinking agent to bond them (A). The two molecules may be the same type of molecule or different types of molecules. Alternatively, the crosslinking method may crosslink two SH groups present in a single molecule, in which case one protein or peptide is crosslinked by reacting it with the crosslinking agent (C).

[0059] The cross-linking agent of the present specification cross-links two thiol groups together. The "thiol group" or "SH group" is usually derived from a cysteine ​​residue constituting a protein or peptide, but may also be an artificially introduced group using an artificial amino acid, for example. The two thiol groups may or may not form a disulfide bond in the natural state or in the state before cross-linking.

[0060] If two thiol groups form a disulfide bond in the native state or before crosslinking, the method may include a step of reducing the disulfide bond to generate a thiol group before carrying out the crosslinking method (B and D). For example, the reduction of the disulfide bond can be carried out using a commercially available reducing agent for protein disulfides, such as tris(2-carbethoxy)phosphine (TCEP), its hydrochloride, dithioethanol (DTT), 2-mercaptoethanol, or cysteine ​​hydrochloride (Cys-HCl). The two thiol groups may be thiol groups present in cysteine ​​residues contained in a single protein or peptide, or may be thiol groups of separate proteins or peptides (including combinations of proteins, peptides, and proteins and peptides). When crosslinking thiol groups that form disulfide bonds within a single protein or peptide, it is desirable that the resulting crosslinked structure have a three-dimensional structure similar to that of the disulfide bond.

[0061] The proteins or peptides to be crosslinked each have at least two SH groups in the case of intramolecular crosslinking and at least one SH group in the case of intermolecular crosslinking. For example, the proteins or peptides may have one or more (e.g., 1 to 10, 2 to 8, 4 to 6, 1, 2, 3, 4, 5, or 6) intramolecular or intermolecular disulfide bonds. For example, the proteins may be 500 Da or more, 1000 Da or more, 2000 Da or more, and / or 500,000 Da or less, 200,000 Da or less, or 100,000 Da or less. For example, such proteins may be enzymes, glycoproteins (such as erythropoietin), cytokines, toxins, adjuvants, structural proteins, antibodies, Fc fusion proteins, antibody fragments (such as F(ab')2, Fab', Fab, Fab3, single-chain Fv (scFv), (tandem) bispecific single-chain Fv (sc(Fv)2), single-chain triple bodies, nanobodies, divalent VHHs, pentavalent VHHs, minibodies, (two-chain) diabodies, tandem diabodies, bispecific tribodies, bispecific bibodies, dual affinity retargeting molecules (DARTs), triabodies (or tribodies), tetrabodies (or [sc(Fv)2]2), or (scFv-SA)4) disulfide-linked Fvs, compact IgG, heavy-chain antibodies, or polymers thereof). Furthermore, for example, the peptide may be a peptide of 5 to 5,000 amino acids, 5 to 1,000 amino acids, 5 to 500 amino acids, 5 to 100 amino acids, 5 to 50 amino acids, or 10 to 40 amino acids. Furthermore, the peptide may be either cyclic or linear. Examples of such peptides include vaccines, microantibodies, and antimicrobial peptides. In this specification, proteins and peptides containing the Fc region of an antibody are referred to as "antibodies, etc."

[0062] The above crosslinking methods may form a single or identical crosslinked structure, or may form multiple crosslinked structures. Crosslinking between multiple proteins or peptides may be performed to crosslink three or more identical or different proteins and / or peptides. Furthermore, intramolecular crosslinking and intermolecular crosslinking may be combined. For example, two types of crosslinking may be performed: intramolecular crosslinking within an Fc-binding peptide (described below) and intermolecular crosslinking between the peptide and the Fc region of an antibody.

[0063] (Method for producing cross-linked proteins or peptides by peptide synthesis) Alternatively, a crosslinking method according to another embodiment is a method for producing a crosslinked protein or peptide in which at least two thiol groups of a protein or peptide represented by the following formula are bound to each other, [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, and Protein / Peptide represents a protein or a peptide] synthesizing a protein or peptide by the Fmoc method; and In said synthesis, in place of at least one cysteine ​​residue, [ka] More specifically, this method involves synthesizing a peptide containing two Cys residues to be crosslinked from the C-terminus by the Fmoc method on a support such as a peptide synthesis resin. The first Cys residue is linked using a normal Cys residue (the SH group may be protected as necessary), and the remaining Cys residues up to the amino acid immediately preceding the other Cys residue are synthesized by the normal Fmoc method. If a Cys residue contained in the peptide being synthesized is protected, the protecting group is deprotected, and then Fmoc-modified cysteine ​​(e.g., Fmoc-modified chloroacetophenone) to which a 1,1-dichloroacetone derivative (e.g., 1,1-dichloroacetone, 1,1-dichloropinacoline, or 2,2-dichloroacetophenone) is linked at the SH group is added and linked (step d in Scheme B). The Fmoc group is removed (step e in Scheme B), and the N-terminal α-amino group of the resulting peptide is coupled to the α-carboxyl group of the acetophenoyl cysteine ​​(step f in Scheme B), resulting in the cysteine ​​with the 1,1-dichloroacetone derivative attached at the SH group being linked to the peptide chain. The remaining amino acids are then linked together using the Fmoc method (step g in Scheme B), completing the cross-linked peptide chain.

[0064] [ka]

[0065] (Method for introducing reactive functional groups) A reactive functional group can be further introduced into the crosslinker obtained by the above method. Thus, in one embodiment, the method for producing a protein or peptide is a method for producing a protein or peptide having a reactive functional group represented by the following formula bound thereto: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] Obtaining a crosslinked protein or peptide by said method; The method comprises reacting the resulting cross-linked protein or peptide with NH2-LZ (L represents a linker, and Z represents a reactive functional group) to introduce a reactive functional group into the cross-linked portion of the cross-linked protein or peptide.

[0066] [ka]

[0067] Alternatively, in another aspect, the method for producing a protein or peptide having a reactive functional group attached thereto can be carried out using an already cross-linked material. Thus, in another aspect, there is provided a method for producing a protein or peptide having a reactive functional group attached thereto, comprising: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] A cross-linked protein or peptide represented by the formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.] with NH-LZ (where L represents a linker and Z represents a reactive functional group), thereby introducing a reactive functional group into the crosslinked portion of the crosslinked protein or peptide.

[0068] [ka]

[0069] In another embodiment, the method for producing a protein or peptide is a method for producing a protein or peptide having a reactive functional group represented by the following formula bound thereto, [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; L represents a linker; Z represents a reactive functional group; and D represents a drug.] synthesizing the protein or peptide by the Fmoc method; and In said synthesis, in place of at least one cysteine ​​residue, [ka] This method can be carried out in the same manner as described above (Method for producing a cross-linked protein or peptide by peptide synthesis), except that the cysteine ​​residue used is the above compound.

[0070] (reactive functional group) Specifically, the reactive functional group can be introduced by reacting a protein or peptide crosslinked with the crosslinking agent according to the present embodiment with an amino compound (NH2-LZ) having the reactive functional group to form an oxime (oxime method). The amino compound having the reactive functional group used can be NH2-X-L'-Z (where X is O, NH, or N(C1-4 alkyl)).

[0071] (Linker) In the present specification, L and L' represent a linker. The term "linker" as used herein is not particularly limited as long as it has a length that allows binding of a reactive functional group bound to the linker or a protein or peptide crosslinked with a drug, and has a structure that does not affect the crosslinking reaction; it may not even be present (it may represent a bond). For example, the linker may contain one or more alkylenes, alkenylenes, and alkynylenes. The alkylenes, alkenylenes, and alkynylenes may be linear, branched, or cyclic. The number of carbon atoms in the alkylenes may be 1 to 20, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 1 to 2, 2, or 1, and the number of carbon atoms in the alkenylenes and alkynylenes may be 2 to 20, 2 to 10, 2 to 6, 2 to 4, 2 to 3, or 2. The linker may also contain one or more -O-, -S-, -SS-, -NH-, -N((C1-C6) alkyl)-, -NH-C(O)-NH-, -C(O)-NH-, -NH-C(O)-, phenylene, heteroarylene, and / or heterocyclene. Furthermore, the linker may contain one or more amino acid moieties, for example, 1 to 100, 1 to 50, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2, or 1 amino acid moiety. In the groups constituting the linker, a hydrogen atom bonded to a carbon atom may be substituted with a halogen atom. When the crosslinker is ultimately conjugated to a drug and the drug is intended to be released under specific conditions (e.g., when the crosslinker is conjugated to a peptide or protein (e.g., a molecule having an Fc region) directed against a specific target (e.g., an antibody-drug conjugate (ADC)) and the drug is intended to be released in a local area enriched in the target), the linker preferably contains a moiety that is cleavable by specific in vivo conditions, preferably conditions characteristic of the local area. Such conditions include, for example, the presence of enzymes and pH. For example, the linker may contain a peptide bond that is cleaved by a protease or esterase (e.g., GGFG, which is cleaved by a lysosomal enzyme; see Marcin Poreba, The FEBS Journal (2020) 287:1936-1969) or an ester.In this case, the linker may contain an amino acid moiety other than the peptide bond that is cleaved by the protease.

[0072] In this specification, the term "alkylene" refers to a divalent group obtained by removing one hydrogen atom from the alkyl group. Examples of the alkylene group include C1 to C20 linear or branched alkylene and C3 to C7 cycloalkylene, such as methylene, ethylene, propylene, butylene, pentylene, hexylene, and cyclohexylene. The alkylene may be substituted with a hydroxyl group, a halogen atom, an amino group, or the like, as necessary. In this case, the number of substituents on the alkylene may be 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 3, 2, and / or 1.

[0073] "Alkenylene" refers to a divalent group obtained by removing two hydrogen atoms from any carbon atom of a straight-chain, branched, or cyclic unsaturated hydrocarbon having one or more carbon-carbon double bonds. Examples of alkenylene include C2-20 alkenylene, such as vinylene, propenylene, isopropenylene, butenylene, pentenylene, hexenylene, heptenylene, octenylene, nonenylene, and decenylene.

[0074] "Alkynylene" refers to a divalent group formed by removing two hydrogen atoms from any carbon atom of a straight-chain or branched unsaturated hydrocarbon having one or more carbon-carbon triple bonds. Examples of alkynylene include C2-20 alkynylene, such as ethynylene, propynylene, butynylene, pentynylene, hexynylene, and phenylethynylene.

[0075] "Heteroarylene" and "heterocyclene" respectively refer to aromatic and non-aromatic 5- to 14-membered divalent monocyclic or fused heterocyclic groups containing at least one heteroatom of one or more types selected from a nitrogen atom, an oxygen atom, and a sulfur atom. The number of heteroatoms contained in the 5- to 14-membered heterocyclic group may be, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2, or 1. The monocyclic heterocyclic group is preferably a 5- or 6-membered ring. The fused heterocyclic group is preferably an 8- to 10-membered ring.Examples of heteroarylene and heterocyclene include piperidylene, piperazylene, morpholylene, quinuclidylene, pyrrolidine, azetidylene, oxetylene, aziridinylene, tropanylene, furylene, tetrahydrofurylene, thienylene, pyrrolylene, pyrrolylene, pyrrolidinylene, dioxolanylene, oxazolylene, oxazolinylene, isoxazolylene, thiazolylene, thiazolinylene, isothiazolylene, imidazolylene, and imidazolinylene. , imidazolidinylene, oxazolidinylene, thiazolidinylene, pyrazolylene, pyrazolinylene, pyrazolidinylene, oxadiazolylene, furazanylene, thiadiazolylene, triazolylene, tetrazolylene, pyranylene, pyridylene, piperidinylene, pyridazinylene, pyrimidinylene, pyrazinylene, piperazinylene, dioxanylene, oxazinylene, morpholinylene, thiazinylene, triazinylene, benzofuranylene, isobenzofuranylene, dihydrobenzofuranylene, Benzofuranylene, dihydroisobenzofuranylene, benzothienylene, isobenzothienylene, dihydrobenzothienylene, dihydroisobenzothienylene, tetrahydrobenzothienylene, quinolylene, isoquinolylene, quinazolinylene, phthalazinylene, pteridinylene, coumarylene, chromonylene, indolylene, isoindolylene, benzimidazoylene, benzofurylene, purinylene, acridinylene, phenoxazinylene, phenothiazinylene, benzoxazinylene, Examples include sazolylene, benzothiazolylene, indazolylene, benzimidazolylene, benzodioxolanylene, benzodioxanylchromenylene, chromanylene, isochromanylene, chromanonylene, cinnolinylene, quinoxalinylene, indolizinylene, quinolidinylene, imidazopyridylene, naphthyridinylene, dihydrobenzoxazinylene, dihydrobenzoxazolinonylene, dihydrobenzoxazinonylene, and benzothioxanylene.

[0076] In this specification, the word "comprise" is used with the intention of including the case of "consisting of" and can be read as "consisting of."

[0077] As used herein, a "reactive functional group" or a group represented by "Z" refers to a group that can react and bind to a peptide, protein, nucleic acid, small molecule drug, or the like under relatively mild conditions. Examples of reactive functional groups include maleimide, thiol or protected thiol, alcohol, acrylate, acrylamide, amine or protected amine, carboxylic acid or protected carboxylic acid, azide, alkyne including cycloalkyne, 1,3-dienes including cyclopentadiene and furan, alpha-halocarbonyl, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, nitrophenyl ester, carbonate, dibenzocyclooctyne (DBCO), tetrazine, methyltetrazine (MTZ), transcyclooctene (TCO), azide, carboxy, tosyl, amino, epoxy, acyl, isothiocyanate, isocyanate, acyl azide, NHS ester, acid chloride, aldehyde, glyoxal, epoxide, Examples of the arylating agent include oxirane, carbonate, arylating agent, imidoester, carbodiimide, acid anhydride, haloacetyl, alkyl halide, maleimide, aziridine, acryloyl derivative, arylating agent, diazoalkane, diazoacetyl compound, carbonyl, ketone, carbodiimide, epoxide, oxirane, carbonyldiimidazole, N,N'-disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, alkyl halide, isocyanate, hydrazine, Schiff base, reductive amination product, Mannich condensation product, diazonium derivative, Mannich condensation product, iodination reaction product, aryl azide, halogenated aryl azide, benzophonone, diazo compound, and diazirine derivative.

[0078] The reactive functional group may be a group that forms a covalent bond with a drug via an amide bond, disulfide bond, thioether bond, thioester bond, hydrazone bond, ester bond, ether bond, or urethane bond. For example, N-succinimidyl ester or N-sulfosuccinimidyl ester is suitable for reacting with a primary amine; p-nitrophenyl ester, dinitrophenyl ester, or pentafluorophenyl ester is suitable for reacting with an amino group; maleimide group, carboxylic acid chloride, pyridyldithio group, nitropyridyldithio group, haloalkyl group, and haloacetyl group is suitable for reacting with a mercapto group; and isocyanate is suitable for reacting with a hydroxy group (Greg T. Hermanson, Bioconjugate Techiniques Second Edition, pp. 234-345).

[0079] Furthermore, reactive functional groups also include reactive functional groups that participate in (are capable of) reactions such as a reaction with an alkoxyamine to form an oxime bond, a Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction ("Click" reaction) with an alkyne or azide, an inverse electron demand Diels-Alder reaction, a Michael reaction, a metathesis reaction, a transition metal-catalyzed cross-coupling reaction, a radical polymerization reaction, an oxidative coupling reaction, an acyl transfer reaction, or a photoclick reaction (Kim CH et al., Curr Opin Chem Biol. 2013 Jun;17(3):412-9).

[0080] As an example, the introduction of a reactive functional group can be carried out by reacting the above-mentioned protein, the above-mentioned peptide, or a fusion product thereof bound to each other via a crosslinker with a hydroxyamine derivative in a buffer solution such as PBS (pH 7.4) at a temperature between 4°C and room temperature for 30 minutes to overnight to introduce a reactive functional group such as an alkyne group.

[0081] [ka]

[0082] (Drug binding method) The crosslinking agent according to this embodiment can be used to introduce a drug (payload) into a protein, peptide, or fusion product thereof. Specifically, the drug (payload) can be introduced into the protein, peptide, or fusion product thereof by reacting and binding the reactive functional group of the protein, peptide, or fusion product thereof into which the reactive functional group has been introduced.

[0083] Thus, in one aspect, the method for producing a conjugate of a protein or peptide and a drug is a method for producing a conjugate of a protein or peptide and a drug represented by the following formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.] Obtaining a protein or peptide having a reactive functional group bound thereto by the method; The method comprises reacting the resulting protein or peptide having the reactive functional group bound thereto with a drug having a functional group capable of reacting with the reactive functional group, thereby binding the drug to the crosslinked portion of the crosslinked protein or peptide.

[0084] Alternatively, the method for producing a conjugate of a protein or peptide with a drug can be carried out using a protein and / or peptide into which a reactive functional group has already been introduced. Thus, in another embodiment, the method for producing a conjugate of a protein or peptide with a drug is a method for producing a conjugate of a protein or peptide with a drug represented by the following formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.] A protein or peptide having a reactive functional group represented by the following formula attached thereto: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] and reacting the reactive functional group with a drug having a functional group capable of reacting with the reactive functional group, thereby binding the drug to the crosslinked portion of the crosslinked protein or peptide.

[0085] [ka]

[0086] As used herein, the term "drug" is not particularly limited as long as it is a drug that is used by being introduced into a protein or peptide, and examples thereof include therapeutic agents, prophylactic agents, targeting agents, labeling agents, and diagnostic agents. Examples of therapeutic or prophylactic agents include anticancer agents such as monomethyl auristatin, auristatin, maytansine, emtansine, doxorubicin, bleomycin, ozogamicin, vedotin, pasdotox, deruxtecan, maytansinol calicheamicin, exatecan, pyrrolobenzodiazepine dimer, duocarmycin, eribulin, SN-38, PNU-159682, emtansine (DM1), mertansine, or derivatives thereof; 90 Examples of such labels include radioisotopes such as Y; drugs that bind to receptors on the blood-brain barrier and enable migration into the central nervous system; targeting agents such as drugs that bind to cancer cells and enable the antibody to migrate into the cells; and detectable labels such as radioactive labels, enzymes, fluorescent labels, bioluminescent labels, and chemiluminescent labeled metals.

[0087] As an example, a drug can be introduced by reacting a protein, peptide, or fusion product thereof to which a maleimide group has been introduced as a reactive functional group by an oxime reaction with the anticancer drug mertansine, to which an azide group has been introduced at the SH group, in a buffer solution such as PBS (pH 7.4) at a temperature between 4°C and room temperature for 30 minutes to overnight.

[0088] [ka]

[0089] (cross-linked proteins or peptides) In one aspect, there is provided a protein and / or peptide cross-linked with the above-mentioned cross-linking agent. In another aspect, there is provided a fusion protein or fusion peptide having the following structure, in which two thiol groups present in separate proteins and / or peptides are linked to each other via the above-mentioned cross-linking agent. In the following, Protein / Peptide A and Protein / Peptide B may be different substances or may be the same substance.

[0090] [ka] wherein A is defined as in formula (I).

[0091] In another aspect, there is provided a protein or peptide having the following structure, in which two thiol groups present in a single protein or peptide are linked to each other via the crosslinker of the present invention.

[0092] [ka] wherein A is defined as in formula (I).

[0093] The crosslinker of this embodiment is bonded to a reactive functional group or a drug, thereby allowing the introduction of the functional group or drug into a desired protein, peptide, or complex thereof. Thus, the crosslinked protein or peptide may be a protein or peptide to which a reactive functional group is bonded via a crosslinker, or a protein or peptide to which a drug is bonded via a crosslinker, and includes the molecules shown below.

[0094] [ka] [In the formula, L represents a linker, Z represents a reactive functional group, and D represents a drug]

[0095] In the present specification, the linker has a branched structure, which allows it to bond to multiple functional groups Z or drugs D. The number of functional groups Z or drugs D bonded to the crosslinker according to this embodiment can be 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. For example, when the number of functional groups Z or drugs D is two, the following structure can be used (L1 and L2 represent linkers, which are defined as above).

[0096] [ka]

[0097] The crosslinking agent of this embodiment can be used to enhance the binding stability of a desired protein, peptide, or complex thereof. For example, the crosslinked protein or peptide includes a molecule represented by the following formula, in which group A may be a hydrogen atom:

[0098] [ka]

[0099] In the above, the linker L, reactive functional group Z, and drug D are the same as those described above. Crosslinked molecules are stabilized and have improved alkali resistance compared to disulfide bonds. The number of bonds via the crosslinker described above may be one or more. When two or more crosslinks are present, they may be within a single protein or peptide, or they may be within separate proteins and / or peptides, or a combination of a crosslink within a single protein or peptide and a crosslink within separate proteins and / or peptides. When two or more crosslinks are present within separate proteins or peptides, they may be between the same two proteins and / or peptides, or multiple proteins and / or peptides may be crosslinked, resulting in three or more proteins and / or peptides being linked by the crosslinks.

[0100] (Cross-linked Fc-binding peptide) The peptide to be cross-linked herein may be an Fc-binding peptide. Accordingly, in another aspect, there is provided an Fc-binding peptide cross-linked with the above-described cross-linking agent, which is represented by the following formula (wherein A, L, Z, and D are as defined above), or a method for producing the same:

[0101] [ka]

[0102] [ka]

[0103] [ka]

[0104] As used herein, the term "Fc-binding peptide" refers to a peptide that specifically binds to the Fc region of IgG. As used herein, the term "IgG Fc region" typically refers to a C-terminal fragment obtained by treating IgG with the protease papain. The Fc-binding peptide is preferably a peptide that binds to a site selected from Lys248, Lys246, Lys338, Lys288, Lys290, Lys360, Lys414, and Lys439 in Fc (according to the Eu numbering system) and / or a region adjacent thereto, preferably Lys248 and / or a region adjacent thereto, or that binds to the binding region of Protein A. For example, the Fc-binding peptide may be a partial peptide of Protein A having Fc-binding ability or a mutant thereof. Specific examples of such peptides are described in WO 2008 / 054030, WO 2013 / 027796, WO 2016 / 186206, WO 2018 / 230257, and Kyohei Muguruma et al., ACS Omega (2019); 4(11):14390-14397. They can be appropriately prepared according to the methods described in each document. In the Fc-binding peptide, preferably, the two cysteine ​​residues closest to the C-terminus and the N-terminus are crosslinked with the crosslinker of the present specification.

[0105] As used herein, "IgG" may refer to IgG from mammals, for example, primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats, and is preferably human IgG (IgG1, IgG2, IgG3, or IgG4). IgG as used herein is preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.

[0106] Specifically, the Fc-binding peptide may be a peptide selected from the following (i) to (iv): (i) a peptide represented by the following formula (I): NH2-(Linker)-(X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )···(I) [In formula (I), (Linker) represents a linker, and 1 to 3 X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and 1 to 3 X 8 each independently represents the same or different amino acid residue, each X 1 , X 2 , X 3 and each X 8 are each independently the same or different and represent any amino acid residue other than C, X 4 is H, R, S, or D, X 5is one amino acid residue selected from K, C, D, E, R, V, F, L, 2-aminosuberic acid, Dpr, Orn, AcOrn, AcDab, Dab, Nle, Nva, Tle, Ala(t-Bu), and Cha; X 6 is E, N, R, or D, X 7 is I or V]; (ii) A peptide represented by the following formula (II), or an amino acid sequence of (II), wherein X 9 ~X 13 Peptides containing an amino acid sequence in which one or several amino acids have been added, deleted, and / or substituted at positions other than: X 9 1-2 NMQCQRRFYEALHDPNLNEEQRNAX 11 IX 12 SIRDDC-(Linker2)-CONH2 (SEQ ID NO: 2) (II) [In formula (II), (Linker2) represents a linker, and X 9 1-2 are GF, AF, VF, LF, IF, MF, PF, FF, WF, KF, Orn-F, CF, DF, EF, βAla-F, 2-aminosuberic acid-F, Dpr-F, and NH2-(PEG) n -CO(n=1 to 50)-F, F, K, Orn, C, D, E, 2-aminosuberic acid residue, and Dpr; X 11 and X 12 are each independently selected from the group consisting of R, H, D, E, S, T, N, Q, Y, and C; (iii) a peptide represented by the following formula (I') or (I''): Z-[(Linker3)-(X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )]···(I') [(X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )-(Linker3)]-Z···(I'') [In formula (I') and formula (I''), Z represents a reactive functional group; (Linker3) represents a linker, 1 to 3 X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and 1 to 3 X 8 each independently represents the same or different amino acid residue, each X 1 , X 2 , X 3 and each X 8 are each independently the same or different and represent any amino acid residue other than C, X 4 is H, R, S, or D, X 5 is one amino acid residue selected from K, C, D, E, R, V, F, L, 2-aminosuberic acid, Dpr, Orn, AcOrn, AcDab, Dab, Nle, Nva, Tle, Ala(t-Bu), and Cha; X 6 is E, N, R, or D, X 7 is I or V] (iv) A peptide comprising an amino acid sequence represented by the following formula (II'), or the amino acid sequence of (II'), wherein X 9 ~X 14 Peptides containing an amino acid sequence in which one or several amino acids have been added, deleted, and / or substituted at positions other than: X 91-2 NMQCQX 14 RFYEALHDPNLNEEQRNAX 11 IX 12 SIRDDC-(Linker2)-NH2 (SEQ ID NO: 3) (II') [In formula (II'), (Linker2) represents a linker, and X 9 1-2 are GF, AF, VF, LF, IF, MF, PF, FF, WF, KF, Orn-F, CF, DF, EF, βAla-F, 2-aminosuberic acid-F, Dpr-F, and NH2-(PEG) n -CO(n=1 to 50)-F, F, K, Orn, C, D, E, 2-aminosuberic acid residue, Dpr, and Acetyl-K; X 11 and X 12 are each independently selected from the group consisting of R, H, D, E, S, T, N, Q, Y, C, and K(Z); X 14 are R, C, K(Z), Z is a reactive functional group].

[0107] In this specification, X m (m is an integer) represents an amino acid. m n " is a sequence of n amino acids X m represents a bond, and n is not specified "X m " is amino acid X m If n is 2 or more, there are multiple X m may each independently be the same or different amino acid. mIn the amino acid sequences described herein, A is an alanine residue, R is an arginine residue, N is an asparagine residue, D is an aspartic acid residue, C is a cysteine ​​residue, Q is a glutamine residue, E is a glutamic acid residue, G is a glycine residue, H is a histidine residue, I is an isoleucine residue, L is a leucine residue, K is a lysine residue, M is a methionine residue, F is a phenylalanine residue, P is a proline residue, S is a serine residue, T is a threonine residue, W is a tryptophan residue, Y is a tyrosine residue, and V is a valine residue. Furthermore, Hcy is homocysteine, Dpr is diaminopropionic acid, Orn is ornithine residue, βAla is β-alanine residue, Dab is 2,4-diaminobutyric acid residue, Nle is norleucine residue, Nva is norvaline residue, Tle is tert-leucine residue, Ala(t-Bu) is tert-butylalanine residue, and Cha is cyclohexylalanine residue. Furthermore, the amino group in a residue having an amino group in its side chain (lysine residue, ornithine residue, 2,4-diaminobutyric acid residue) may be acetylated as needed. In this specification, acetylated forms of natural and artificial amino acids are sometimes referred to by the prefix Ac in the amino acid symbols described above. However, unless such interpretation is particularly inconsistent, it is understood that acetylated forms may be included even if not indicated as Ac. In this specification, K(Z) refers to a functional group-bound lysine residue, and preferably, K(Z) is K(Azide). K (Azide) represents an azide-linked lysine residue.

[0108] In the present specification, the Fc-binding peptide may be a carrier-binding peptide intended to bind an antibody to a carrier by binding to the carrier, or a drug-binding peptide intended to bind a drug to an antibody via the peptide. In the case of a carrier-binding peptide, the above-mentioned peptide (i) or (ii) is preferred.

[0109] In formula (I), Linker is (GSGGS)1-3 , (SGSGS) 1-3 , (GGGGS) 1-3 , or (PEG) 2-10 (Preferably, (PEG)4) or is not present. In addition, for binding to a carrier, an amino group may be bonded to the carboxyl terminal (-COOH) at the C-terminus of formula (I) to form a (-C(=O)NH2) group, and optionally, a Linker (as defined above) may be inserted between the carboxyl terminal and the amino group. When a Linker is present at the C-terminus, the Linker on the N-terminus side may not be present. That is, (X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )-(Linker)-NH2. In addition, in formula (I), the N-terminal amino group may be acetylated (in this case, a Lys residue is introduced into the N-terminal Linker at an appropriate position near the N-terminus).

[0110] Preferred examples of the peptide represented by formula (I) include the following peptides: [1]X 1 1-3 is an amino acid sequence represented by (S, G, F, or none)-(D, G, A, S, P, Hcy, or none)-(S, D, T, N, E, or R). [2]X 1 1-3 is D, GPD, R, GPR, SPD, GDD, GPS, SDD, RGN, G-Hcy-D, RGP, or GPD. [3]X 1 1-3 is D or GPD. [4]X 2 is A, S, or T. [5]X 2 is A or T. [6]X 2But it is A. [7]X 3 is Y or W. [8]X 3 But it is Y. [9]X 4 But it's H.

[10] X 5 is one amino acid residue selected from A, R, K, C, D, E, L, 2-aminosuberic acid, Dpr, R, F, 2-aminosuberic acid, Dpr, AcOrn, AcDab, Dab, Nle, Nva, Ala(t-Bu), and Cha.

[11] X 5 ,K,R,AcOrn,.

[12] X 5 is one amino acid residue selected from V, Dab, F, R, L, Nva, Nle, Ala(t-Bu), and Cha.

[13] X 5 is one amino acid residue selected from F, R, L, Nva, Nle, Ala(t-Bu), and Cha.

[14] X 5 is one amino acid residue selected from L, Ala(t-Bu), and Cha.

[15] X 6 is E or N.

[16] X 6 But it is E.

[17] X 7 But it's V.

[18] X 8 1-3 is (S, T, or D)-(H, G, Y, T, N, D, F, Hcy, or none)-(Y, F, H, M, or none).

[19] X 8 1-3 is T, TFH, S, SFH, THH, TFY, TYH, or T-Hcy-H.

[20] X 8 1-3 is T or TFH.

[0111] The peptide represented by formula (I) may be any one or a combination of two or more of the above conditions, and may be, for example, a peptide that satisfies the conditions described below: [8] and [9]; [8] and

[17] ; [9] and

[17] ; [8], [9] and

[17] ; or a combination of any one of these with any one of

[10] to

[14] .

[0112] More specifically, the following peptides can be mentioned (hereinafter referred to as X 5 is the same as above, and may have an NH-(Linker)- group at the N-terminus, and may have an -NH- group or an NH-(Linker)- group at the C-terminus): 1) DCAYHX 5 GELVWCT (SEQ ID NO: 4) 2)GPDCAYHX 5 GELVWCTFH (SEQ ID NO: 5) 3) RCAYHX 5 GELVWCS (SEQ ID NO: 6) 4)GPRCAYHX 5 GELVWCSFH (SEQ ID NO: 7) 5) SPDCAYHX 5 GELVWCTFH (SEQ ID NO: 8) 6)GDDCAYHX 5 GELVWCTFH (SEQ ID NO: 9) 7) GPS CAYHX 5 GELVWCTFH (SEQ ID NO: 10) 8)GPDCAYHX 5 GELVWCSFH (SEQ ID NO: 11) 9)GPDCAYHX 5 GELVWCTHH (SEQ ID NO: 12) 10)GPDCAYHX 5 GELVWCTFY (SEQ ID NO: 13) 11)SPDCAYHX 5 GELVWCTFY (SEQ ID NO: 14) 12)SDDCAYHX 5 GELVWCTFY (SEQ ID NO: 15) 13)RGNCAYHX 5 GQLVWCTYH (SEQ ID NO: 16) 14)G-Hcy-DCAYHX 5 GELVWCT-Hcy-H (SEQ ID NO: 17) 15)RRGPDCAYHX 5 GELVWCTFH (SEQ ID NO: 18) 16)DCTYHX 5 GNLVWCT (SEQ ID NO: 19) 17)DCAYHX 5 GNLVWCT (SEQ ID NO: 20) 18)DCTYHX 5 GELVWCT (SEQ ID NO: 21) 19) DCAWHX 5 GELVWCT (SEQ ID NO: 22) 20) DCTYTX 5 GNLVWCT (SEQ ID NO: 23) 21)DCAYTX 5 GNLVWCT (SEQ ID NO: 24) 22) DCSYTX 5 GNLVWCT (SEQ ID NO: 25) 23)DCTWTX 5 GNLVWCT (SEQ ID NO: 26) 24)DCTYHX 5 GNLVWCT (SEQ ID NO: 27) 25) DCTYRX 5 GNLVWCT (SEQ ID NO: 28) 26)DCTYSX 5 GNLVWCT (SEQ ID NO: 29) 27) DCTYTX 5 GNLVWCT (SEQ ID NO: 30) 28) DCTYTX 5 GELVWCT (SEQ ID NO: 31) 29) DCTYTX 5 GRLVWCT (SEQ ID NO: 32) 30)DCTYTX 5 GDLVWCT (SEQ ID NO: 33) 31) DCTYTX 5 GNLIWCT (SEQ ID NO: 34) 32) DCAYHRGELVWCT (SEQ ID NO: 35) 33) GPDCAYHRGELVWCTFH (SEQ ID NO: 36) 34) RCAYHRGELVWCS (SEQ ID NO: 37) 35) GPRCAYHRGELVWCSFH (SEQ ID NO: 38) 36) SPDCAYHRGELVWCTFH (SEQ ID NO: 39) 37) GDDCAYHRGELVWCTFH (SEQ ID NO: 40) 38) GPSCAYHRGELVWCTFH (SEQ ID NO: 41) 39) GPDCAYHRGELVWCSFH (SEQ ID NO: 42) 40) GPDCAYHRGELVWCTHH (SEQ ID NO: 43) 41) GPDCAYHRGELVWCTFY (SEQ ID NO: 44) 42) SPDCAYHRGELVWCTFY (SEQ ID NO: 45) 43) SDDCAYHRGELVWCTFY (SEQ ID NO: 46) 44) DCTYHRGNLVWCT (SEQ ID NO: 47) 45) DCAYHRGNLVWCT (SEQ ID NO: 48) 46) DCTYHRGELVWCT (SEQ ID NO: 49) 47) DCAWHRGELVWCT (SEQ ID NO: 50) 48) DCTYTNGNLVWCT (SEQ ID NO: 51) 49) DCAYTNGNLVWCT (SEQ ID NO: 52) 50) DCSYTNGNLVWCT (SEQ ID NO: 53) 51) DCTWTNGNLVWCT (SEQ ID NO: 54) 52) DCTYHNGNLVWCT (SEQ ID NO: 55) 53) DCTYRNGNLVWCT (SEQ ID NO: 56) 54) DCTYSNGNLVWCT (SEQ ID NO: 57) 55) DCTYTRGNLVWCT (SEQ ID NO: 58) 56) DCTYTNGELVWCT (SEQ ID NO: 59) 57) DCTYTNGRLVWCT (SEQ ID NO: 60) 58) DCTYTNGDLVWCT (SEQ ID NO: 61) 59) DCTYTNGNLIWCT (SEQ ID NO: 62)

[0113] As an example, a peptide having the following structure can be used as a carrier bond: GSGGS-GPDCAYHRGELVWCTFH-NH2 (PEG)4-GPDCAYHRGELVWCTFH-NH2 GSGGS-DCAYHRGELVWCT-NH2 (PEG)4-DCAYHRGELVWCT-NH2

[0114] In formula (II), Linker2 is (GSGGS) 1-3 , (SGSGS) 1-3 , (GGGGS) 1-3 , or (PEG) 2-10 -Lys (preferably PEG)4-Lys) or is absent. 2 ) may be acetylated to form a (CH3-C(=O)-NH-) group. Linker2 may also be bound to the amino terminus (in this case, a Lys residue is introduced into the N-terminal Linker at an appropriate position near the N-terminus), in which case C-terminal Linker2 may or may not be present.

[0115] Preferred examples of the peptide having the amino acid sequence represented by formula (II) include the following peptides:

[21] X 9 However, GF, AF, βAlaF, NH2-(PEG) n -CO(n=2 to 10)- is selected from the group consisting of F, F, K, Orn, C, and Dpr.

[22] X 9 is selected from the group consisting of GF, F, and K.

[23] X 11 and X 12 are each independently selected from the group consisting of R, H, and E.

[24] X 11 and X 12 is R.

[0116] More specifically, examples of peptides having the amino acid sequence represented by the above formula (II) include the following peptides: 60) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 63), 61) GFNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 64), 62) KNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 65), 63) GFNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 66), 64) KNMQCQKRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 67), or 66) GKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC (SEQ ID NO: 68).

[0117] For example, a peptide having the following structure can be used as a peptide to be bound to a carrier. Acetyl-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-SGSGSK-NH2 Acetyl-FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDC-(PEG)4-Lys-NH2

[0118] The peptide for carrier binding has at least one amino group (-NH) for covalent binding to a carrier. Such amino group is preferably the N-terminal amino group, but may also be the side chain amino group of a lysine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, Dpr, or arginine residue near the N-terminus or C-terminus (e.g., located in a linker) as long as it is capable of binding to a carrier.

[0119] In addition, in the case of a drug-binding peptide, the above-mentioned peptide (iii) or (iv) is preferred.

[0120] The peptide represented by formula (I') above may have a functional group at the C-terminus instead of at the N-terminus. That is, the peptide represented by formula (I') above may be a peptide represented by the following formula (I''): [(X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )-(Linker3)]-Z···(I'') [In formula (I″), Z represents a functional group, and [(X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )-(Linker3)], (Linker3) represents a linker, and 1 to 3 X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and 1 to 3 X 8 each independently represents the same or different amino acid residue, each X 1 , X 2 , X 3 and each X 8 are each independently the same or different and represent any amino acid residue other than C, X 4 is H, R, S, or T, X 5 is one amino acid residue selected from K, C, D, E, R, V, F, L, 2-aminosuberic acid, Dpr, Orn, AcOrn, AcDab, Dab, Nle, Nva, Tle, Ala(t-Bu), and Cha; X 6 is E, N, R, or D, X 7 is I or V.]

[0121] In formula (I') and formula (I''), Linker3 is RRRGS, EEGGS or (PEG) 1-8 (Preferably, (PEG)4) or absent. In addition, an amino group may be bonded to the terminal (-COOH) of the C-terminal amino acid of formula (I') to form a (-C(=O)NH2) group. In addition, an acetyl group may be bonded to the terminal (-NH2) of the N-terminal amino acid of formula (I'') to form a (CH3-C(=O)-NH-) group.

[0122] The peptides having the amino acid sequences represented by the formula (I') and the formula (I'') include Z-(Linker3)-(X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 ) or (X 1 1-3 )-C-(X 2 )-(X 3 )-(X 4 )-(X 5 )-G-(X 6 )-L-(X 7 )-WC-(X 8 1-3 )-(Linker3)-Z. Preferred amino acid sequences are the same as the preferred amino acid sequences for the peptide represented by formula (I) above.

[0123] As an example of a drug-binding peptide, the following peptides can be mentioned: Acetyl-K(Z)-RRRGS-GPDCAYHKGELVWCTFH-NH2 Acetyl-K(Z)-EEGGS-GPDCAYHKGELVWCTFH-NH2 Acetyl-K(Z)-(PEG)4-GPDCAYHKGELVWCTFH-NH2 マレイミド-RRRGS-GPDCAYHKGELVWCTFH-NH2 マレイミド-EEGGS-GPDCAYHKGELVWCTFH-NH2 マレイミド-(PEG)4-GPDCAYHKGELVWCTFH-NH2 DBCO-RRRGS-GPDCAYHKGELVWCTFH-NH2 DBCO-EEGGS-GPDCAYHKGELVWCTFH-NH2 DBCO-(PEG)4-GPDCAYHKGELVWCTFH-NH2 テトラジン-RRRGS-GPDCAYHKGELVWCTFH-NH2 テトラジン-EEGGS-GPDCAYHKGELVWCTFH-NH2 テトラジン-(PEG)4-GPDCAYHKGELVWCTFH-NH2 TCO-RRRGS-GPDCAYHKGELVWCTFH-NH2 TCO-EEGGS-GPDCAYHKGELVWCTFH-NH2 TCO-(PEG)4-GPDCAYHKGELVWCTFH-NH2 Acetyl-K(Z)RRRGS-DCAYHKGELVWCT-NH2 Acetyl-K(Z)EEGGS-DCAYHKGELVWCT-NH2 Acetyl-K(Z)-(PEG)4-DCAYHKGELVWCT-NH2 マレイミド-RRRGS-DCAYHKGELVWCT-NH2 マレイミド-EEGGS-DCAYHKGELVWCT-NH2 マレイミド-(PEG)4-DCAYHKGELVWCT-NH2 DBCO-RRRGS-DCAYHKGELVWCT-NH2 DBCO-EEGGS-DCAYHKGELVWCT-NH2 DBCO-(PEG)4-DCAYHKGELVWCT-NH2 Tetrazine-RRRGS-DCAYHKGELVWCT-NH2 Tetrazine-EEGGS-DCAYHKGELVWCT-NH2 Tetrazine-(PEG)4-DCAYHKGELVWCT-NH2 TCO-RRRGS-DCAYHKGELVWCT-NH2 TCO-EEGGS-DCAYHKGELVWCT-NH2 TCO-(PEG)4-DCAYHKGELVWCT-NH2

[0124] In formula (II'), Linker2 is SGSGSK, SRRCR, SRRK(Z)R, SRRCRRCRRC, SRRK(Z)RRK(Z)RRK(Z), or (PEG) 1-8 -Lys (preferably (PEG)4-Lys) or absent. 2 ) may be acetylated to form a (CH3-C(=O)-NH-) group. Furthermore, the Cys residue (C) contained in the linker may be linked to another functional molecule via a maleimide group, if necessary.

[0125] Preferred examples of the peptide having the amino acid sequence represented by formula (II') include the following peptides: [a]X 9 However, GF, AF, βAlaF, NH2-(PEG) n -CO(n=1 to 50)- is selected from the group consisting of F, F, K, Orn, C, Dpr, and Acetyl-K. [b]X 9 is selected from the group consisting of GF, F, and Acetyl-K. [b]X 11 and X 12 are each independently selected from the group consisting of R, H, and E. [c]X 11 But it is R. [d]X 12is R or K(Z) (preferably, Z is azide).

[0126] More specifically, examples of peptides having the amino acid sequence represented by the above formula (II') include the peptides described in 60) to 66) above (however, the lysine residues contained therein may have functional groups bonded thereto, if necessary): FNMQQQCRFYEALHDPNLNEEQRNARICSIRDDP-SRRCRRCRRC-NH2 ACetyl-KNMQQQCRFYEALHDPNLNEEQRNARICSIRDDP-SRRCRRCRRC-NH2 GFNMQQQCRFYEALHDPNLNEEQRNARICSIRDDP-SRRCRRCRRC-NH2 FNMQCQZRFYEALHDPNLNEEQRNARIRSIRDDC-NH2 ACetyl-KNMQCQZRFYEALHDPNLNEEQRNARIRSIRDDC-NH2 GFNMQCQK(Z)RFYEALHDPNLNEEQRNARIRSIRDDC-SRRK(Z)R-NH2 FNMQCQK(Z)RFYEALHDPNLNEEQRNARIRSIRDDC-NH2 ACetyl-KNMQCQK(Z)RFYEALHDPNLNEEQRNARIRSIRDDC-SRRK(Z)R-NH2 GFNMQCQK(Z)RFYEALHDPNLNEEQRNARIRSIRDDC-SRRK(Z)RRK(Z)RRK(Z)-NH2 ACetyl-KNMQCQK(Z)RFYEALHDPNLNEEQRNARIRSIRDDC-SRRK(Z)RRK(Z)RRK(Z)-NH2 GFNMQCQK(Z)RFYEALHDPNLNEEQRNARIRSIRDDC-SRRK(Z)RRK(Z)RRK(Z)-NH2

[0127] Other Fc-binding peptides include the following peptides (Figure 4). 1) CAWHLGELVWC (SEQ ID NO: 70) 2) DCAWHLGELVWCT (SEQ ID NO: 71) 3) DCAWHLGELVFCT (SEQ ID NO: 72) 4) DCAWHLGELVXCT (SEQ ID NO: 73) X=1-naphtoyl, 2-naphtoyl, benzyl, or benzothiophene 5) CDCAWHLGELVWCTC (SEQ ID NO: 74) 6) CAYHLGELVWC (SEQ ID NO: 75) 7) DCAYHLGELVWCTF(2-Pya) (SEQ ID NO: 76)

[0128] For example, the drug-binding peptide of the present specification may have a reactive functional group (preferably an azide group) bound to its N-terminus or C-terminus (preferably the N-terminus), optionally via a linker. For example, the peptide may have one to three (preferably two) glutamic acid residues further bound to its N-terminus and / or C-terminus, and the reactive functional group (e.g., an azide group) at the end of the peptide. A peptide having an azide group can be click-reacted with another functional molecule having dibenzylcyclooctyne (DBCO), alkyne, or TCO to link the other functional molecule to the peptide. Alternatively, the peptide can be bound to another functional substance by other methods known to those skilled in the art, such as the reaction of a maleimide group with a sulfhydryl group.

[0129] The peptides of the present specification may be bound to other functional molecules. For example, such other molecules can be bound via the reactive functional group (e.g., to the amino terminus, etc.) or, if an amino acid (e.g., a lysine residue) in the peptide has a reactive functional group, to the reactive functional group (e.g., an azide group that the lysine residue has as a substituent), or to a Cys residue in the peptide (e.g., a Cys residue in Linker2) via a maleimide group.

[0130] Other functional molecules that can be conjugated to the peptides of the present invention include, but are not limited to, labeling substances or pharmaceutical agents, including peptides, proteins, nucleic acids, or small molecule drugs. Any molecule that can utilize the antigen specificity or other properties of Fc molecules can be conjugated as the other molecule. Such substances include anticancer drugs, small molecule drugs, radioactive labels, fluorescent labels, nucleic acid drugs, gene therapy drugs, peptide drugs, antibodies such as IgA or VHH, etc.

[0131] The drug-binding peptide has at least one amino group (-NH2) for covalent binding to an antibody, which may be the amino terminal amino group or a side chain amino group of a lysine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, or arginine residue.

[0132] Because crosslinking using the above-mentioned crosslinking agent results in strong alkali resistance, the crosslinking method according to this embodiment can be used to improve the alkali resistance of a protein or peptide having two or more SH groups, or a fusion product thereof. The crosslinking method according to this embodiment may be, for example, a method for improving the alkali resistance of a disulfide bond, or a method for improving the alkali resistance or stability of a protein or peptide having a disulfide bond in its molecule. Specifically, a method for improving the alkali resistance of a protein or peptide is provided, which comprises bonding two thiol groups in the protein or peptide by the above-mentioned method.

[0133] (Molecules having the Fc region of IgG bound to a cross-linked Fc-binding peptide) The Fc-binding peptide can bind to a molecule having the Fc region of IgG. Thus, a conjugate according to another embodiment is a conjugate between an Fc-binding peptide intramolecularly crosslinked with the above-mentioned crosslinking agent and a molecule having the Fc region of IgG. Such conjugates include a conjugate in which an Fc-binding peptide crosslinked with the above-mentioned crosslinking agent containing a drug is bound to a molecule having the Fc region of IgG; and a conjugate in which an Fc-binding peptide crosslinked with the above-mentioned crosslinking agent containing a reactive functional group is bound to a molecule having the Fc region of IgG.

[0134] Furthermore, the cross-linking agent according to this embodiment may cross-link an Fc-binding peptide and a molecule having the Fc region of IgG. Specifically, the Fc-binding peptide may be linked to an SH group of a cysteine ​​residue contained in an antibody or the like via a cross-linking agent. The Fc-binding peptide in such an antibody or the like-Fc-binding peptide cross-linked product may be further intramolecularly cross-linked. For example, a reactive functional group / drug may be bound to the Fc-binding peptide via intramolecular cross-linking, and the Fc-binding peptide may then be further cross-linked to a molecule having an Fc region, such as an antibody. Thus, a complex according to another embodiment is a complex of an Fc-binding peptide and a molecule having an IgG Fc region, cross-linked with the cross-linking agent. This complex includes a complex in which a molecule having an IgG Fc region and an Fc-binding peptide are cross-linked with the cross-linking agent containing a drug; and a complex in which a molecule having an IgG Fc region and an Fc-binding peptide are cross-linked with the cross-linking agent containing a reactive functional group.

[0135] [ka]

[0136] As used herein, "a molecule having the Fc region of IgG" refers to a peptide, protein, or other complex containing the Fc region of IgG, and includes wild-type or artificial IgG or variants thereof, as well as fusions of the Fc region of IgG with other substances (active ingredients, drugs, proteins, low-molecular-weight compounds, medium-molecular-weight compounds, high-molecular-weight compounds, matrices, lipids, liposomes, nanoparticles, DDS vehicles, nucleic acids, and / or peptides), such as Fc fusion proteins, and molecules consisting of only the Fc region. For example, when the Fc molecule is an Fc fusion protein, examples of proteins or peptides fused to the Fc include receptors, cytokines, interleukins, blood coagulation factor VIII, CTLA4, human lactoferrin, TNF receptors, LFA-3, or portions thereof (preferably the target-binding portion).

[0137] The complex can be produced by contacting an Fc-binding peptide cross-linked with the above-mentioned cross-linking agent with a molecule having the Fc region of IgG. Thus, a method for producing a molecule having the Fc region of IgG fused with a drug comprises contacting a molecule having the Fc region of IgG with an Fc-binding peptide cross-linked with the above-mentioned cross-linking agent. Alternatively, the complex can be produced by binding an Fc-binding peptide to a molecule having the Fc region of IgG, followed by intramolecular and / or intermolecular cross-linking with the above-mentioned cross-linking agent.

[0138] Therefore, a method for producing a molecule having an IgG Fc region is a method for producing a molecule having an IgG Fc region bound to a cross-linked Fc-binding peptide, comprising the steps of: The Fc-binding peptide bound to a molecule having the Fc region of IgG is reacted with the crosslinking agent to convert two thiol groups in the Fc-binding peptide to the following groups: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] This includes bonding the components to each other via a

[0139] Furthermore, a method for producing a molecule having the Fc region of IgG includes the steps of: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, L represents a linker, and Z represents a reactive functional group] A method for producing a molecule having an IgG Fc region bound to A cross-linked Fc-binding peptide represented by the formula: [ka] [In the formula, A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] and reacting a molecule having an IgG Fc region bound to the Fc region with NH2-LZ (L represents a linker, and Z represents a reactive functional group) to introduce a reactive functional group into the cross-linking portion of the cross-linked Fc-binding peptide.

[0140] Methods for binding an Fc-binding peptide to an antibody or the like can be carried out with reference to WO 2013 / 027796, WO 2018 / 092867, WO 2020 / 075670, and the like.

[0141] In the Fc-binding peptide, a lysine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, or arginine residue (preferably a lysine residue), or the amino group of the amino acid at position 1, may be optionally modified with a moiety for covalently binding to an antibody, and when the peptide binds to an antibody or the like, it may be covalently bound to the antibody or the like via this moiety. Herein, an Fc-binding peptide modified with such a moiety may be referred to as a "CCAP reagent." Herein, the "moiety for covalently binding to an antibody" refers to a chemical structure for covalently linking the Fc-binding peptide to a molecule having the Fc region of IgG, and can be a compound structure having at least one site capable of binding to a desired amino acid (e.g., a lysine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, or arginine residue). Compounds that provide a moiety for covalently binding to such antibodies include DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DMA (dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate dihydrochloride), DMS (dimethyl suberimidate dihydrochloride), DTBP (dimethyl 3,3'-dithiobispropionimidate dihydrochloride), and DSP (dithiobis(succinimidyl propionate)), with DSG, DSS, or DSP being preferred. For example, succinimidyl groups such as DSS and DSG react with primary amines present in the side chains of lysine residues and the N-terminus of polypeptides. Therefore, by blocking the N-terminus of an Fc-binding peptide and then reacting it with DSS or DSG, only the side chains of lysine residues of IgBP can be specifically modified with DSS or DSG. Crosslinking between an Fc-binding peptide and IgG can be achieved, for example, by using the above-mentioned X 5 , X 9 , X 11 , X 12 , X 14 and Lys248 or Lys246, preferably Lys248, of the Fc region of IgG.

[0142] The binding of the Fc-binding peptide, which is a CCAP reagent, to an antibody or the like is not particularly limited as long as it is performed under conditions that allow a crosslinking reaction. For example, the Fc-binding peptide can be reacted with the antibody or the like by mixing them in an appropriate buffer at room temperature (e.g., about 15°C to 30°C). The mixing step may be performed with the addition of an appropriate amount of a catalyst that promotes the crosslinking reaction, as needed. The mixing ratio of the Fc-binding peptide to the antibody or the like in the mixing step can be, for example, a molar ratio of Fc-binding peptide:antibody or the like of 1:1 to 20:1, preferably 2:1 to 20:1 or 5:1 to 10:1. The mixing time (reaction time) in the mixing step can be, for example, 1 minute to 5 hours, preferably 10 minutes to 2 hours or 15 minutes to 1 hour. The resulting conjugate may be further purified, as needed.

[0143] Since the Fc region of IgG and the like usually consists of two symmetric heavy chain constant regions, there can be two binding sites for an Fc-binding peptide. Therefore, one to two Fc-binding peptides, and preferably one, can bind to one molecule having the IgG Fc region.

[0144] (Cross-linked Fc-binding peptide-bound carrier) Furthermore, Fc-binding peptides can be bound to a support such as a column and used in purifying antibodies, etc. Because the crosslinking method of the present invention improves alkali resistance, the use of an Fc-binding peptide crosslinked with the crosslinking agent of the present invention makes it possible to reuse the support after washing with alkali once or multiple times. Thus, one embodiment provides a support to which an Fc-binding peptide intramolecularly crosslinked with the crosslinking agent is bound. Binding of a peptide to a support can be carried out, for example, by reacting the peptide with a support having a functional group capable of reacting with an amino group. The reaction is carried out under conditions that allow sufficient binding between the two, for example, by contacting them in a buffer solution at room temperature for 1 to 5 hours (preferably 2.5 to 3.5 hours).

[0145] Supports include gels (e.g., column gels), particles, beads, nanoparticles, microparticles, macrobeads, membranes, microplates, and arrays, and are made of materials such as magnetic substances, latex, agarose, glass, cellulose, Sepharose, nitrocellulose, polystyrene, and other polymeric materials. Preferably, the support is a column gel (column chromatography). Examples of support that can be used include HiTrap NHS-activated HP (GE Healthcare).

[0146] Also provided is a method for purifying a molecule having the Fc region of IgG using the above-mentioned carrier. In one embodiment, the method for purifying an antibody, etc., includes contacting a liquid containing an antibody, etc., with the above-mentioned carrier to bind the antibody, etc., to the carrier, washing to remove components that do not bind to the carrier, and eluting and recovering the components that bind to the carrier.

[0147] The contact between the antibody-containing solution and the carrier is carried out under conditions that allow sufficient contact between the two. For example, when the carrier is a column, the contact is carried out by injecting the antibody-containing solution into the column. Components that do not bind to the carrier can be removed by a conventional method, for example, by washing the carrier to which the antibody-bound solution is attached with a buffer solution (pH approximately 7.0). The recovery of the antibody-bound solution can be carried out at a pH of 2.5 or higher, but a weak acidity is desirable to prevent denaturation of the antibody, and the pH is preferably 3.6 or higher, for example, a pH of 3.6 to 4.3. Alternatively, when beads are used as the carrier, the antibody can be recovered by contacting the antibody with the carrier, recovering the beads by centrifugation or the like, and resuspending them in an eluate.

[0148] (Medical composition) In another aspect, there is provided a medical composition, particularly a therapeutic, prophylactic, or diagnostic agent, containing as an active ingredient a peptide and / or protein cross-linked by the above-mentioned cross-linking agent, or a molecule having an IgG Fc region bound to an Fc-binding peptide cross-linked by the cross-linking agent of the present invention. The peptide or protein cross-linked by a cross-linking agent to which a therapeutic or prophylactic agent is bound can be used as a medical (therapeutic or prophylactic) composition.

[0149] In the above, when the peptide and / or protein, or the drug D bound via a cross-linking agent, is a drug that functions as a therapeutic drug, a prophylactic drug, or a vaccine, the cross-linked peptide and / or protein, or the antibody or the like bound thereto, can be used for therapeutic or prophylactic purposes.

[0150] In the above, when the peptide and / or protein, or the drug D bound via a cross-linking agent, is a drug that functions as a label, the cross-linked peptide and / or protein, or the antibody or the like bound thereto, can be used for diagnostic or detection purposes.

[0151] When the composition is a therapeutic or preventive composition, the agent is a therapeutic or preventive agent, and when the composition is a diagnostic agent, the agent is a labeling substance. Target diseases of the medical composition can be appropriately determined by selecting the peptide, protein, antibody, etc. or binding agent to be used, and examples thereof include cancer, inflammatory diseases, infectious diseases, and neurodegenerative diseases.

[0152] For example, medical compositions can be used as injections, including intravenous, subcutaneous, intradermal, intramuscular, and drip infusion injections. Such injections can be prepared according to known methods, for example, by dissolving, suspending, or emulsifying the active ingredient in a sterile aqueous or oily liquid typically used for injections. The prepared injection solution is typically filled into an appropriate ampule, vial, or syringe. Alternatively, a lyophilized formulation can be prepared by adding an appropriate excipient to the active ingredient, and then dissolved in water for injection, physiological saline, or the like, at the time of use to prepare an injection solution. While oral administration of proteins such as antibodies is generally considered difficult due to their degradation in the digestive tract, oral administration is possible with the use of antibody fragments or modified antibody fragments and innovative formulations. Examples of oral formulations include capsules, tablets, syrups, and granules.

[0153] The medical composition is preferably prepared in a dosage unit form that corresponds to the dosage of the active ingredient. Examples of such dosage unit forms include injections (ampoules, vials, pre-filled syringes), which may typically contain 5 to 500 mg, 5 to 100 mg, or 10 to 250 mg of the active ingredient or drug per dosage unit form.

[0154] The administration route of the medical composition may be local or systemic. There are no particular limitations on the administration method, and as described above, the composition may be administered parenterally or orally. Parenteral administration routes include subcutaneous, intraperitoneal, intravenous, or intraarterial injection or infusion into the blood or spinal fluid, with administration into the blood being preferred. The medical or diagnostic composition may be administered temporarily, or continuously or intermittently. For example, the composition may be administered continuously for 1 minute to 2 weeks. The administration regimen of the medical composition is not particularly limited as long as the dosage and timing of administration achieve the desired therapeutic or preventive effect, and can be determined appropriately depending on the symptoms, sex, age, etc. For example, a single dose of the active ingredient is typically about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, and more preferably about 0.1 to 5 mg / kg body weight, administered by intravenous injection about 1 to 10 times per month, preferably about 1 to 5 times per month, before and / or after the onset of clinical symptoms of the above-mentioned diseases. Similar amounts can also be administered for other parenteral and oral administrations.

[0155] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Note that all documents cited throughout this specification are incorporated herein by reference in their entirety.

[0156] Example 1: Preparation of 1,1-dichloroacetone-bridged cyclic peptide The starting peptide [sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1): IgGBP-longGS or IgGBP-LGS] was synthesized by solid-phase peptide synthesis (Fmoc method) at Eurofins. 5 mg (1.95 μmol) of the starting peptide was dissolved in 1500 μL of DMF, and TCEP·HCl (1.12 mg, 3.9 μmol, 2 equiv. mol) dissolved in 2 mL of PBS (pH 7.4) was added. The reduction reaction was carried out at room temperature for 30 minutes with stirring. Subsequently, 1,1-Dichloro-2-propanone (0.495 mg, 3.9 μmol, 2 equiv. mol) dissolved in 120 μL of acetonitrile was added and stirred at room temperature. After 1 hour, the completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030). The reaction solution was purified by HPLC (C18 reverse-phase column) to obtain the Fmoc-cyclized peptide (2 mg, 0.76 μmol, 40% yield). To deprotect the Fmoc protecting group, 2% piperidine was added. After 10 minutes, the completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030). The reaction solution was directly purified by HPLC (C18 reverse-phase column) to obtain the cyclized peptide (1 mg, 0.38 μmol, 20% yield). The molecular weight of the final purified peptide was confirmed by LC-MS analysis (Shimadzu, LC-MS8030), and the peptide was lyophilized.

[0157] (Example 2) Preparation of 1,1-dichloropinacoline-bridged cyclic peptide The starting peptide [sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1)] was synthesized by solid-phase peptide synthesis (Fmoc method) at Eurofins. 10 mg (3.9 μmol) of the starting peptide was dissolved in 1500 μL of DMF. TCEP·HCl (2.24 mg, 7.8 μmol, 2 equiv. mol) dissolved in 2 mL of PBS (pH 7.4) was added and the reduction reaction was carried out at room temperature with stirring for 30 minutes. 1,1-Dichloropinacoline (1.32 mg, 7.8 μmol, 2 equiv. mol) dissolved in 120 μL of acetonitrile was then added and stirred at room temperature. After 1 hour, completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030). The reaction solution was purified by HPLC (C18 reverse-phase column) to obtain the Fmoc-cyclized peptide (7 mg, 2.7 μmol). To remove the Fmoc protecting group, 2% piperidine was added. After 10 minutes, the completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030). The reaction solution was directly purified by HPLC (C18 reverse-phase column) to obtain the cyclized peptide (5 mg, 2.04 μmol, 50% yield). The molecular weight of the final purified peptide was confirmed by LC-MS analysis (Shimadzu, LC-MS8030) and lyophilized.

[0158] (Example 3) Preparation of 2,2-dichloroacetophenone-bridged cyclic peptide The starting peptide [sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1)] was synthesized by solid-phase peptide synthesis (Fmoc method) at Eurofins. 2.5 mg (1.00 μmol) of the starting peptide was dissolved in 1500 μL of DMF. TCEP·HCl (0.506 mg, 2.0 μmol, 2 equiv. mol) dissolved in 2 mL of PBS (pH 7.4) was added and the reduction reaction was carried out at room temperature with stirring for 30 minutes. Then, 2,2-dichloroacetophenone (0.38 mg, 2.0 μmol, 2 equiv. mol) dissolved in 120 μL of acetonitrile was added and stirred at room temperature. After 1 hour, completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030). The reaction solution was purified by HPLC (C18 reverse-phase column) to obtain approximately 2 mg of Fmoc-cyclized peptide. To remove the Fmoc protecting group, 2% piperidine was added. After 10 minutes, the completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030). The reaction solution was directly purified by HPLC (C18 reverse-phase column) to obtain the cyclized peptide (1.2 mg, 0.49 μmol, 49% yield). The molecular weight of the final purified peptide was confirmed by LC-MS analysis (Shimadzu, LC-MS8030) and lyophilized.

[0159] Example 4: Measurement of binding affinity of cross-linked cyclic peptides Affinity analysis was performed as follows. First, a CM5 sensor chip was activated by injecting an equal volume mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 0.1 M sulfo-N-hydroxysuccinimide (sulfo-NHS) onto the chip at a flow rate of 10 μl / ml. IgG was then immobilized on the chip at pH 5.5 (10 mM Na-acetate). Binding reactions were monitored by injecting peptides at concentrations of 15.625, 31.2, 62.5, 125, 250, and 500 nM for 180 seconds in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, 3 mM EDTA, pH 7.4) at a flow rate of 50 μl / ml. For dissociation measurements, buffer solution alone was injected for 600 seconds. Interaction parameters were analyzed using BIAevaluation T100 software.

[0160] The IgG-binding peptide derivatives evaluated for comparison are shown in Figure 1. The affinity of these peptides for human IgG1 is shown in Table 1. The affinity of the original IgG-binding peptide containing a disulfide bond was 8.2 nM in terms of Kd value, whereas the affinity of the 1,3-dichloroacetone-bridged cyclic peptide was 4.9 μM, approximately 500-fold lower. On the other hand, the Kd value of the 1,1-dichloroacetone-bridged cyclic peptide was 45.6 nM, approximately 5-fold lower than that of the original peptide. The Kd value of the 1,1-dichloropinacoline-bridged cyclic peptide was 112 nM, approximately 14-fold lower than that of the original peptide. The Kd value of the 2,2-dichloroacetophenone-bridged cyclic peptide was 6.4 nM, similar to that of the original peptide.

[0161] [Table 1]

[0162] Example 5 Preparation of peptide-immobilized column and purification of IgG 5 mL of 1 mM hydrochloric acid was applied to a 1 mL NHS-activated prepacked column, and the isopropanol solution was removed. Next, a 10.0 mg / mL peptide solution (dissolved in 100 μL of DMSO) was diluted 10-fold with coupling solution (20 mM carbonate buffer, 50 mM sodium chloride, pH 8.3), and 1 mL of the diluted solution was applied and immobilized at room temperature for 4 hours. Unreacted NHS was then blocked with 5 mL of 1 M Tris (pH 8.0) at room temperature for 1 hour. The column was then washed with 5 mL of 0.1 N NaOH. Finally, 10 mL of PBS solution (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.4) was applied and used for chromatographic evaluation.

[0163] The prepared IgG-binding peptide-immobilized column was connected to a BioLogic LP (Bio-Rad) liquid chromatography system and equilibrated with PBS. Next, 1 mg / mL human serum-derived IgG (Sigma-Aldrich) dissolved in PBS was pumped through the column at a flow rate of 1 mL / min for 1 minute. The column was then washed with PBS, and the adsorbed IgG was eluted by pumping an elution solution (100 mM glycine buffer, pH 2.8). The elution of IgG from the column was detected by absorbance at 280 nm.

[0164] (Example 6) Measurement of dynamic binding capacity (DBC) of peptide-immobilized column and evaluation of alkaline resistance 1 A column with 1 mg of immobilized peptide was prepared using the same method as above. After equilibrating the column with PBS, 1 mg / mL human serum-derived IgG (Sigma-Aldrich) dissolved in PBS was applied at a flow rate of 1 mL / min (retention time 1 min). DBC was calculated from the amount of added protein at the point when 10% of the absorbance at 280 nm of the applied sample was leaked.

[0165] Next, 5 mL of 0.1 N sodium hydroxide solution was pumped through this 1 mL column with 1 mg of peptide immobilized. The column was then washed with PBS. This cycle was repeated 30 times for the 1,1-dichloroacetone-bridged cyclic peptide and 10 times for the 1,1-dichloropinacoline-bridged cyclic peptide. Alkaline resistance was evaluated by measuring DBC at a flow rate of 1 mL / min on the first to fifth and tenth cycles, and also on the 20th and 30th cycles for the 1,1-dichloroacetone-bridged cyclic peptide. Based on these results, the DBC variation rate was calculated according to Table 2, with the DBC immediately after column preparation taken as 100%.

[0166] [Table 2]

[0167] The initial DBC values ​​of each peptide column were 17.24 mg / mL-column for the original peptide and 2.3 mg / mL-column for the 1,3-dichloroacetone cross-linked peptide, while they were 12.8 mg / mL-column for the 1,1-dichloroacetone cross-linked peptide and 16.70 mg / mL-column for the 1,1-dichloropinacoline cross-linked peptide. These results demonstrate that the IgG adsorption performance of the new peptide columns is superior to that of conventional cross-linked cyclic peptides.

[0168] (Example 7) Measurement of dynamic binding capacity (DBC) of peptide-immobilized column and evaluation of alkaline resistance 2 5 mL of 0.1 M sodium hydroxide solution was pumped onto the prepared 1 mL column with 1 mg of peptide immobilized, followed by washing with 5 mL of PBS. This constitutes one cycle. After 1 to 30 NaOH aqueous solution / PBS washes, DBC measurements were performed at a flow rate of 1 mL / min at the designated times (1st, 2nd, 3rd, 4th, 5th, 10th, 20th, and 30th washes).

[0169] The results are shown in Figure 2. With the original IgG-binding peptide containing a disulfide bond, alkaline washing reduced the amount of antibody bound to the column (not shown), but with the 1,1-dichloroacetone cross-linked peptide and the 1,1-dichloropinacoline cross-linked peptide, the reduction in the amount of antibody bound to the column was extremely small.

[0170] The rate of change in antibody binding amount was compared in terms of DBC 10%, and the results are shown in Figure 3. With the original IgG-binding peptide-immobilized column with disulfide bonds, the DBC decreased to less than 50% after five alkaline washes, whereas with the 1,1-dichloroacetone cross-linked peptide, the DBC remained above 90% after 30 washes, and with the 1,1-dichloropinacoline cross-linked peptide, it remained above 85% after 10 washes (Figure 3). This demonstrates that the 1,1-dichloroacetone cross-linked peptide and the 1,1-dichloropinacoline cross-linked peptide clearly possess high alkaline resistance. Meanwhile, the DBC value itself was 17.24 mg / ml for the original column immobilized with IgG-bound peptides having disulfide bonds, whereas the 1,1-dichloroacetone cross-linked peptide column had a value of 12.8 mg / ml, or approximately 74%, and the 1,1-dichloropinacoline cross-linked peptide column had a value of 16.70 mg / ml, or approximately 96%. However, because DBC generally varies significantly depending on the amount of immobilized ligand (peptide) and the flow rate, it is likely that columns using the 1,1-dichloroacetone cross-linked peptide and 1,1-dichloropinacoline cross-linked peptide used in this study can be made into columns suitable for practical use by finding optimal conditions.

[0171] (Example 8) Introduction of reactive functional groups

[0172] [ka]

[0173] 0.201 μmol of the prepared 1,1-dichloroacetone-bridged cyclic peptide was dissolved in 400 μl of DMF, and a 100-fold volume of 0-2-propynylhydroxylamine hydrochloride (20.1 μmol) solution, previously dissolved in 1.0 ml of 0.2 M NaHCO3 buffer (pH 8.3), was added. The reaction mixture was stirred at room temperature for 24 hours. Completion of the reaction was confirmed by LC-MS analysis (Shimadzu, LC-MS8030), and the reaction solution was directly purified by HPLC (C18 reverse-phase column) to obtain the alkyne-functionalized bridged cyclized peptide (0.10 μmol, 50% yield). The molecular weight of the final purified peptide was confirmed by LC-MS analysis (Shimadzu, LC-MS8030), and the peptide was lyophilized. The affinity of the resulting compound was measured as in Example 4 (Measurement of Binding Affinity of Bridged Cyclic Peptides). The Kd value was 334 nM, approximately 42-fold lower than that of the original peptide.

[0174] Example 9: Cross-linking of VHH antibodies with 1,1-dichloroacetone 100 μl (95 μg, 56 × 10) of 950 μg / ml anti-CD89 VHH antibody (IgARC25) -10 Urea solution was added to a 5M (11.2 nmol) PBS solution (pH 7.4) and left at room temperature for 1 hour. TCEP·HCl (11.2 nmol, 2 equiv. mol) was then added and the reduction reaction was carried out at room temperature for 30 minutes with stirring. 1,1-Dichloroacetone (11DCA, 11.2 nmol, 2 equiv. mol) dissolved in 120 μL of acetonitrile was then added and stirred at room temperature. After 1 hour, completion of the reaction was confirmed by LC-MS analysis (Waters Bio-Accord SYSTEM). The molecular weight of IgARC25 before crosslinking was 14,028 Da, but after 11DCA treatment it increased by 57 Da to 14,085 Da, confirming crosslinking by 11DCA.

[0175] (Example 10) Preparation of peptides with cross-linked structures by peptide synthesis 1,1-dichloroacetone derivative compounds (1,1-dichloroacetone, 1,1-dichloropinacoline, 2,2-dichloroacetophenone) represented by the following formula: [ka] In the disulfide bond crosslinking reaction using Fmoc, a peptide containing two N-terminal Fmoc-modified Cys residues (or a peptide obtained by reducing a disulfide bond-containing peptide modified with Fmoc at the N-terminus) is reacted with 1.0-2.0 equivalents of a 1,1-dichloroacetone derivative compound in PBS (Scheme A, Step a). Finally, the N-terminal Fmoc residue is removed and the peptide is deprotected (Scheme A, Step b). However, we have devised a different method for crosslinking peptides during peptide synthesis using the Fmoc method, as shown in Scheme B. A peptide containing one of the two Cys residues to be crosslinked is synthesized on a peptide synthesis resin using the Fmoc method, starting from the C-terminus, up to the one just before the remaining Cys residue. After deprotecting the Cys residues in the peptide (Scheme B, Step c), Fmoc-chloroacetophenoylcysteine ​​is added for linkage (Scheme B, Step d). The Fmoc group is removed (step e in Scheme B), and the N-terminal α-amino group of the resulting peptide is coupled with the α-carboxyl group of the acetophenoyl cysteine ​​(step f in Scheme B). The remaining amino acids are then linked together using the Fmoc method (step g in Scheme B). Finally, the target peptide is obtained by cleavage from the resin and Fmoc deprotection.

[0176] [ka]

[0177] (Synthesis of Fmoc-allylated chloroacetophenoyl cysteine) The Fmoc-chloroacetophenoyl cysteine ​​(hereinafter referred to as Compound 5) used in this method was synthesized by the following method, except that the α-carboxyl group of the Fmoc-chloroacetophenoyl cysteine ​​was protected with an allyl group.

[0178] [ka]

[0179] Under an argon atmosphere, N,N-diisopropylethylamine (1.60 mL, 9.38 mmol, 1.1 equiv.) was added to a solution of compound 1 (5.00 g, 8.53 mmol) and allyl bromide (1.44 mL, 17.1 mmol, 2 equiv.) in acetonitrile (85 mL), and the mixture was stirred at room temperature for 16 hours. After quenching the reaction with water, the reaction mixture was concentrated and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium bicarbonate, 2N aqueous hydrochloric acid, and saturated brine, and then dried over sodium sulfate. The residue obtained after concentration was purified using silica gel column chromatography (hexane:ethyl acetate = 4:1) to give compound 2 (5.03 g, 94%).

[0180] [ka]

[0181] Compound 2 (5.03 g, 8.03 mmol) and triisopropylsilane (5.45 mL, 26.5 mmol, 3.3 equivalents) were added to a 1:1 trifluoroacetic acid:dichloromethane solution (30 mL) and stirred at room temperature for 1 hour. After completion of the reaction, the mixture was azeotroped with toluene, and the residue obtained by concentration was purified using silica gel column chromatography (hexane:ethyl acetate = 4:1) to give compound 3 (1.94 g, 63%).

[0182] [ka]

[0183] Under an argon atmosphere, a solution of compound 3 (1.00 g, 2.63 mmol) and phenacyl chloride (814 mg, 5.27 mmol, 2 equivalents) in dichloromethane (26 mL) was mixed with triethylamine (0.401 mL, 2.90 mmol, 1.1 equivalents) and stirred at room temperature for 30 minutes. After quenching the reaction with water, the mixture was extracted with dichloromethane, washed with saturated brine, and then dried over sodium sulfate. The residue obtained by concentrating the mixture was purified using silica gel column chromatography (hexane:ethyl acetate = 2:1) to give compound 4 (1.17 g, 89%). 1H NMR (400MHz, CDCl3, ppm): δ7.96(d,J=7.8Hz,2H),7.76(d,J=7.3Hz,2H),7.64-7.57(m,3 H),7.47(dd,J=7.6,8.0Hz,2H),7.40(dd,J=7.3,7.8Hz,2H),7.31(ddd,J=1.4,7.3,7.3H z,2H),5.94-5.84(m,2H),5.33(d,J=16.9Hz,1H),5.24(dd,J=1.4,10.1Hz,1H),4.71-4. 64(m,3H),4.40-4.38(m,2H),4.23(dd,J=6.9,6.9Hz,1H),3.90(s,2H),3.16-3.03(m,2H) HRMS (FAB-TOF) m / z: [(M+H) + ]calcd for C 29 H 28 N1O5S1502.1688;found502.1690.

[0184] [ka]

[0185] Under an argon atmosphere, compound 4 (204 mg, 0.401 mmol) and N-chlorosuccinimide (59.6 mg, 0.447 mmol, 1.1 equivalents) in a 1:1 carbon tetrachloride:dichloromethane solution (4 mL) were added and stirred at room temperature for 1 hour. Water was added to quench the reaction, and the aqueous layer was extracted with dichloromethane. The organic layer was washed with saturated brine and then dried over sodium sulfate. The residue obtained by concentration was purified using silica gel column chromatography (dichloromethane only) to give compound 5 (210 mg, 97%). 1H NMR (400MHz, CDCl3, ppm): δ7.99(d,J=7.8Hz,2H),7.76(d,J=7.3Hz,2H),7.63-7.57(m,3 H),7.48(dd,J=7.8,7.8Hz,2H),7.39(dd,J=7.3,7.8Hz,2H),7.30(ddd,J=1.0,7.3,7.3H z,2H),6.37(d,J=25.6Hz,1H),5.95-5.84(m,1H),5.63-5.58(m,1H),5.38-5.24(m,2H), 4.76-4.60(m,3H),4.39(d,J=6.9Hz,2H),4.21(dd,J=6.9,6.9Hz,1H),3.51-3.14(m,2H) HRMS (FAB-TOF) m / z: [(M+H) + ]calcd for C 29 H 27 N1O5S2Cl1S1536.1296;found 536.1298.

[0186] (Reactivity of Fmoc-allylated chloroacetophenoyl cysteine ​​with Cys) The following reaction was carried out to verify whether the synthesized Fmoc-allylated chloroacetophenoyl cysteine ​​(compound 5) has reactivity with the thiol of Cys.

[0187] [ka]

[0188] Under an argon atmosphere, triethylamine (0.017 mL, 0.121 mmol, 1.1 equiv.) was added to a dichloromethane solution (1.1 mL) of compound 5 (59.0 mg, 0.110 mmol) and Fmoc-Cys-OAllyl (0.046 mg, 0.121 mmol, 1.1 equiv.) and stirred at room temperature for 10 minutes. After adding water to quench the reaction, the aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated brine and dried over sodium sulfate. The residue obtained by concentration was purified using silica gel column chromatography (hexane:ethyl acetate = 3:1) to give compound 6 (93.8 mg, 97%). 1H NMR(400MHz, CDCl3, ppm): δ7.96(d,J=7.8Hz,2H),7.75(dd,J=2.7,7.8Hz,4H),7.59-7.53 (m,5H),7.41(dd,J=7.8,7.8Hz,2H),7.39-7.35(m,4H),7.30-7.26(m,4H),5.91-5.80(m,2 H),5.70(dd,J=7.8,22.0Hz,2H),5.61(s,1H),5.31(d,J=17.4Hz,2H),5.22(d,J=11.6Hz, 2H),4.69-4.59(m,6H),4.41-4.29(m,4H),4.19(dd,J=7.3,7.3Hz,2H),3.33-2.97(m,4H), HRMS (FAB-TOF) m / z: [(M+Na) + ]calcd for C 50 H 46 N2O9S2Na 905.2542;found 905.2542.

[0189] These results demonstrate that Fmoc-allylated chloroacetophenoylcysteine ​​(compound 5) is reactive with the thiol of Cys, and demonstrate that the reaction shown in step d of Scheme B above proceeds. These results demonstrate that the synthesis of novel cross-linked peptides can be carried out using Scheme B above.

[0190] Example 11: Cross-linking of oxytocin Oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 77), intramolecular SS bond, molecular weight: 1007.19) was crosslinked using 1,1-dichloroacetone. 10 mg (9.92 μmol) of the acetate salt of oxytocin SS-oxidized (oxytocin-OX) (Toronto Research Chemicals) shown in Formula A below was dissolved in 3 mL of 0.1 M HEPES-HCl buffer (pH 8.0). 10.2 mL of 0.1 M HEPES-HCl buffer (pH 8.0) containing 57.1 mg (200 μmol) of TCEP hydrochloride (Tris(2-carboxyethyl)phosphine hydrochloride) as a reducing agent was added, and the mixture was stirred for 1 hour. To this was added 2.77 mg (21.8 μmol) of 1,1-dichloroacetone (2.2 times the molar ratio to oxytocin) dissolved in 0.722 mL of acetonitrile, and the mixture was stirred for 1 hour.

[0191] [ka]

[0192] The resulting reaction product was analyzed by LC-MS as follows: After diluting the reaction product 5-fold with 0.1% formic acid, 20 μL of the diluted solution was analyzed using an Acquity UPLC / SQ detector system (Waters) connected to a Peptide BEH-C18 column (130 Å, 1.7 μm, 2.1 × 100 mm, Waters) (flow rate: 0.2 mL / min, elution: linear gradient from 4% CH3CN to 70% CH3CN containing 0.1% formic acid, column temperature: 25°C).

[0193] The LC-MS analysis results of oxytocin-OX before and after the addition of TCEP hydrochloride and the reaction product are shown in Figures 5A and 5B, respectively. The measured mass of the oxytocin-OX peak was 1006.3, which was almost identical to the theoretical mass of oxytocin-OX (1007.19). On the other hand, the peak of the 1,1-dichloroacetone crosslinked product eluted later than the original peak, with a mass of 1060.4. This mass was almost identical to the theoretical mass of oxytocin-dichloroacetone crosslinked (oxytocin-DA) shown in Formula A1 (1063.26), indicating that the crosslinked product of the desired structure was obtained.

[0194] [ka]

[0195] Example 12: Cross-linking of vasopressin Vasopressin (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 (SEQ ID NO: 78), intramolecular SS bond, molecular weight: 1084.24) was crosslinked using 1,1-dichloroacetone. 5.5 mg (5.07 μmol) of vasopressin SS oxidized acetate (Tokyo Chemical Industry Co., Ltd.) shown in the following formula B (vasopressin-OX) was dissolved in 1.65 mL of 0.1 M phosphate buffer (pH 8.0), and 24.6 mg (86.0 μmol) of TCEP hydrochloride was dissolved in 4.4 mL of 0.1 M phosphate buffer (pH 8.0) and mixed and stirred for 1 hour. 1.42 mg (11.2 μmol) of 1,1-dichloroacetone (2.2 times the molar ratio to vasopressin) dissolved in 0.371 mL of acetonitrile was added and stirred for 1 hour.

[0196] [ka]

[0197] The resulting reaction product was analyzed by LC-MS as in Test Example 11. The LC-MS analysis results for vasopressin-OX before the addition of TCEP hydrochloride and for the reaction product are shown in Figures 6A and 6B, respectively. The measured mass of the vasopressin-OX peak was 1083.7, which closely matched the theoretical value of 1084.24 for vasopressin-OX. Meanwhile, the peaks for the 1,1-dichloroacetone crosslinked reaction product eluted later than the original peak, with masses of 1139.0 and 1121.6. The mass of 1139.0 closely matched the theoretical value of 1140.30 for dichloroacetone-crosslinked vasopressin (vasopressin-DA) shown in Formula B1 below, indicating that the sample contained a crosslinked product of the desired structure. On the other hand, the mass of 1121.6 is 17.4 smaller than that of 1139.0. This suggests that vasopressin-DA shown in formula B1 was reduced by TCEP to form the dichloroacetone-bridged reduced form of vasopressin (vasopressin-DA-R) shown in formula B2 below, and then further dehydrated to form the dichloroacetone-bridged reduced and dehydrated form of vasopressin (vasopressin-DA-RDH, theoretical mass: 1124.3) shown in formula B3.

[0198] [ka]

[0199] (Example 13) Acquisition of resistance to proteases by cross-linking of oxytocin To evaluate the stabilization of peptides by crosslinking, oxytocin crosslinked with 2,2-dichloroacetophenone was used to evaluate its resistance to protease degradation by α-chymotrypsin (derived from bovine pancreas, MP Biomedicals). Crosslinking of oxytocin with 2,2-dichloroacetophenone was performed in the same manner as in Example 11, except that 1,1-dichloroacetone was replaced with 2,2-dichloroacetophenone. The target product was isolated by reverse-phase HPLC. Specifically, 10 mg (10 μmol) of oxytocin acetate was dissolved in 3 mL of 0.1 M HEPES-HCl buffer (pH 8.0) and mixed with 2 mL of 0.1 M HEPES-HCl buffer (pH 8.0) containing 57.4 mg (200 μmol) of TCEP·HCl. The mixture was stirred for 1 hour. Next, 8.3 mg (44 μmol) of 2,2-dichloroacetophenone dissolved in 0.44 mL of acetonitrile was added, mixed, and stirred at room temperature for 1 hour. The obtained sample was applied to an InertSustain C18 column (5 μm, 14 × 250 mm, GL Sciences) connected to an LC-Forte (YMC) at a flow rate of 5 mL / min. Elution was performed with a linear gradient of 4% to 70% containing 0.1% formic acid. After collecting the target product, the acetonitrile was removed under negative pressure and the product was lyophilized.

[0200] The structure of the 2,2-dichloroacetophenone-bridged form of oxytocin (oxytocin-DP) is shown in Formula A2.

[0201] [ka]

[0202] For comparison, oxytocin-OX and the reduced form of oxytocin SH (oxytocin-RD) shown in Formula A3 below were used. Specifically, a 0.5 mg / mL solution of oxytocin in 0.1 M phosphate buffer (pH 7.0) was used as oxytocin-OX. Another 0.5 mg / mL solution of oxytocin in 0.1 M phosphate buffer (pH 7.0) containing 0.5 mg / mL TCEP was added. The resulting substance after 30 minutes was used as oxytocin-RD. To 200 μL of these solutions, 10 μL of 1 mg / mL α-chymotrypsin (1 / 10 weight ratio to oxytocin) was added, incubated at 37°C, and analyzed by reverse-phase HPLC. For blanks, phosphate buffer was added instead of α-chymotrypsin.

[0203] In addition, purified oxytocin-DP was dissolved at 0.5 mg / mL in 0.1 M phosphate buffer (pH 7.0) containing 0.5 mg / mL TCEP, and the substance obtained after 30 minutes was used as a sample. α-Chymotrypsin was added to the sample and analyzed by reverse-phase HPLC.

[0204] [ka]

[0205] As shown in Figure 7, the peak of oxytocin-OX remained unchanged even after 15 minutes to 2 hours, whereas the peak of oxytocin-RD disappeared after only 10 minutes, as shown in Figure 8. The broad peak eluted around 15 minutes is thought to be derived from α-chymotrypsin.

[0206] Figure 9 shows the reversed-phase HPLC elution chromatogram of a sample prepared from oxytocin-DP. Two peaks, A and B, were observed. Mass analysis revealed that peak A had a mass of 1126.7 and peak B had a mass of 1106.5. These values ​​closely matched the theoretical mass of oxytocin dichloroacetone phenone-bridged (oxytocin-DP) (see formula A4 below) (1125.34) and the theoretical mass of oxytocin-DP-RDH (see formula A6 below) (1109.33), which was obtained by dehydration of its reduced form (oxytocin-DP-R, formula A5 below). This indicated that the sample was a mixture of oxytocin-DP and oxytocin-DP-RDH. The reaction was monitored by adding α-chymotrypsin in a 1 / 10 weight ratio to this sample. Unlike the case of oxytocin-RD, no decrease in the two peaks was observed after the addition of α-chymotrypsin, as in the case of oxytocin-OX.

[0207] To examine the change in each molecular species after the addition of α-chymotrypsin, the relative peak area (%) of the blank was plotted against time. Figure 10 shows the results. The peak of oxytocin-RD rapidly disappeared immediately after the addition of α-chymotrypsin, suggesting extremely low resistance to proteases. However, its disulfide-crosslinked form, oxytocin-OX, showed no decomposition even after the addition of α-chymotrypsin, demonstrating its high protease resistance. This indicates that disulfide-crosslinked forms contribute significantly to the resistance. On the other hand, even the dichloroacetophenone-crosslinked forms (oxytocin-DP and oxytocin-DP-RDH) showed high resistance to α-chymotrypsin digestion in the presence of the reducing agent TCEP. This indicates that this crosslinking method significantly contributes to peptide stability, particularly protease resistance.

[0208] [ka]

[0209] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to explain the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0210] This application is based on Japanese Patent Application No. 2020-186833 filed on November 9, 2020, and Japanese Patent Application No. 2021-82739 filed on May 14, 2021. The entire specifications, claims, and drawings of Japanese Patent Application No. 2020-186833 and Japanese Patent Application No. 2021-82739 are incorporated herein by reference. [Industrial Applicability]

[0211] The present invention is useful for cross-linking peptides and proteins.

Claims

1. A crosslinking agent for proteins or peptides, comprising a compound represented by the following formula (I): 【Chemistry 1】 [In the formula, Hal represents a halogen atom, and the two halogen atoms may be the same or different, and A represents a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, or a phenyl group]

2. 1. A method for producing a cross-linked protein or peptide, comprising binding at least two thiol groups of a single or separate protein or peptide represented by the following formula: 【Chemistry 2】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.] The protein or peptide is reacted with the cross-linking agent of claim 1 to convert the two thiol groups to the following groups: 【Transformation 3】 [In the formula, A is a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] The method includes bonding the components to each other via a

3. The method according to claim 2, wherein the protein or peptide is a protein or peptide in which all or some of the at least two thiol groups form a disulfide bond, and the method comprises generating two thiol groups by reducing the disulfide bond.

4. A method for improving the alkaline tolerance of a protein or peptide, comprising binding thiol groups in the protein or peptide by the method of claim 2 or 3 to obtain a crosslinked protein or peptide.

5. 1. A method for producing a protein or peptide having a reactive functional group represented by the following formula attached thereto, 【Chemistry 4】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] Obtaining a crosslinked protein or peptide by the method of claim 2 or 3, The resulting cross-linked protein or peptide and NH ２ -L-Z (L represents a linker, and Z represents a reactive functional group), thereby introducing a reactive functional group into the crosslinked portion of the crosslinked protein or peptide.

6. 1. A method for producing a protein or peptide having a reactive functional group represented by the following formula attached thereto, 【Transformation 5】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] A cross-linked protein or peptide represented by the formula: 【Transformation 6】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.] and NH ２ -L-Z (L represents a linker, and Z represents a reactive functional group), thereby introducing a reactive functional group into the crosslinked portion of the crosslinked protein or peptide.

7. A method for producing a conjugate of a drug with a protein or peptide represented by the following formula: 【Transformation 7】 [In the formula, A is a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.] Obtaining a protein or peptide having a reactive functional group bound thereto by the method according to claim 5 or 6, The method comprises reacting the resulting protein or peptide having the reactive functional group bound thereto with a drug having a functional group capable of reacting with the reactive functional group, thereby binding the drug to the crosslinked portion of the crosslinked protein or peptide.

8. A method for producing a conjugate of a drug with a protein or peptide represented by the following formula: 【Transformation 8】 [In the formula, A is a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.] A protein or peptide having a reactive functional group represented by the following formula attached thereto: 【Chemistry 9】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.] and reacting the reactive functional group with a drug having a functional group capable of reacting with the reactive functional group, thereby attaching the drug to the cross-linked portion of the cross-linked protein or peptide.

9. The method of any one of claims 5 to 8, wherein the linker comprises a moiety that is cleavable by a protease.

10. The method according to any one of claims 2 to 9, wherein the protein or peptide is a peptide of 5 to 50 amino acids.

11. The method according to any one of claims 2 to 10, wherein the thiol groups to be crosslinked are thiol groups present within a single protein or peptide.

12. The method of claim 11, wherein the protein or peptide is an Fc-binding peptide.

13. The method according to any one of claims 2 to 10, wherein the thiol groups to be crosslinked are thiol groups present in an isolated protein or peptide.

14. A cross-linked protein or peptide represented by the formula: 【Chemistry 10】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; and Protein / Peptide A and Protein / Peptide B may be the same or different.]

15. A protein or peptide having a reactive functional group attached thereto, represented by the following formula: 【Chemistry 11】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and Z represents a reactive functional group.]

16. A conjugate of a protein or peptide and a drug represented by the following formula: 【Chemistry 12】 [In the formula, A is a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; Protein / Peptide A and Protein / Peptide B may be the same or different; L represents a linker; and D represents a drug.]

17. 17. The protein or peptide of claim 15, or the conjugate of claim 16, wherein the linker comprises a moiety that is cleavable by a protease.

18. The protein, peptide or complex according to any one of claims 14 to 17, wherein the protein or peptide is a peptide of 5 to 50 amino acids.

19. The protein, peptide or complex according to any one of claims 14 to 18, wherein the thiol groups to be crosslinked are thiol groups present within a single protein or peptide.

20. 20. The protein, peptide or complex of claim 19, wherein the protein or peptide is an Fc-binding peptide.

21. The protein, peptide, or complex according to claim 20, wherein the amino group of a lysine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, arginine residue, or the amino acid at position 1 in the Fc-binding peptide is modified with DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DMA (dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate dihydrochloride), DMS (dimethyl suberimidate dihydrochloride), DTBP (dimethyl 3,3'-dithiobispropionimidate dihydrochloride), or DSP (dithiobis(succinimidyl propionic acid)).

22. The protein, peptide or complex according to any one of claims 14 to 18, wherein the thiol groups to be crosslinked are thiol groups present in separate proteins or peptides.

23. A method for producing a molecule having an IgG Fc region bound to a cross-linked Fc-binding peptide, comprising contacting the molecule with the protein, peptide, or complex described in claim 20 or 21.

24. 1. A method for producing a molecule having an IgG Fc region bound to a cross-linked Fc-binding peptide, comprising: An Fc-binding peptide bound to a molecule having an IgG Fc region is reacted with the crosslinking agent according to claim 1 to convert two thiol groups in the Fc-binding peptide to the following groups: 【Chemistry 13】 [In the formula, A is a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] The method includes bonding the components to each other via a

25. An Fc-binding peptide having a reactive functional group Z represented by the following formula attached thereto: 【Chemistry 14】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, L represents a linker, and Z represents a reactive functional group] A method for producing a molecule having an IgG Fc region bound to A cross-linked Fc-binding peptide represented by the formula: 【Chemistry 15】 [In the formula, A is a hydrogen atom, a phenyl group, a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group] a molecule having an IgG Fc region bound to NH ２ -L-Z (L represents a linker, and Z represents a reactive functional group), thereby introducing a reactive functional group into the cross-linking moiety of the cross-linked Fc-binding peptide.

26. An Fc-binding peptide to which a drug D represented by the following formula is bound: 【Chemistry 16】 A method for producing a molecule having an IgG Fc region bound to An Fc-binding peptide having a reactive functional group Z represented by the following formula attached thereto: 【Chemistry 17】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, L represents a linker, and Z represents a reactive functional group] and reacting a molecule having an IgG Fc region to which the reactive functional group Z is bound with a drug having a functional group that can react with the reactive functional group Z, thereby binding the drug to the cross-linked portion of the cross-linked protein or peptide.

27. The method according to any one of claims 23 to 26, further comprising covalently binding the Fc-binding peptide to a molecule having the Fc region of IgG.

28. A molecule having the Fc region of IgG bound to the protein, peptide or complex described in claim 20 or 21.

29. The molecule of claim 28 , wherein the Fc-binding peptide is covalently linked to a molecule having an IgG Fc region.

30. A carrier having the peptide of claim 20 or 21 bound thereto.

31. A method for purifying a molecule having an IgG Fc region, comprising: Contacting a liquid containing a molecule having an IgG Fc region with the carrier according to claim 30; Washing to remove components that do not bind to the carrier; and and recovering molecules having the Fc region of the IgG by eluting the components bound to the carrier.

32. A method for producing a crosslinked protein or peptide, in which at least two thiol groups of a protein or peptide represented by the following formula are bound to each other, comprising the steps of: [Chemistry 18] [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group, and Protein / Peptide represents a protein or a peptide] synthesizing the protein or peptide by the Fmoc method; and In said synthesis, in place of at least one cysteine ​​residue, 【Chemistry 19】 A method comprising using a compound represented by the formula:

33. 1. A method for producing a protein or peptide having a reactive functional group represented by the following formula attached thereto, 【Chemistry 20】 [In the formula, A represents a hydrogen atom, a phenyl group, or a C1-6 alkyl group optionally substituted with a halogen atom, a phenyl group, or a C1-6 alkyl group; Protein / Peptide represents a protein or peptide; L represents a linker; Z represents a reactive functional group; and D represents a drug.] synthesizing the protein or peptide by the Fmoc method; and In said synthesis, in place of at least one cysteine ​​residue, 【Chemistry 21】 A method comprising using a compound represented by the formula:

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