Patent Application
20240391950
The present disclosure relates to a method for producing a crosslinked peptide, and the like.
Protein pharmaceuticals and peptide pharmaceuticals are one of biopharmaceutical products currently drawing the most attention. In particular, antibody pharmaceuticals, mainly IgG antibodies, and peptide pharmaceuticals have been recently utilized in the pharmaceutical field, and have been increasingly important for industrial and pharmaceutical applications.
The inventor has heretofore reported the ability of purification of IgG by a column where a peptide ligand containing an IgG peptide composed of 17 residues and cyclized by a disulfide bond and containing a specified sequence is immobilized, in order to construct an alternative system to a protein A column used for purification of an antibody pharmaceutical product (Patent Literature 1). However, such an IgG peptide column has the problem of being low in alkaline resistance of a disulfide bond present in the peptide and thus unable to be repeatedly utilized. The inventor has repeatedly studied solutions for increasing alkali resistance in order to solve the problem of chemical weakness of a disulfide bond in a peptide or protein, against alkali washing, and as a result, has found an IgG-binding peptide dramatically increased in resistance against alkali washing due to crosslinking between thiol groups in a cysteine residue in a peptide or protein by use of 1,3-dichloroacetone (Patent Literature 2). However, this crosslinking, although imparts strong alkali resistance, has the new problem of causing a low affinity (Kd=4.9 μM) with IgG and the loss of functionality. Then, a crosslinked structure has been re-designed and thus tried to be improved so as to allow for retention of alkali resistance without any considerable deterioration in functionality, unlike crosslinking by dichloroacetone, and as a result, it has been found that one thiol in disulfide is changed to a methyl group to convert an SS bond into a CH3—S bond, thereby providing a crosslinked structure close to practical one, in which the crosslinked structure, although is still slightly low in affinity, has a Kd value of 340 nM and has alkali resistance (Patent Literature 3). However, such a method involves chlorinating homoserine in a peptide once and then reacting the resultant with a thiol group of cysteine, and thus has the problem of being low in production yield.
An objective of the present disclosure is to provide a method for producing a crosslinked structure with a high yield that is high in alkali resistance and retained in functionality without any considerable deterioration in affinity, by re-design of a crosslinked structure.
The inventors have further studied improvements of structures of crosslinking agents, and as a result, has succeeded in producing an IgG-binding peptide retaining alkali resistance and a high affinity (Kd=45 nM) with IgG by using of 1,1-dichloroacetone and its derivative that are usually not used in an aqueous solution due to its high reactivity, as a crosslinking agent. This disclosure has succeeded in providing a crosslinking technique that can be practically used for pharmaceutical products, that imparts a high yield, and that can allow the function of a protein or peptide to be kept with alkali resistance being exhibited.
A crosslinking agent for a protein or a peptide according to a first aspect of the present disclosure is represented by the following formula (I).
In the formula, A is a hydrogen atom, a C1 to 6 alkyl group optionally substituted with a phenyl group or a halogen atom, or a phenyl group.
A method for producing a crosslinked protein or peptide according to a second aspect of the present disclosure is a method for producing a crosslinked protein or peptide in which at least two thiol groups in a single or separated protein(s) or peptide(s) are bound as represented by the following formula:
The protein or the peptide may be a protein or a peptide wherein all or a part of the at least two thiol groups form a disulfide bond, and the method may comprise reducing the disulfide bond to thereby generate two thiol groups.
A method for improving alkali resistance of a protein or a peptide according to a third aspect of the present disclosure comprises binding a thiol groups in the protein or the peptide to obtain a crosslinked protein or peptide by the method according to the second aspect of the present disclosure.
A method for producing a protein or a peptide according to a fourth aspect of the present disclosure is a method for producing a protein or a peptide bound to a reactive functional group which is represented by the following formula:
A method for producing a protein(s) or a peptide(s) according to a fifth aspect of the present disclosure is a method for producing a protein(s) or a peptide(s) bound to a reactive functional group which is represented by the following formula:
A method for producing a complex of a protein(s) or peptide(s) and a drug according to a sixth aspect of the present disclosure is a method for producing a complex of a protein(s) or peptide(s) and a drug represented by the following formula:
A method for producing a complex of a protein(s) or a peptide(s) and a drug according to a seventh aspect of the present disclosure is a method for producing a complex of a protein(s) or a peptide(s) and a drug represented by the following formula:
In the methods according to the fourth to seventh aspects of the present disclosure, the linker may contain a moiety cleavable by a proteolytic enzyme.
In the methods according to the second to seventh aspects of the present disclosure, the protein or the peptide may be a peptide of 5 to 50 amino acids.
In the methods according to the second to seventh aspects of the present disclosure, the thiol groups for crosslinking may be present in a single protein or peptide.
In the methods according to the second to seventh aspects of the present disclosure, the protein or peptide may be an Fc-binding peptide.
In the methods according to the second to seventh aspects of the present disclosure, the thiol groups for crosslinking may be present in a separated proteins or peptides.
A crosslinked protein(s) or peptide(s) according to an eighth aspect of the present disclosure is represented by the following formula.
A protein(s) or a peptide(s) bound to a reactive functional group according to a ninth aspect of the present disclosure is represented by the following formula:
A complex of a protein(s) or a peptide(s) and a drug according to a tenth aspect of the present disclosure is represented by the following formula:
In the protein(s) or peptide(s) according to the eighth and ninth aspects of the present disclosure, and the complex according to the tenth aspect of the present disclosure, the linker may contain a moiety cleavable by a proteolytic enzyme.
In the proteins or peptides according to the eighth and ninth aspects of the present disclosure, and the complex according to the tenth aspect of the present disclosure, the protein or the peptide may be a peptide of 5 to 50 amino acids.
In the protein or the peptides according to the eighth and ninth aspects of the present disclosure, and the complex according to the tenth aspect of the present disclosure, the thiol groups for crosslinking may be present in a single protein or peptide.
In the proteins or the peptides according to the eighth and ninth aspects of the present disclosure, and the complex according to the tenth aspect of the present disclosure, the protein or the peptide may be an Fc-binding peptide.
An amino group in a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, an arginine residue or an amino acid at the 1-position in the Fc-binding peptide, 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)).
In the proteins or the peptides according to the eighth and ninth aspects of the present disclosure, and the complex according to the tenth aspect of the present disclosure, the thiol groups for crosslinking may be present in a separated proteins or peptides.
A method for producing an IgG-Fc region-containing molecule bound to a crosslinked Fc-binding peptide(s) according to an eleventh aspect of the present disclosure comprises contacting an IgG-Fc region-containing molecule and the protein or the peptide according to the eighth or ninth aspect of the present disclosure or the complex according to the tenth aspect of the present disclosure.
A method for producing an IgG-Fc region-containing molecule bound to a crosslinked Fc-binding peptide(s) according to a twelfth aspect of the present disclosure comprises
A method for producing an IgG-Fc region-containing molecule bound to an Fc-binding peptide having a reactive functional group Z,
A method for producing an IgG-Fc region-containing molecule bound to an Fc-binding peptide having a drug D,
The methods according to the eleventh to fourteenth aspects of the present disclosure may each further comprise covalently binding the Fc-binding peptide and the IgG-Fc region-containing molecule.
An IgG-Fc region-containing molecule according to a fifteenth aspect of the present disclosure is to which the proteins or the peptides according to the eighth and ninth aspects of the present disclosure or the complex according to the tenth aspect of the present disclosure is bound.
In the molecule according to the fifteenth aspect of the present disclosure, the Fc-binding peptide and the IgG-Fc region-containing molecule may be covalently bound.
A carrier according to a sixteenth aspect of the present disclosure is to which the peptide according to the eighth or ninth aspect of the present disclosure is bound.
A method for purifying an IgG-Fc region-containing molecule according to a seventeenth aspect of the present disclosure comprises
A method for producing a crosslinked protein or peptide according to an eighteenth aspect of the present disclosure is a method for producing a crosslinked protein or peptide in which at least two thiol groups are bound as,
A method for producing a protein or a peptide according to a nineteenth aspect of the present disclosure is a method for producing a protein or a peptide bound to a reactive functional group or drug represented by the following formula:
An SH—SH bond in an amino acid obtained by the method of the present disclosure can form a crosslinked structure at a high yield. The bond is excellent in alkali resistance, and thus is not cleaved even if exposed under alkaline conditions. Accordingly, a protein and a peptide having strong alkali resistance can be provided, and these can be used as a protein and a peptide which can be used or washed under alkaline conditions. Furthermore, such a protein and a peptide keep the structures originally possessed therein by the crosslinked structures and are not impaired in functions, and thus can be used for stabilization of various peptides and proteins.
A crosslinking agent for crosslinking a protein or peptide in one aspect is represented by the following formula (I).
In the formula, Hal represents a halogen atom, two halogen atoms are optionally the same or different, and A is a hydrogen atom, a C1 to 6 alkyl group optionally substituted with a phenyl group or a halogen atom, or a phenyl group.
Herein, the “alkyl group” means a straight, branched, or cyclic saturated hydrocarbon group. Herein, C represents the number of carbon atoms, and “C1 to 6 alkyl group” represents an alkyl group having 1 to 6 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, a n-propyl group, an i-propyl group, a 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.
The number of substituents in the C1 to 6 alkyl group in the 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, for example, trifluoromethane can be exemplified.
The “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, preferably a fluorine atom, a chlorine atom, and a bromine atom, more preferably a fluorine atom or a chlorine atom.
Preferably, two Hals are the same halogen atoms, and both may be, for example, fluorine atoms, chlorine atoms, or bromine atoms.
Examples of the crosslinking agent can include 1,1-dichloroacetone, 1,1-dichloropinacolin, and 2,2-dichloroacetophenone.
When A is an alkyl chain, dichlorination can be made by synthesis according to the Document of Gallucci et al. (Gallucci, R. R. and Going, R., Journal of Organic Chemistry, 1981, vol. 46, #12, p. 2532-2538). On the other hand, when A is phenyl, dichlorination can be made by synthesis according to the Document of Zhang et al. (Shao-Lin Zhang, Zheng Yang, Xiaohui Hu, Kin Yip Tam, Bioorganic & Medicinal Chemistry Letters, vol. 28, Issue 21, 15 Nov. 2018, p. 3441-3445).
In one aspect, the method for producing a crosslinked protein or peptide is a method for producing a crosslinked protein or peptide where at least two thiol groups in a single or separated protein or peptide are bound, the crosslinked protein or peptide being represented by the following formula:
wherein A is a hydrogen atom, a C1 to 6 alkyl group optionally substituted with a phenyl group or a halogen atom, a phenyl group, or a C1 to 6 alkyl group, Protein/Peptide represents a protein or peptide, and Protein/Peptide A and Protein/Peptide B are optionally the same or different;
The method for producing a crosslinked protein can be represented by, for example, the following reaction scheme.
The crosslinking reaction between the crosslinking agent and the thiol groups can be performed by adding the crosslinking agent dissolved in acetonitrile, to a protein or peptide solution where an SH group is generated by cleavage of a disulfide bond due to addition of TCEP-HCl, if necessary, dissolved in buffer such as PBS and reduction under stirring at room temperature for 30 minutes to one day, and then stirring the mixture at room temperature for 30 minutes to one day. The reaction solution can be, if necessary, purified with HPLC or the like. The protein or peptide serving as a raw material may be, if necessary, protected with Fmoc, and in this case, a base such as piperidine is added in order to deprotect an Fmoc protection group in the protein or peptide, after the reaction. The reaction solution can be, if necessary, further purified with HPLC (C18 reverse-phase column). Whether or not a crosslinked protein or peptide is generated can be confirmed by confirmation of the molecular weight with, for example, LC-MS analysis.
The crosslinking agent according to the present embodiment may impart crosslinking of SH groups present in two proteins or peptides separated, and in this case, such two proteins or peptides are reacted with the crosslinking agent and thus bound (A). Such two molecules are optionally the same or different kinds of molecules. Alternatively, the present crosslinking method may involve crosslinking two SH groups present in one molecule, and in this case, one protein or peptide is reacted with the crosslinking agent to thereby perform crosslinking (C).
The crosslinking agent herein imparts crosslinking of two thiol groups. The “thiol group” or the “SH group” is usually derived from a cysteine residue constituting a protein or peptide, and may be a group artificially introduced, for example, by use of an artificial amino acid. The two thiol groups, which are in the natural state or the state before crosslinking, may or may not form a disulfide bond.
When the two thiol groups, which are in the natural state or the state before crosslinking, form a disulfide bond, a step of reducing the disulfide bond to generate thiol groups before the crosslinking method may be included (B and D). For example, the disulfide bond can be reduced by using, for example, a commercially available reducing agent for protein disulfide, such as tris(2-carboethoxy)phosphine (TCEP), a hydrochloride thereof, dithioethanol (DTT), 2-mercaptoethanol, and cysteine hydrochloride (Cys-HCl). The two thiol groups may be thiol groups present in a cysteine residue contained in a single protein or peptide, or may be thiol groups of separated proteins or peptides (including proteins, peptides, and a combination of a protein and a peptide). When thiol groups forming a disulfide bond in a single protein or peptide are crosslinked, the structure after the crosslinking desirably provides the same steric structure as in the disulfide bond.
The protein or peptide to be crosslinked each has at least two SH groups in the case of intramolecular crosslinking, or at least one SH group in the case of intermolecular crosslinking. The protein or peptide may have, for example, one or more (for example, 1 to 10, 2 to 8, 4 to 6, 1, 2, 3, 4, 5, or 6) disulfide bonds in a molecule or between molecules. For example, the protein may be a protein of 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, the protein may be an enzyme, a glycoprotein (erythropoietin or the like), cytokine, toxin, an adjuvant, a structure protein, an antibody, an Fc fusion protein, an antibody fragment (F(ab′)2, Fab′, Fab, Fab3, single-stranded Fv(scFv), (tandem) bispecific single-stranded Fv(sc(Fv)2), a single-stranded triplebody, a nanobody, a divalent VHH, a pentavalent VHH, a minibody, a (double-stranded) diabody, a tandem diabody, a bispecific tribody, a bispecific bibody, a dual affinity retargeting molecule (DART), a triabody (or tribody), a tetrabody (or [sc(Fv)2]2), or (scFv-SA)4) disulfide bond Fv, compact IgG, a heavy chain antibody, or a polymer thereof). The peptide may be, for example, a peptide of 5 to 5000 amino acids, 5 to 1000 amino acids, 5 to 500 amino acids, 5 to 100 amino acids, 5 to 50 amino acids, or 10 to 40 amino acids. The peptide may be either cyclic or linear. Examples of the peptide include vaccine, a micro antibody, and an antibacterial peptide. Herein, the protein and the peptide each containing an antibody Fc region are referred to as “antibody or the like”.
The crosslinking method may form a single or same crosslinked structure, or a plurality of crosslinked structures. A plurality of proteins or peptides may be crosslinked and thus the same or different three or more proteins and/or peptides may be crosslinked. Such crosslinking may be made by a combination of intramolecular crosslinking and intermolecular crosslinking, and, for example, may be made by two kinds of crosslinking, intramolecular crosslinking in an Fc-binding peptide described below and intermolecular crosslinking between the peptide and the antibody Fc region.
Alternatively, a crosslinking method according to another aspect is a method for producing a crosslinked protein or peptide where at least two thiol groups in a protein or peptide are bound, the crosslinked protein or peptide being represented by the following formula:
More specifically, the present method involves synthesizing a peptide containing two Cyses to be crosslinked, from the C-terminal, on a carrier such as a peptide synthesis resin according to an Fmoc method. The first Cys is bound by using common Cys (if necessary, an SH group is optionally protected), and subjected to synthesis up to the previous amino acid of the remaining other Cys, according to a common Fmoc method. If Cys contained in a peptide being synthesized is protected, such a protection group is deprotected and then Fmoc-cysteine (example: Fmoc-chloroacetophenoyl cysteine) where a 1,1-dichloroacetone derivative (for example, 1,1-dichloroacetone, 1,1-dichloropinacolin, or 2,2-dichloroacetophenone) is bound on an SH group is added and linked (step d in scheme B). Fmoc is deprotected (step e in scheme B) and an α-amino group at the N-terminal of the peptide generated and an α-carboxyl group at the acetophenoyl cysteine side are coupled (step f in scheme B), and thus the cysteine where the 1,1-dichloroacetone derivative is bound on an SH group is bound to a peptide chain. The remaining amino acid is synthesized by linking according to an Fmoc method (step g in scheme B), and thus a peptide chain crosslinked is completed.
A reactive functional group can be further introduced into the crosslinking agent obtained by the method. Accordingly, a method for producing a protein or peptide in one aspect is a method for producing a protein or peptide where a reactive functional group is bound, the protein or peptide being represented by the following formula:
Alternatively, in another aspect, the method for producing a protein or peptide where a reactive functional group is bound can be performed by use of a crosslinked substance. Accordingly, in another aspect, the method may be a method for producing a protein or peptide where a reactive functional group is bound:
A method for producing a protein or peptide of another aspect is a method for producing a protein or peptide where a reactive functional group is bound, the protein or peptide being represented by the following formula:
The present method can be performed by the same method as described above (method for producing a crosslinked protein or peptide by peptide synthesis) except that the cysteine residue used is the above compound.
Specifically, the reactive functional group can be introduced by reacting a protein or peptide crosslinked by the crosslinking agent according to the present embodiment, and an amino compound (NH2-L-Z) having the reactive functional group, to thereby form an oxime (oxime method). The amino compound having the reactive functional group, here used, can be NH2—X-L′-Z (wherein X is O, NH, or N(C1 to 4 alkyl)).
Herein, L and L′ each represent a linker. Herein, the “linker” is not particularly limited as long as it has a length allowing for binding of the reactive functional group or a drug, to be bound to the linker, and the crosslinked protein or peptide and has a structure having no influence on any crosslinking reaction, and may be absent (may represent a bond). As one example, the linker may contain one or more alkylenes, alkenylenes, and alkynylenes. Such alkylenes, alkenylenes, and alkynylenes may be each straight, branched, or cyclic. The number of carbon atoms in the alkylene can be 1 to 20, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 1 to 2, 2, or 1, and the numbers of carbon atoms in the alkenylene and the alkynylene can be each 2 to 20, 2 to 10, 2 to 6, 2 to 4, 2 to 3, or 2. The linker may contain one or more —O—, —S—, —S—S—, —NH—, —N((C1 to C6)alkyl)-, —NH—C(O)—NH—, —C(O)—NH—, —NH—C(O)—, phenylenes, heteroarylenes, and/or heterocyclenes. Furthermore, the linker may contain one or more amino acid moieties, and may be contain, 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. A hydrogen atom bound to a carbon atom may be replaced by a halogen atom in a group constituting the linker. When an objective is to finally bind the present crosslinking agent to a drug and then release the drug under specified conditions (for example, the present crosslinking agent is intended to be bound (for example, antibody-drug complex (ADC)) to a peptide or protein (for example, an Fc region-containing molecule) directional to a specified target to release a drug at a local rich in the target), the linker contains a moiety cleavable preferably under specified conditions in vivo, preferably, conditions characteristic of the local. Examples of such conditions include the presence of an enzyme, and pH. For example, the linker may contain a peptide bond to be cleaved by protease or esterase (for example, GGFG to be cleaved by a lysosome 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 to be cleaved by protease.
Herein, the “alkylene” represents a divalent group obtained by removing one hydrogen atom from the alkyl group. Examples of the alkylene group include a C1 to 20 straight or branched alkylene and a C3 to 7 cycloalkylene, and examples thereof include a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, and a cyclohexylene group. The alkylene is, if necessary, optionally substituted with a hydroxyl group, a halogen atom, and an amino group, and in this case, the number of substituents in the alkylene 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.
The “alkenylene” means a divalent group obtained by removing two hydrogen atoms from any carbon atom of a straight, branched, or cyclic unsaturated hydrocarbon having one or more carbon-carbon double bonds. Examples of the alkenylene include C2 to 20 alkenylene, and examples thereof include vinylene, propenylene, isopropenylene, butenylene, pentenylene, hexenylene, heptenylene, octenylene, nonenylene, and decenylene.
The “alkynylene” means a divalent group obtained by removing two hydrogen atoms from any carbon atom of a straight or branched unsaturated hydrocarbon having one or more carbon-carbon triple bonds. Examples of the alkynylene include C2 to 20 alkynylene, and examples thereof include ethynylene, propynylene, butynylene, pentynylene, hexynylene, and phenyl ethynylene.
The “heteroarylene” and the “heterocyclene” respectively mean aromatic and non-aromatic 5- to 14-membered divalent monocyclic or fused heterocyclic groups each containing at least one of one or more hetero atoms selected from a nitrogen atom, an oxygen atom and a sulfur atom. The number of hetero atoms 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- to 6-membered ring. The fused heterocyclic group is preferably an 8- to 10-membered ring. Examples of the heteroarylene and the heterocyclene can include piperidylene, piperazylene, morpholylene, quinuclidylene, pyrrolidylene, azetidylene, oxetylene, aziridinylene, tropanylene, furylene, tetrahydrofurylene, thienylene, pyrrolylene, pyrrolynylene, pyrrolidinylene, dioxolanylene, oxazolylene, oxazolinylene, isoxazolylene, thiazolylene, thiazolinylene, isothiazolylene, imidazolylene, imidazolinylene, imidazolidinylene, oxazolidinylene, thiazolidinylene, pyrazolylene, pyrazolinylene, pyrazolidinylene, oxadiazolylene, furazanylene, thiadiazolylene, triazolylene, tetrazolylene, pyranylene, pyridylene, piperidinylene, pyridazinylene, pyrimidinylene, pyrazinylene, piperazinylene, dioxanylene, oxazinylene, morpholinylene, thiadinylene, triazinylene, benzofuranylene, isobenzofuranylene, dihydrobenzofuranylene, dihydroisobenzofuranylene, benzothienylene, isobenzothienylene, dihydrobenzothienylene, dihydroisobenzothienylene, tetrahydrobenzothienylene, quinolylene, isoquinolylene, quinazolinylene, phthalazinylene, pteridinylene, cumarylene, chromonylene, indolylene, isoindolylene, benzimidazoylene, benzofurylene, purinylene, acridinylene, phenoxazinylene, phenothiazinylene, benzoxazolylene, benzothiazolylene, indazolylene, benzimidazolylene, benzodioxolanylene, benzodioxanyl chromenylene, chromanylene, isochromanylene, chromanonylene, cinnolinylene, quinoxalinylene, indolizinylene, quinolidinylene, imidazopyridylene, naphthylidinylene, dihydrobenzoxazinylene, dihydrobenzoxazolinonylene, dihydrobenzoxazinonylene, and benzothioxanylene.
Herein, the term “including” is designated to mean the inclusion of the case “composed of/consisting of”, and can be read as “composed of/consisting of”.
Herein, the “reactive functional group” or the group represented by “Z” means a group capable of reacting and binding with a peptide, a protein, nucleic acid, a low-molecular pharmaceutical, or the like under relatively mild conditions. Examples of the reactive functional group can 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-diene including cyclopentadiene and furan, alpha-halocarbonyl, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, nitrophenyl ester, carbonate, dibenzocyclooctyne (DBCO), tetrazine, methyltetrazine (MTZ), trans-cyclooctene (TCO), azide, carboxy, tosyl, amino, epoxy, acyl, isothiocyanate, isocyanate, acyl azide, NHS ester, acid chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, an arylating agent, imide ester, carbodiimide, acid anhydride, haloacetyl, alkyl halide, maleimide, aziridine, an acryloyl derivative, diazoalkane, a diazoacetyl compound, carbonyl, ketone, carbodiimide, epoxide, oxirane, carbonyldiimidazole, N,N′-disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, isocyanate, hydrazine, Schiff base, reductive amination product, a Mannich condensation product, a diazonium derivative, an iodination reaction product, arylazide, arylazide halide, benzophenone, a diazo compound, and a diaziridine derivative.
The reactive functional group may be a group imparting a covalent bond by an amide bond, a disulfide bond, a thioether bond, a thioester bond, a hydrazone bond, an ester bond, an ether bond, or a urethane bond, in a bound form with a drug. For example, the reactive functional group suitable for reaction with primary amine is N-succinimidyl ester or N-sulfosuccinimidyl ester, the reactive functional group suitable for reaction with an amino group is p-nitrophenyl ester, dinitrophenyl ester or pentafluorophenyl ester, the reactive functional group suitable for reaction with a mercapto group is a maleimide group, carboxylic acid chloride, a pyridyldithio group, a nitropyridyldithio group, a haloalkyl group, and a haloacetyl group, and the reactive functional group suitable for reaction with a hydroxy group is an isocyanate group (isocyanate) (Greg t. Hermanson, Bioconjugate Techiniques Second Edition, p 234-345).
The reactive functional group further encompasses also a reactive functional group involving in (capable of) reaction that forms an oxime bond by reaction with alkoxyamine, Huisgen's 1,3-dipolar cyclization addition reaction (“Click” reaction) that is reaction with alkyne or azide by a Cu(I) catalyst, inverse-electron-demand Diels-Alder reaction, Michael reaction, metathesis reaction, cross-coupling reaction by a transition metal catalyst, radical polymerization reaction, oxidative-coupling reaction, transacylation reaction, or photoclick reaction (Kim C H et al, Curr Opin Chem Biol. 2013 June; 17(3):412-9).
As one example, reactive functional group introduction can be performed by introducing a reactive functional group such as an alkyne group, due to reaction of the protein or the peptide or its fused product bound via the crosslinking agent, and a hydroxyamine derivative, in a buffer such as PBS (pH 7.4) under an environment at 4° C. to ordinary temperature for 30 minutes to overnight.
The crosslinking agent according to the present embodiment can be utilized to thereby introduce a drug (payload) into a protein or peptide or its fused product. Specifically, a reactive functional group of a protein or peptide or its fused product where the reactive functional group is introduced, and a drug (payload) can be reacted and bound to thereby introduce the drug (payload) into the protein or peptide or its fused product.
Accordingly, in one aspect, a method for producing a complex of a protein or peptide and a drug is a method for producing a complex of a protein or peptide and a drug, the complex being represented by the following formula:
Alternatively, the method for producing a complex of a protein or peptide and a drug could be carried out by use of a protein and/or peptide where a reactive functional group is introduced. Accordingly, a method for producing a complex of a protein or peptide and a drug in another aspect is a method for producing a complex of a protein or peptide and a drug, the complex being represented by the following formula:
Herein, the “drug” is not particularly limited as long as it is a drug to be introduced into a protein or peptide and then utilized, and examples thereof include a therapeutic agent, a prophylactic agent, a targeting agent, a labeling agent, or a diagnostic agent. Examples of the therapeutic agent or the prophylactic agent can include anticancer agents such as monomethyl auristatin, auristatin, maytansine, emtansine, doxorubicin, bleomycin, ozogamicin, vedotin, pasudotox, deruxtecan, maytansinol, calicheamicin, exatecan, a pyrrolobenzodiazepine dimer, duocarmycin, eribulin, SN-38, PNU-159682, emtansine (DM1), mertansine or derivatives thereof, radioisotopes such as 90Y; targeting agents such as a drug capable of binding to a receptor on the blood-brain barrier and then transferring to the central nerve and a drug capable of binding to cancer cells and then transferring antibodies into cells; and detectable labels such as a radioactive label, an enzyme, a fluorescent label, a bioluminescent label, and a chemiluminescent label metal.
As one example, the drug can be introduced by reaction of a protein or peptide or its fused product where a maleimide group as the reactive functional group is introduced by oxime reaction in advance, with one obtained by introduction of an azide group to an SH group of mertansine being an anticancer agent, as the drug, in a buffer such as PBS (pH 7.4) under an environment at 4° C. to ordinary temperature for 30 minutes to overnight.
One aspect provides crosslinked protein and/or peptide by the crosslinking agent. Another aspect provides a fused protein or fused peptide having the following structure, in which two thiol groups present in separated protein and/or peptide are bound to each other via the crosslinking agent. Hereinafter, Protein/Peptide A and Protein/Peptide B are optionally the same substance as or different substances from each other.
In the formula, A is defined as in formula (I).
Another aspect provides a protein or peptide having the following structure where two thiol groups present in a single protein or peptide are bound to each other via the crosslinking agent of the present disclosure.
In the formula, A is defined as in formula (I).
The crosslinking agent according to the present embodiment can be bound with the reactive functional group or the drug to thereby introduce the functional group or the drug to a desired protein, peptide, or complex thereof. Accordingly, the crosslinked protein and peptide may be a protein or peptide where the reactive functional group is bound via the crosslinking agent, or may a protein or peptide where the drug is bound via the crosslinking agent, and has a molecule represented below.
In the formula, L represents the linker, Z represents the reactive functional group, and D represents the drug.
Herein, the linker can have a branched structure to thereby bind with a plurality of functional groups Z or drugs D. The number of functional groups Z or drugs D bound to the crosslinking agent according to the present 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 2, the following structure can be adopted (L1 and L2 each represent a linker; The linker is defined as described above.).
The crosslinking agent according to the present embodiment can be used for the purpose of an increase in binding stability of a desired protein, peptide, or complex thereof. For example, in the crosslinked protein or peptide, the group A may be a hydrogen atom, and a molecule represented below is contained.
In the above, the linker L, the reactive functional group Z, and the drug D are the same as described above. The molecule crosslinked is a molecule improved in alkali resistance and stabilized as compared with a disulfide bond. The number of such binding sites via the crosslinking agent may be one, or two or more. When two or more such crosslinking sites are present, crosslinking in a single protein or peptide may be made, crosslinking in separated protein and/or peptide may be made, or crosslinking in a single protein or peptide and crosslinking in separated protein and/or peptide may be combined. When two or more such crosslinking sites in a separated protein or peptide are present, crosslinking between the same two proteins and/or peptides may be made, or binding by crosslinking among three or more proteins and/or peptides by crosslinking between a plurality of proteins and/or peptides may be made.
The peptide to be crosslinked herein may be an Fc-binding peptide. Accordingly, another aspect provides an Fc-binding peptide represented by the following formula (wherein A, L, Z, and D are defined as described above) and crosslinked via the crosslinking agent, or a production method thereof.
Herein, the “Fc-binding peptide” means a peptide to be specifically bound to an Fc region of IgG. Herein, the “Fe region of IgG” typically means a fragment at the C-terminal, obtained as a product formed by treatment of IgG with papain being a proteolytic enzyme. The Fc-binding peptide is preferably a peptide to be bound to a site selected from Lys248, Lys246, Lys338, Lys288, Lys290, Lys360, Lys414, and Lys439 of Fc according to the Eu numbering, and/or an adjacent region thereof, preferably Lys248 and/or an adjacent region thereof, or to be bound to a binding region of protein A. For example, the Fc-binding peptide may be a partial peptide of protein A having an Fc-binding ability, or a mutant thereof. Specific examples of such a peptide are described in International Publication No. WO 2008/054030, International Publication No. WO 2013/027796, International Publication No. WO 2016/186206, International Publication No. WO 2018/230257, and Kyohei Muguruma et al., ACS Omega (2019); 4(11): 14390-14397, and such a peptide can be appropriately prepared according to a method described in each of these Documents. In the Fc-binding peptide, preferably, two cysteine residues, cysteine closest to the C-terminal and cysteine closest to the N-terminal, are crosslinked via the crosslinking agent herein.
Herein, the “IgG” may be IgG from mammals, for example, primates such as humans and chimpanzees, lab animals such as rats, mice, and rabbits, livestock animals such as pigs, cattle, horses, sheep, and goats, and pet animals such as dogs and cats, and is preferably human IgG (IgG1, IgG2, IgG3 or IgG4). Herein, the IgG is preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and is particularly preferably human IgG1, IgG2, or IgG4.
Specifically, the Fc-binding peptide may be a peptide selected from the following (i) to (iv):
NH2-(Linker)-(X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X7)—W—C—(X81-3) (I)
Z-[(Linker3)-(X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X)—W—C—(X81-3)] (I′)
[(X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X7)—W—C—(X81-3)-(Linker3)]-Z (I″)
Herein, Xm (m is an integer) represents an amino acid. “Xmn” represents binding of n of amino acids Xm, and “Xm” where no n is designated represents the presence of one amino acid Xm. When n is 2 or more, a plurality of Xm may be each independently the same or different amino acids. A case where n is “p-q” represents the presence of p to q of amino acids Xm. In 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. Hcy is homocysteine, Dpr is diaminopropionic acid, Orn is an ornithine residue, βAla is a β-alanine residue, Dab is a 2,4-diaminobutyric acid residue, Nle is a norleucine residue, Nva is a norvaline residue, Tle is a tert-leucine residue, Ala(t-Bu) is a tert-butylalanine residue, and Cha is a cyclohexylalanine residue. In a residue (lysine residue, ornithine residue, 2,4-diaminobutyric acid residue) containing an amino group at a side chain, the amino group may be, if necessary, acetylated. Herein, each acetylated form of natural and artificial amino acids may also be designated with the prefix Ac in the designation of such an amino acid, and such an amino acid is interpreted to be capable of encompassing such an acetylated form even if not designated with Ac, unless such an interpretation is particularly inconsistent. Herein, K(Z) means a functional group-bound lysine residue, and K(Z) is preferably K(Azide). K(Azide) represents an azide-bound lysine residue.
Herein, the Fc-binding peptide may be a peptide for carrier-binding, which is for binding with a carrier to thereby bind an antibody and the carrier, or may be a peptide for drug-binding, which is for binding a drug to an antibody via the peptide. In the case of the peptide for carrier-binding, the peptide (i) or (ii) is preferable.
In formula (I), Linker is (GSGGS)1-3, (SGSGS)1-3, (GGGGS)1-3, or (PEG)2-10 (preferably, (PEG)4), or absent. For binding with a carrier, an amino group may be bound to the carboxyl terminal (—COOH) at the C-terminal of formula (I) to provide a (—C(═O)NH2) group, or Linker (defined as described above) may be arbitrarily inserted between the carboxyl terminal and an amino group. When Linker is present at the C-terminal, no Linker at the N-terminal side may be present. In other words, (X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X7)—W—C—(X81-3)-(Linker)-NH2 may also be formed. The amino group at the N-terminal of formula (I) may be acetylated (in this case, a Lys residue is introduced to an appropriate position in the vicinity of the N-terminal in Linker at the N-terminal side).
Examples of the peptide represented by formula (I) preferably include the following peptides.
The peptide represented by formula (I) may be a peptide satisfying any one, or a combination of two or more of the above conditions, or may be, for example, a peptide satisfying the following conditions: [8] and [9]; [8] and [17]; [9] and [17]; [8] and [9] and [17]; or a combination thereof with any one of [10] to [14].
More specifically, examples thereof can include the following peptides (hereinafter, X5 is the same as described above, an NH2-(Linker)-group may be present at the N-terminal, and an —NH2 group or an NH2-(Linker)-group may be present at the C-terminal):
As one example, a peptide having the following structure can be used for carrier binding.
In formula (II), Linker 2 is (GSGGS)1-3, (SGSGS)1-3, (GGGGS)1-3 or (PEG)2-10-Lys (preferably, PEG)4-Lys), or absent. The terminal (—NH2) of the N—terminal amino acid of formula (II) may be acetylated and thus in the form of a (CH3—C(═O)—NH—) group. Linker 2 may be bound to the amino terminal (in this case, a Lys residue is introduced to an appropriate position in the vicinity of the N-terminal in Linker at the N-terminal side), and in this case, Linker 2 at the C-terminal may be present or absent.
Examples of the peptide containing the amino acid sequence represented by formula (II) preferably include the following peptides.
Examples of the peptide containing the amino acid sequence represented by formula (II) more specifically can include the following peptides:
For example, a peptide having the following structure can be used as the peptide for carrier-binding.
The peptide for carrier-binding has at least one amino group (—NH2) for a covalent bond with a carrier. Such an amino group is preferably an amino group at the N-terminal, and may be any side-chain amino group of a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, a 2-aminosuberic acid, Dpr, and an arginine residue in the vicinity of the N-terminal or the C-terminal (for example, positioned in the linker) as long as binding with a carrier can be made.
In the case of the peptide for drug-binding, the peptide (iii) or (iv) is preferable.
The peptide represented by formula (I′) may contain a functional group at the C-terminal, instead of a functional group at the N-terminal. In other words, the peptide represented by formula (I′) may be a peptide represented by the following formula (I″):
[(X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X7)—W—C—(X81-3)-(Linker3)]-Z (I″)
In formula (I′) and formula (I″), Linker 3 is RRRGS, EEGGS or (PEG)1-8 (preferably, (PEG)4), or absent. An amino group may be bound to the terminal (—COOH) of the C-terminal amino acid of formula (I′) and thus in the form of a (—C(═O)NH2) group. An acetyl group may be bound to the terminal (—NH2) of the N-terminal amino acid of formula (I″) and thus in the form of a (CH3—C(═O)—NH—) group.
The peptides having the amino acid sequences represented by formula (I′) and formula (I″) may be each Z-(Linker3)-(X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X7)—W—C—(X81-3) or (X11-3)—C—(X2)—(X3)—(X4)—(X5)-G-(X6)-L-(X7)—W—C—(X81-3)-(Linker3)-Z. A preferable amino acid sequence is the same as a preferable amino acid sequence in the peptide represented by formula (I).
Examples of the peptide for drug-binding can include the following peptides.
In formula (II′), Linker 2 is SGSGSK, SRRCR, SRRK(Z)R, SRRCRRCRRC, SRRK(Z)RRK(Z)RRK(Z), or (PEG)1-8-Lys (preferably, (PEG)4-Lys), or absent. The terminal (—NH2) of the N-terminal amino acid of formula (II′) may be acetylated and thus in the form of a (CH3—C(═O)—NH—) group. Other functional molecule may also be, if necessary, bound to a Cys residue (C) contained in the linker via a maleimide group.
Examples of the peptide containing the amino acid sequence represented by formula (II′) preferably include the following peptides.
Examples of the peptide containing the amino acid sequence represented by formula (II′) more specifically can include the peptides described in 60) to 66) (where a functional group may be, if necessary, bound to a lysine residue contained):
Other examples of the Fc-binding peptide can include the following peptides (
For example, a reactive functional group (preferably azide group) may be, if necessary, bound to the N-terminal or C-terminal (preferably, N-terminal) of the peptide for drug-binding herein, via a linker. For example, a reactive functional group (for example, azide group) is contained at the terminal of a peptide where 1 to 3 (preferably, 2) glutamic acids are further bound at the N-terminal and/or C-terminal. A peptide having an azide group can be subjected to click reaction with other functional molecule having Dibenzylcyclooctyne (DBCO), alkyne, and TCO, to result in linking of such other functional molecule to the peptide. The binding of the peptide and such other functional substance can also be performed by other method known by those skilled in the art, for example, reaction of a maleimide group and a sulfhydryl group.
Other functional molecule may also be bound to the peptide herein. For example, such other molecule can be bound via the reactive functional group (for example, to the amino terminal or the like), can be bound to the reactive functional group (for example, azide group as a substituent in a lysine residue) in the case where an amino acid (for example, lysine residue) in the peptide has the reactive functional group, or can be bound to a Cys residue (for example, Cys residue in Linker 2) in the peptide, via a maleimide group.
Other functional molecules capable of being bound to the peptide herein include a label substance or a medical drug each containing a peptide, protein, nucleic acid, or low-molecular pharmaceutical, but are not limited thereto. Any molecule where antigen specificity and other properties of an Fc molecule can be applied can be bound as such other molecule. Examples of such a substance include an anticancer agent, a low-molecule pharmaceutical product, a radiation label, a fluorescent label, a nucleic acid pharmaceutical, a gene therapy drug, a peptide pharmaceutical, and an antibody such as IgA or VHH.
The peptide for carrier-binding has at least one amino group (—NH2) for a covalent bond with an antibody. Such an amino group is preferably an amino group at the amino terminal, and may be any side-chain amino group of a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, and an arginine residue.
The crosslinking by the crosslinking agent is strong in resistance to alkali, and thus the crosslinking method according to the present embodiment can be a method involving improving alkali resistance of a protein or peptide or its fused product having two or more SH groups. The crosslinking method according to the present embodiment may be, for example, a method for improving alkali resistance of a disulfide bond, a method for improving alkali resistance of a protein or peptide having a disulfide bond in its molecule, or a method for enhancing stability. Specifically, a method for improving alkali resistance of a protein or peptide, including binding two thiol groups in the protein or peptide by the method, is provided.
(IgG-Fc Region-Containing Molecule where Crosslinked Fc-Binding Peptide is Bound)
The Fc-binding peptide can be bound with an IgG-Fc region-containing molecule. Accordingly, a complex according to another embodiment is a complex of the Fc-binding peptide intramolecularly crosslinked by the crosslinking agent and the IgG-Fc region-containing molecule. The complex encompasses a complex where the Fc-binding peptide crosslinked by the crosslinking agent containing the drug is bound to the IgG-Fc region-containing molecule; and a complex where the Fc-binding peptide crosslinked by the crosslinking agent containing the reactive functional group is bound to the IgG-Fc region-containing molecule.
The crosslinking agent according to the present embodiment may provide crosslinking between the Fc-binding peptide and the IgG-Fc region-containing molecule. Specifically, the Fc-binding peptide may be bound with an SH group of a cysteine residue contained in the antibody or the like, via the crosslinking agent. The Fc-binding peptide in such an antibody or the like-Fc-binding peptide crosslinked product may be further intramolecularly crosslinked, and, for example, the reactive functional group/drug may be bound to the Fc-binding peptide by intramolecular crosslinking and the Fc-binding peptide may be further crosslinked with a molecule containing an Fc region of the antibody or the like. Accordingly, a complex according to another embodiment is a complex of the Fc-binding peptide and the IgG-Fc region-containing molecule, crosslinked by the crosslinking agent. The complex encompasses a complex where the IgG-Fc region-containing molecule and the Fc-binding peptide are crosslinked by the crosslinking agent containing the drug; and a complex where the IgG-Fc region-containing molecule and the Fc-binding peptide are crosslinked by the crosslinking agent containing the reactive functional group.
Herein, the “IgG-Fc region-containing molecule” means a peptide, a protein, or other complex, containing an Fc region of IgG, and encompasses, in addition to wild-type or artificial IgG and a mutant thereof, a fused product of an Fc region of IgG and other substance (active component, drug, protein, low-molecular compound, medium-molecular compound, high-molecular compound, matrix, lipid, liposome, nanoparticle, vehicle for DDS, nucleic acid and/or peptide), typified by an Fc fusion protein, and a molecule composed of only an Fc region. For example, when an Fc molecule is an Fc fusion protein, examples of the protein or peptide to be fused with Fc include a receptor, cytokine, interleukin, factor VIII, CTLA4, human lactoferrin, a TNF receptor, or LFA-3, or a portion thereof (preferably target-bound portion).
The complex can be produced by contacting the Fc-binding peptide crosslinked by the crosslinking agent, with the IgG-Fc region-containing molecule. Accordingly, the method for producing the IgG-Fc region-containing molecule where the drug is fused includes contacting the IgG-Fc region-containing molecule with the Fc-binding peptide crosslinked by the crosslinking agent. Alternatively, the complex can be produced by binding the Fc-binding peptide to the IgG-Fc region-containing molecule and then performing intramolecular and/or intermolecular crosslinking by the crosslinking agent.
Accordingly, the method for producing the IgG-Fc region-containing molecule is a method for producing an IgG-Fc region-containing molecule to which a crosslinked Fc-binding peptide is bound, comprising:
The method for producing the IgG-Fc region-containing molecule is a method for producing an IgG-Fc region-containing molecule where an Fc-binding peptide where a reactive functional group Z is bound, the Fc-binding peptide being represented by the following formula:
The method for binding the Fc-binding peptide with the antibody or the like can be performed with reference to International Publication No. WO 2013/027796, International Publication No. WO 2018/092867, International Publication No. WO 2020/075670, and/or the like.
An amino acid in a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, an arginine residue (preferably, lysine residue) or an amino acid at the 1-position in the Fc-binding peptide may be optionally modified with a moiety for covalent binding with the antibody, and may be covalently bound with the antibody or the like on the moiety when the peptide is bound with the antibody or the like. Herein, the Fc-binding peptide modified with the moiety is sometimes referred to as “CCAP reagent”. Herein, the “moiety for covalent binding with the antibody” refers to a chemical structure for linking the Fc-binding peptide and the IgG-Fc region-containing molecule by a covalent bond, and can be a chemical structure having at least one site capable of binding with a desired amino acid (for example, a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, diaminopropionic acid, or an arginine residue). Examples of the compound imparting the moiety for covalent binding with the antibody can 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 propionic acid)), and DSG, DSS, or DSP is preferable. For example, a succinimidyl group such as DSS or DSG is reactive with a primary amine present in a side chain of a lysine residue and the N-terminal of a polypeptide, and thus only a side chain of a lysine residue of IgBP can be specifically modified with DSS or DSG by blocking the N-terminal of the Fc-binding peptide and then performing reaction with DSS or DSG. The crosslinking between the Fc-binding peptide and IgG can site-specifically occur, for example, between an amino acid residue of X5, X9, X11, X12, or X14 of the Fc-binding peptide, and Lys248 or Lys246 in an Fc region of IgG, preferably Lys248.
The binding between the Fc-binding peptide as a CCAP reagent and the antibody or the like is not particularly limited as long as the binding is performed under conditions allowing for the occurrence of crosslinking reaction, and can be made by, for example, reaction of the Fc-binding peptide and the antibody or the like by mixing in an appropriate buffer at room temperature (for example, about 15° C. to 30° C.). The mixing step may be performed by, if necessary, addition of a proper amount of a catalyst promoting the crosslinking reaction. The mixing ratio between the Fc-binding peptide and the antibody or the like in the mixing step can be 1:1 to 20:1, preferably 2:1 to 20:1 or 5:1 to 10:1, for example, in terms of the molar ratio of Fc-binding peptide:antibody or the like. 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 bound product can be, if necessary, further purified.
An Fc region of IgG or the like usually forms a heavy-chain constant region that is a symmetric pair of two, and thus two sites where the Fc-binding peptide is to be bound can be present. Accordingly, one to two peptides, preferably one peptide of the Fc-binding peptide can be bound to one molecule of the IgG-Fc region-containing molecule.
The Fc-binding peptide can be bound to a carrier of a column or the like and thus used for purification of the antibody or the like. The crosslinking method in the present disclosure results in an enhancement in resistance to alkali, and thus the Fc-binding peptide crosslinked by the crosslinking agent of the present disclosure can be used to thereby reuse a carrier by washing with alkali once or several times. Accordingly, one aspect provides a carrier where the Fc-binding peptide intramolecularly crosslinked by the crosslinking agent is bound. The binding of the peptide to the carrier can be performed by, for example, reaction of the peptide with a carrier having a functional group reactive with an amino group. The reaction is performed under conditions imparting sufficient binding of both, and can be performed by, for example, contacting them in a buffer at room temperature for 1 to 5 hours (preferably, 2.5 to 3.5 hours).
Examples of the carrier include forms of gel (for example, gel for columns), particles, beads, nanoparticles, fine particles, macrobeads, membrane, microplate, and array, and examples of the material thereof include a magnetic substance, latex, agarose, glass, cellulose, sepharose, nitrocellulose, polystyrene, and other polymer material. The carrier is preferably a gel for columns (column chromatography). The carrier here used can be, for example, HiTrap NHS-activated HP (GE Healthcare).
A method for purifying the IgG-Fc region-containing molecule by use of the carrier is also provided. A method for purifying the antibody or the like in one aspect includes contacting an antibody or the like-containing liquid with the carrier to thereby bind the antibody or the like to the carrier, washing and removing a component not bound to the carrier, and eluting and recovering a component bound to the carrier.
The contacting between the antibody or the like-containing liquid and the carrier is performed under conditions so that both can be sufficiently contacted. For example, when the carrier is a column, the contacting is performed by injecting the antibody or the like-containing liquid into the column. The removal of the component not bound to the carrier can be performed by an ordinary method, and can be performed by, for example, washing the carrier bound to the antibody or the like, with a buffer (pH about 7.0). The recovery of the antibody or the like bound to the carrier can be performed at a pH of 2.5 or more, and the degree of acidity is desirably weak in order to prevent the antibody or the like from being denatured and the pH is preferably 3.6 or more and can be, for example, 3.6 to 4.3. Alternatively, when beads are used in the carrier, the antibody or the like can be recovered by contacting the antibody or the like and the carrier, then recovering the beads by centrifugation or the like and re-suspending them in an eluate.
Another aspect provides a medical composition containing a peptide and/or a protein crosslinked by the crosslinking agent or an IgG-Fc region-containing molecule where an Fc-binding peptide crosslinked by the crosslinking agent of the present disclosure is bound, as an active ingredient, in particular, a therapeutic drug, a prophylactic drug, or a diagnostic product. A peptide or protein crosslinked by a crosslinking agent where a drug having a therapeutic or prophylactic effect is bound can be used in a medical (therapeutic or prophylactic) composition.
When the above peptide and/or protein, or drug D bound via the crosslinking agent are/is each a therapeutic drug, a prophylactic drug, or a drug serving as a vaccine, the peptide and/or protein crosslinked, or the antibody or the like where the peptide and/or protein crosslinked are/is bound can be used for therapy or prophylaxis.
When the above peptide and/or protein, or drug D bound via the crosslinking agent are/is each a drug serving as a label, the peptide and/or protein crosslinked, or the antibody or the like where the peptide and/or protein crosslinked are/is bound can be used for diagnosis or detection.
When the above composition is a therapeutic or prophylactic composition, the drug is a therapeutic agent or prophylactic agent, and when the above composition is a diagnostic product, the drug is a label substance. The target disease of the medical composition can be appropriately set by selecting the peptide, the protein, or the antibody or the like to be used, and the drug to be bound, and examples thereof include cancer, inflammatory disease, infection, and neurodegenerative disease.
For example, the medical composition can be utilized as an injection preparation, and encompasses dosage forms such as an intravenous injection preparation, a subcutaneous injection preparation, an intradermal injection preparation, an intramuscular injection preparation, and a drip injection preparation. Such an injection preparation can be prepared by, for example, dissolving, suspending or emulsifying an active ingredient in a sterile aqueous or oily liquid commonly used in injection preparations, according to a known method. An injection solution prepared is usually packed in an appropriate ampule, vial or syringe. The injection solution can also be obtained by adding an appropriate excipient to an active ingredient to thereby prepare a freeze-dried formulation and dissolving the formulation in an injection solvent, normal saline or the like before using. While oral administration of a protein such as an antibody is generally difficult due to decomposition by the digestive system, such oral administration can also be made by originality and ingenuity of an antibody fragment and an antibody fragment modified, and a dosage form. Examples of an oral administration formulation can include a capsule, a tablet, a syrup and a granule.
The medical composition is suitably prepared into a dosage form of a dose unit adapted to the amount of an active component administered. The dosage form of the dose unit is, for example, an injection preparation (ampule, vial, or prefilled syringe), and may usually contain 5 to 500 mg, 5 to 100 mg, 10 to 250 mg of an active ingredient or a drug per dosage form of the dose unit.
The administration route of the medical composition may be local or systemic. The administration method is not particularly limited, and is parenteral or oral administration as described above. Examples of the parenteral administration route include subcutaneous administration, intraperitoneal administration, administration into the blood (intravenous or intraarterial), or injection or dripping into the spinal fluid, and administration into the blood is preferable. The medical or diagnostic composition may be temporarily administered, or may be continuously or intermittently administered. For example, the administration can also be continuous administration for 1 minute to 2 weeks. The dosage regimen of the medical composition is not particularly limited as long as the dose and the time of administration are set so as to impart a desired therapeutic effect or prophylactic effect, and can be appropriately determined depending on the symptom, the sex, the age, and the like. For example, a single dose of the active ingredient is advantageously administered approximately once to ten times a day, preferably once to five times a day as usually about 0.01 to 20 mg/kg body weight, preferably about 0.1 to 10 mg/kg body weight, further preferably about 0.1 to 5 mg/kg body weight of an intravenous injection, before and/or after the development of clinical symptoms of the disease. Also in cases of other parenteral administration and oral administration, the administration can be made in a similar dose thereto.
Hereinafter, the present disclosure is more specifically described with reference to Examples, but the present disclosure is not limited by these Examples. The entire of the Documents cited throughout the present specification is herein incorporated by reference.
A raw material peptide [sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1): IgGBP-longGS or IgGBP-LGS] was synthesized on commission (Eurofins) by a solid-phase peptide synthesis method (Fmoc method). In 1500 μL of DMF was dissolved 5 mg (1.95 μmol) of the raw material peptide, TCEP-HCl (1.12 mg, 3.9 μmol, 2 equivalent moles) dissolved in 2 ml of PBS (pH 7.4) in advance was added thereto, and reduction reaction was performed under stirring at room temperature for 30 minutes. Thereafter, 1,1-Dichloro-2-propanone (0.495 mg, 3.9 μmol, 2 equivalent moles) dissolved in 120 μL of acetonitrile was added, and the resultant was stirred at room temperature. After 1 hour, the termination of the reaction was confirmed by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and the reaction solution was purified by HPLC (C18 reverse-phase column), to thereby obtain an Fmoc-cyclized peptide (2 mg, 0.76 μmol, 40% yield). Thereafter, 2% piperidine was added for deprotection of an Fmoc protection group, the termination of the reaction was confirmed after 10 minutes by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and the reaction solution was directly purified by HPLC (C18 reverse-phase column), to thereby obtain a cyclized peptide (1 mg, 0.38 μmol, 20% yield). The peptide finally purified was subjected to confirmation of the molecular weight by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and then freeze-dried.
A raw material peptide [sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1)] was synthesized on commission (Eurofins) by a solid-phase peptide synthesis method (Fmoc method). In 1500 μL of DMF was dissolved 10 mg (3.9 μmol) of the raw material peptide, TCEP-HCl (2.24 mg, 7.8 μmol, 2 equivalent moles) dissolved in 2 ml of PBS (pH 7.4) in advance was added thereto, and reduction reaction was performed under stirring at room temperature for 30 minutes. Thereafter, 1,1-dichloropinacolin (1.32 mg, 7.8 μmol, 2 equivalent moles) dissolved in 120 μL of acetonitrile was added, and the resultant was stirred at room temperature. After 1 hour, the termination of the reaction was confirmed by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and the reaction solution was purified by HPLC (C18 reverse-phase column), to thereby obtain an Fmoc-cyclized peptide (7 mg, 2.7 μmol). Thereafter, 2% piperidine was added for deprotection of an Fmoc protection group, the termination of the reaction was confirmed after 10 minutes by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and the reaction solution was directly purified by HPLC (C18 reverse-phase column), to thereby obtain a cyclized peptide (5 mg, 2.04 μmol, 50% yield). The peptide finally purified was subjected to confirmation of the molecular weight by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and then freeze-dried.
A raw material peptide [sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1)] was synthesized on commission (Eurofins) by a solid-phase peptide synthesis method (Fmoc method). In 1500 μL of DMF was dissolved 2.5 mg (1.00 μmol) of the raw material peptide, TCEP-HCl (0.506 mg, 2.0 μmol, 2 equivalent moles) dissolved in 2 ml of PBS (pH 7.4) in advance was added thereto, and reduction reaction was performed under stirring at room temperature for 30 minutes. Thereafter, 2,2-dichloroacetophenone (0.38 mg, 2.0 μmol, 2 equivalent moles) dissolved in 120 μL of acetonitrile was added, and the resultant was stirred at room temperature. After 1 hour, the termination of the reaction was confirmed by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and the reaction solution was purified by HPLC (C18 reverse-phase column), to thereby obtain about 2 mg of an Fmoc-cyclized peptide. Thereafter, 2% piperidine was added for deprotection of an Fmoc protection group, the termination of the reaction was confirmed after 10 minutes by LC-MS analysis (LC-MS8030 manufactured by SHIIMIADZU CORPORATION), and the reaction solution was directly purified by HPLC (C18 reverse-phase column), to thereby obtain a cyclized peptide (1.2 mg, 0.49 μmol, 49% yield). The peptide finally purified was subjected to confirmation of the molecular weight by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and then freeze-dried.
The affinity analysis was performed by the following method. First, a CM5 sensor chip was installed on BIAcoreT200 (GE healthcare), and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and a 0.1 M sulfo-N-hydroxysuccinimide (sulfo-NHS) solution mixed in equivalent amounts were injected to the sensor chip at a flow rate of 10 μl/ml, to thereby activate the sensor chip. Thereafter, IgG was immobilized onto the sensor chip under a condition of a pH of 5.5 (10 mM Na acetate). An HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, 3 mM EDTA, pH 7.4) was used in measurement, and 15.625, 31.2, 62.5, 125, 250, and 500 nM of peptides were each injected at a flow rate of 50 μl/ml for 180 seconds, to thereby monitor binding reaction. When dissociation reaction was measured, only the buffer was injected for 600 seconds. The interaction parameter was analyzed with BIAevaluation T100 software.
IgG-binding peptide derivatives compared and evaluated are illustrated in
Five mL of 1 mM hydrochloric acid was sent to an NHS-activated prepacked column having a volume of 1 mL, and an isopropanol solution in the column was removed. Next, a 10.0 mg/mL peptide solution (dissolved in 100 μL of DMSO) was diluted with a coupling solution (20 mM carbonate buffer, 50 mM sodium chloride, pH 8.3) by 10-fold, 1 mL of the dilution liquid was sent, and immobilization was performed at room temperature for 4 hours. Thereafter, unreacted NHS was blocked by 5 mL of 1 M Tris (pH 8.0) at room temperature for 1 hour. Thereafter, washing with 5 mL of an aqueous 0.1 N NaOH solution was made. Finally, 10 mL of a PBS solution (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.4) was sent, and used in chromatography evaluation.
The IgG-binding peptide-immobilized column produced was connected to a BioLogic LP (manufactured by Bio-Rad Laboratories Inc.) liquid chromatographic system, and equilibrated with PBS. Next, 1 mg/mL human serum-derived IgG (manufactured by Sigma-Aldrich Co. LLC) dissolved in PBS was sent at a flow rate of 1 mL/min for 1 minute. Furthermore, the column was washed with PBS, and an elution solution (100 mM glycine buffer, pH 2.8) was sent, to thereby elute IgG as an adsorption component. The elution of IgG from the column was detected by the absorbance at 280 nm.
A column where the amount of immobilization of a peptide was 1 mg was produced by the same method as described above. The column produced was equilibrated with PBS, and 1 mg/mL human serum-derived IgG (manufactured by Sigma-Aldrich Co. LLC) dissolved in PBS was sent at a flow rate of 1 mL/min (retention time 1 min). DBC was indicated as one determined from the amount of a protein added at the time of point where 10% of the absorbance at 280 nm of the sample added was leaked.
Next, 5 mL of an aqueous 0.1 N sodium hydroxide solution was sent to the 1-mL column where 1 mg of the peptide was immobilized. Thereafter, washing with PBS was performed. This cycle was repeated 30 times for the 1,1-dichloroacetone-crosslinked cyclic peptide, and was repeated 10 times for the 1,1-dichloropinacolin-crosslinked cyclic peptide. DBC measurement was performed at a flow rate of 1 mL/min at the 1st to 5th and 10th times of the cycles, and further performed at the 20th and 30th times of the cycles with respect to the 1,1-dichloroacetone-crosslinked cyclic peptide, and thus alkali resistance was evaluated. The variation in DBC was determined based on the measurement results under the assumption that DBC immediately after column production was 100% according to Table 2.
In this regard, while the initial value of DBC of each peptide column was 17.24 mg/mL-column in the case of the original peptide and was 2.3 mg/mL-column in the case of the 1,3-dichloroacetone-crosslinked peptide, the value of DBC was 12.8 mg/mL-column in the case of the 1,1-dichloroacetone-crosslinked peptide and was 16.70 mg/mL-column in the case of the 1,1-dichloropinacolin-crosslinked peptide. Accordingly, it was revealed that IgG adsorption performance was high as compared with a conventional crosslinked cyclic peptide.
Five mL of a 0.1 M sodium hydroxide solution was sent to the produced 1-mL column where the amount of immobilization of a peptide was 1 mg, and thereafter washing with 5 mL of PBS was performed. These operations were defined as one cycle and treatment of washing with aqueous NaOH solution/washing with PBS was performed 1 to 30 times, and thereafter DBC measurement was performed at a flow rate of 1 mL/min at the intended number of times (1, 2, 3, 4, 5, 10, 20, 30 times).
The results are illustrated in
In 400 μl of DMF was dissolved 0.201 μmol of the prepared 1,1-dichloroacetone-crosslinked cyclic peptide, and a 100-fold volume of an O-2-Propynylhydroxylamine hydrochloride (20.1 μmol) solution obtained by dissolution in 1.0 ml of a 0.2 M NaHCO3 buffer (pH 8.3) in advance was added thereinto. The reaction liquid was stirred at room temperature for 24 hours. The termination of the reaction was confirmed by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and the reaction solution was directly purified by HPLC (C18 reverse-phase column), to thereby obtain an alkyne functional group-introduced, crosslinked and cyclized peptide (0.10 μmol, 50% yield). The peptide finally purified was subjected to confirmation of the molecular weight by LC-MS analysis (LC-MS8030 manufactured by SHIMADZU CORPORATION), and then freeze-dried. The resulting compound was subjected to the same affinity measurement as in Example 4 (Measurement of binding affinity of crosslinked cyclic peptide), and as a result, the Kd value was 334 nM and was reduced by about 42-fold as compared with that of the original peptide.
A urea solution was added to 100 μl (95 μg, 56×10−10 mol) of a PBS solution (pH 7.4) of a 950 μg/ml anti-CD89 VHH antibody (IgARC25) so as to have a final concentration of 5 M, and the mixture was left to still stand at room temperature for 1 hour. Thereafter, TCEPTCEP-HCl (11.2 nmol, 2 equivalent moles) was added, and reduction reaction was performed under stirring at room temperature for 30 minutes. Thereafter, 1,1-Dichloroacetone (11DCA, 11.2 nmol, 2 equivalent moles) dissolved in 120 μL of acetonitrile was added, and the mixture was stirred at room temperature. After 1 hour, the termination of the reaction was confirmed by LC-MS analysis (Bio-Accord SYSTEM manufactured by Waters Corporation). As a result, the molecular weight of IgARC25 was 14,028 Da before crosslinking, and was 14,085 Da after treatment with 11DCA and was found to be increased by 57 Da, and crosslinking by 11DCA was thus confirmed.
In crosslinking reaction of a disulfide bond by a 1,1-dichloroacetone derivative compound (1,1-dichloroacetone, 1,1-dichloropinacolin, 2,2-dichloroacetophenone) represented by the following formula,
a peptide having two Cyses each modified by Fmoc at the N-terminal (or a peptide obtained by reduction of a peptide modified by Fmoc at the N-terminal and having a disulfide bond) and 1.0 to 2.0 equivalents of the 1,1-dichloroacetone derivative compound were reacted in PBS to thereby perform crosslinking (step a in scheme A), and finally Fmoc at the N-terminal was removed and deprotected (step b in scheme A) for preparation, as shown in the following formula. On the other hand, a method not according to this method was designed where crosslinking reaction was performed in peptide synthesis by am Fmoc method, as shown in scheme B. A peptide containing one of two Cyses to be crosslinked was synthesized from the C-terminal to the previous one of the other Cys, on a peptide synthesis resin by an Fmoc method. The protection group of Cys in the peptide was deprotected (step c in scheme B), and then Fmoc-chloroacetophenoyl cysteine was added and linked (step d in scheme B). Fmoc was deprotected (step e in scheme B), and the α-amino group at the N-terminal of the peptide generated, and the α-carboxyl group at the acetophenoyl cysteine side were coupled (step f in scheme B). The remaining amino acids were subjected to linking and synthesis by an Fmoc method (step g in scheme B), and finally an objective peptide was obtained by cleavage from the resin and deprotection of Fmoc.
Fmoc-chloroacetophenoyl cysteine (hereinafter, compound 5) used in the present method was synthesized by the following method. In the following, the α-carboxyl group of the Fmoc-chloroacetophenoyl cysteine was protected by an allyl group.
Under an argon stream, N,N-diisopropylethylamine (1.60 mL, 9.38 mmol, 1.1 equivalents) was added to an acetonitrile solution (85 mL) of a compound 1 (5.00 g, 8.53 mmol) and allyl bromide (1.44 mL, 17.1 mmol, 2 equivalents), and the mixture was stirred at room temperature for 16 hours. After termination of the reaction by addition of water, the reaction liquid was concentrated and then extracted with ethyl acetate, and the organic layer was washed with an aqueous saturated sodium hydrogen carbonate solution, an aqueous 2 N hydrochloric acid solution and saturated saline, and then dried by sodium sulfate. The resultant was concentrated and the resulting residue was purified with silica gel column chromatography (hexane:ethyl acetate=4:1), to thereby obtain a compound 2 (5.03 g, 94%).
The compound 2 (5.03 g, 8.03 mmol) and triisopropylsilane (5.45 mL, 26.5 mmol, 3.3 equivalents) were added to a solution of trifluoroacetic acid and dichloromethane at 1:1 (30 mL), and the mixture was stirred at room temperature for 1 hour. After completion of the reaction and then azeotropy with toluene, the residue obtained by concentration was purified with silica gel column chromatography (hexane: ethyl acetate=4:1), to thereby obtain a compound 3 (1.94 g, 63%).
Under an argon stream, triethylamine (0.401 mL, 2.90 mmol, 1.1 equivalents) was added to a dichloromethane solution (26 mL) of the compound 3 (1.00 g, 2.63 mmol) and phenacyl chloride (814 mg, 5.27 mmol, 2 equivalents), and the mixture was stirred at room temperature for 30 minutes. After termination of the reaction by addition of water, extraction with dichloromethane was made, and the organic layer was washed with saturated saline and then dried by sodium sulfate. The resultant was concentrated and the resulting residue was purified with silica gel column chromatography (hexane:ethyl acetate=2:1), to thereby obtain a compound 4 (1.17 g, 89%).
1H NMR (400 MHz, CDCl3, ppm): δ 7.96 (d, J=7.8 Hz, 2H), 7.76 (d, J=7.3 Hz, 2H), 7.64-7.57 (m, 3H), 7.47 (dd, J=7.6, 8.0 Hz, 2H), 7.40 (dd, J=7.3, 7.8 Hz, 2H), 7.31 (ddd, J=1.4, 7.3, 7.3 Hz, 2H), 5.94-5.84 (m, 2H), 5.33 (d, J=16.9 Hz, 1H), 5.24 (dd, J=1.4, 10.1 Hz, 1H), 4.71-4.64 (m, 3H), 4.40-4.38 (m, 2H), 4.23 (dd, J=6.9, 6.9 Hz, 1H), 3.90 (s, 2H), 3.16-3.03 (m, 2H)
HRMS (FAB-TOF) m/z: [(M+H)+] calcd for C29H28N1O5S1 502.1688; found 502.1690.
Under an argon stream, a solution of carbon tetrachloride and dichloromethane at 1:1 (4 mL), of the compound 4 (204 mg, 0.401 mmol) and N-chlorosuccinimide (59.6 mg, 0.447 mmol, 1.1 equivalents), was added, and stirred at room temperature for 1 hour. After termination of the reaction by addition of water, the aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated saline and then dried by sodium sulfate. The resultant was concentrated and the resulting residue was purified with silica gel column chromatography (only dichloromethane), to thereby obtain a compound 5 (210 mg, 97%).
1H NMR (400 MHz, CDCl3, ppm): δ 7.99 (d, J=7.8 Hz, 2H), 7.76 (d, J=7.3 Hz, 2H), 7.63-7.57 (m, 3H), 7.48 (dd, J=7.8, 7.8 Hz, 2H), 7.39 (dd, J=7.3, 7.8 Hz, 2H), 7.30 (ddd, J=1.0, 7.3, 7.3 Hz, 2H), 6.37 (d, J=25.6 Hz, 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=6.9 Hz, 2H), 4.21 (dd, J=6.9, 6.9 Hz, 1H), 3.51-3.14 (m, 2H)
HRMS (FAB-TOF) m/z: [(M+H)+] calcd for C29H27N1O5S2Cl1S1 536.1296; found 536.1298.
Whether or not the Fmoc-allylated chloroacetophenoyl cysteine (compound 5) synthesized had reactivity with thiol of Cys was tested by the following reaction.
Under an argon stream, triethylamine (0.017 mL, 0.121 mmol, 1.1 equivalents) was added to a dichloromethane solution (1.1 mL) of the compound 5 (59.0 mg, 0.110 mmol) and Fmoc-Cys-OAllyl (0.046 mg, 0.121 mmol, 1.1 equivalents), and the mixture was stirred at room temperature for 10 minutes. After termination of the reaction by addition of water, the aqueous layer was extracted with dichloromethane, and the organic layer was washed with saturated saline and then dried by sodium sulfate. The resultant was concentrated and the resulting residue was purified with silica gel column chromatography (hexane:ethyl acetate=3:1), to thereby o btain a compound 6 (93.8 mg, 97%).
1H NMR (400 MHz, CDCl3, ppm): δ 7.96 (d, J=7.8 Hz, 2H), 7.75 (dd, J=2.7, 7.8 Hz, 4H), 7.59-7.53 (m, 5H), 7.41 (dd, J=7.8, 7.8 Hz, 2H), 7.39-7.35 (m, 4H), 7.30-7.26 (m, 4H), 5.91-5.80 (m, 2H), 5.70 (dd, J=7.8, 22.0 Hz, 2H), 5.61 (s, 1H), 5.31 (d, J=17.4 Hz, 2H), 5.22 (d, J=11.6 Hz, 2H), 4.69-4.59 (m, 6H), 4.41-4.29 (m, 4H), 4.19 (dd, J=7.3, 7.3 Hz, 2H), 3.33-2.97 (m, 4H),
HRMS (FAB-TOF) m/z: [(M+Na)+] calcd for C50H46N2O9S2Na, 905.2542; found 905.2542.
The above results indicated that the Fmoc-allylated chloroacetophenoyl cysteine (compound 5) had reactivity with thiol of Cys, and the progress of the reaction shown in step d in scheme B was demonstrated. It was verified from the above results that synthesis of a novel crosslinked peptide in scheme B could be carried out.
Crosslinking of oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 77), intramolecular SS bond, molecular weight: 1007.19) was performed with 1.1-dichloroacetone. Ten mg (9.92 μmol) of acetate (manufactured by Toronto Research Chemicals) in an oxytocin SS oxidized form (oxytocin-OX), shown by the following formula A, was dissolved in 3 mL of a 0.1 M HEPES-HCl buffer (pH 8.0), 10.2 mL of a 0.1 M HEPES-HCl buffer (pH 8.0) where 57.1 mg (200 μmol) of TCEP hydrochloride (Tris(2-carboxyethyl)phosphine Hydrochloride) as a reducing agent was dissolved was mixed, and the mixture was stirred for 1 hour. Thereto was added 2.77 mg (21.8 μmol) (molar ratio to oxytocin:2.2) of 1,1-dichloroacetone dissolved in 0.722 mL of acetonitrile, and the mixture was stirred for 1 hour.
The reaction product obtained was analyzed by LC-MS as follows. The reaction product was diluted with 0.1% formic acid by 5-fold, and thereafter 20 μL thereof was subjected to analysis (flow rate: 0.2 mL/min, elution: linear gradient from 4% CH3CN containing 0.1% formic acid, to 70% CH3CN, column temperature: 25° C.) with an Acquity UPLC/SQ detector system (manufactured by Waters Corporation) jointed with a Peptide BEH-C18 column (130 Å, 1.7 μm, 2.1×100 mm, manufactured by Waters Corporation).
The respective results of oxytocin-OX before addition of TCEP hydrochloride, and the reaction product, analyzed with LC-MS, are illustrated in
Crosslinking of vasopressin (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 (SEQ ID NO: 78), intramolecular SS bond, molecular weight: 1084.24) was performed with 1.1-dichloroacetone. In 1.65 mL of a 0.1 M phosphate buffer (pH 8.0) was dissolved 5.5 mg (5.07 μmol) of acetate (manufactured by Tokyo Chemical Industry Co., Ltd.) of a vasopressin SS oxidized form (vasopressin-OX), shown by the following formula B, 4.4 mL of a 0.1 M phosphate buffer (pH 8.0) where 24.6 mg (86.0 μmol) of TCEP hydrochloride was dissolved was mixed, and the mixture was stirred for 1 hour. Thereto was added 1.42 mg (11.2 μmol) (molar ratio to vasopressin:2.2) of 1,1-dichloroacetone dissolved in 0.371 mL of acetonitrile, and the mixture was stirred for 1 hour.
The reaction product obtained was analyzed by LC-MS in the same manner as in Example 11. The respective results of vasopressin-OX before addition of TCEP hydrochloride, and the reaction product, analyzed with LC-MS, are illustrated in
For evaluation of stability of a peptide by crosslinking reaction, resistivity of α-chymotrypsin (derived from bovine pancreas, manufactured by MP Biomedicals) to protease decomposition was evaluated with oxytocin crosslinked by 2,2-dichloroacetophenone. Crosslinking of oxytocin by 2,2-dichloroacetophenone was performed in the same manner with replacement of 1.1-dichloroacetone in Example 11 by 2,2-dichloroacetophenone, and an objective product was taken by reverse-phase HPLC. Specifically, 10 mg (10 μmol) of oxytocin acetate was dissolved in 3 mL of a 0.1 M HEPES hydrochloric acid buffer (pH 8.0), the solution was mixed with 2 mL of a 0.1 M HEPES hydrochloric acid buffer (pH 8.0) containing 57.4 mg (200 μmol) of TCEP-HCl, and 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 and mixed, and the mixture was stirred at room temperature for 1 hour. The resulting sample was applied to an InertSustain C18 column (5 μm, 14×250 mm, manufactured by GL Sciences Inc.) (flow rate 5 mL/min) connected to LC-Forte (manufactured by YMC). The elution was performed at a linear gradient from 4% to 70% (containing 0.1% formic acid). An objective product was taken, and freeze-dried after removal of acetonitrile at a negative pressure.
A structure of a 2,2-dichloroacetophenone crosslinked form (oxytocin-DP) of oxytocin is shown by formula A2.
Evaluation was made with oxytocin-OX and an oxytocin SH reduced form (oxytocin-RD) shown by the following formula A3 used as comparison subjects. In other words, an oxytocin solution obtained by dissolution in a 0.1 M phosphate buffer (pH 7.0) at 0.5 mg/mL was adopted as oxytocin-OX, and a substance obtained after a lapse of 30 minutes from addition of an oxytocin solution obtained by dissolution in a 0.1 M phosphate buffer (pH 7.0) containing 0.5 mg/mL TCEP, at 0.5 mg/mL, was adopted as oxytocin-RD. To 200 μL of each of these solutions was 10 μL ( 1/10 on weight ratio to oxytocin) of 1 mg/mL α-chymotrypsin, and the resultant was subjected to incubation at 37° C. and analysis by reverse-phase HPLC. In the case of blank, a phosphate buffer was added instead of α-chymotrypsin.
A substance obtained after a lapse of 30 minutes from dissolution of 0.5 mg/mL oxytocin-DP purified, in a 0.1 M phosphate buffer (pH 7.0) containing 0.5 mg/mL TCEP, was adopted as a sample, and similarly α-chymotrypsin was added and analysis by reverse-phase HPLC was made.
While no change of any peak was observed even after a lapse of 15 minutes to 2 hours in the case of oxytocin-OX, as illustrated in
The foregoing describes some example embodiments for explanatory purposes. Although the foregoing discussion has presented specific embodiments, persons skilled in the art will recognize that changes may be made in form and detail without departing from the broader spirit and scope of the invention. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense. This detailed description, therefore, is not to be taken in a limiting sense, and the scope of the invention is defined only by the included claims, along with the full range of equivalents where such claims are entitled.
This application claims the benefits of Japanese Patent Application No. 2020-186833, filed on Nov. 9, 2020, and Japanese Patent Application No. 2021-82739 filed on May 14, 2021, the entire disclosures of which are incorporated by reference herein.
The present disclosure is useful for crosslinking a peptide and a protein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-186833 | Nov 2020 | JP | national |
| 2021-082739 | May 2021 | JP | national |
This application is the U.S. National Stage of PCT/JP2021/039054, filed Oct. 22, 2021, which claims priority to JP 2020-186833, filed Nov. 9, 2020 and JP 2021-082739, filed May 14, 2021. The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 3, 2023, is named sequence.txt and is 26,409 bytes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/039054 | 10/22/2021 | WO |
