WO2022097500A1 - ペプチド架橋剤及び当該架橋剤で架橋された架橋ペプチド - Google Patents

ペプチド架橋剤及び当該架橋剤で架橋された架橋ペプチド Download PDF

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WO2022097500A1
WO2022097500A1 PCT/JP2021/039054 JP2021039054W WO2022097500A1 WO 2022097500 A1 WO2022097500 A1 WO 2022097500A1 JP 2021039054 W JP2021039054 W JP 2021039054W WO 2022097500 A1 WO2022097500 A1 WO 2022097500A1
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Prior art keywords
peptide
protein
group
crosslinked
alkyl group
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PCT/JP2021/039054
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English (en)
French (fr)
Japanese (ja)
Inventor
祐ニ 伊東
浩 中山
モハンマド アブドゥール ラフィーク
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Kagoshima University NUC
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Kagoshima University NUC
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Priority to US18/035,914 priority Critical patent/US20240391950A1/en
Priority to JP2022560709A priority patent/JP7782851B2/ja
Priority to CN202180075697.7A priority patent/CN116710138A/zh
Priority to IL302587A priority patent/IL302587A/en
Priority to KR1020237018809A priority patent/KR20230107276A/ko
Priority to EP21889053.1A priority patent/EP4242220A4/en
Application filed by Kagoshima University NUC filed Critical Kagoshima University NUC
Priority to CA3197961A priority patent/CA3197961A1/en
Priority to AU2021373429A priority patent/AU2021373429A1/en
Priority to MX2023005411A priority patent/MX2023005411A/es
Publication of WO2022097500A1 publication Critical patent/WO2022097500A1/ja
Anticipated expiration legal-status Critical
Priority to ZA2023/05421A priority patent/ZA202305421B/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/04—Saturated compounds containing keto groups bound to acyclic carbon atoms
    • C07C49/16—Saturated compounds containing keto groups bound to acyclic carbon atoms containing halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/80—Ketones containing a keto group bound to a six-membered aromatic ring containing halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/16—Oxytocins; Vasopressins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to a method for producing a crosslinked peptide and the like.
  • Protein drugs and peptide drugs are one of the biopharmacy currently attracting the most attention.
  • antibody drugs and peptide drugs centered on IgG antibodies have come to be used in the pharmaceutical field, and their importance in industrial and pharmaceutical use is increasing.
  • the present inventor comprises a specific sequence in which an IgG peptide consisting of 17 residues is cyclized with a disulfide bond in order to construct an alternative system for a protein A column used for purification of an antibody drug. It has been reported that IgG can be purified by a column on which a peptide ligand is immobilized (Patent Document 1). However, the IgG peptide column has a problem that it cannot be repeatedly used because the disulfide bond existing in the peptide has low resistance to alkalinity. Therefore, the present inventor has repeatedly studied means for increasing alkali resistance in order to solve the problem that disulfide bonds in peptides and proteins are chemically weak against alkaline washing.
  • the present invention provides a method for producing a crosslinked structure having high alkali resistance and maintaining functionality in a high yield by further redesigning the crosslinked structure without a large decrease in affinity. Is the subject.
  • the present inventor uses 1,1-dichloroacetone and its derivatives, which are usually highly reactive and are not used in aqueous solution, as the cross-linking agent to improve alkali resistance and IgG.
  • 1,1-dichloroacetone and its derivatives which are usually highly reactive and are not used in aqueous solution
  • we succeeded in producing an IgG-binding peptide having a high affinity (Kd 45 nM).
  • cross-linking agent for a protein or peptide according to the first aspect of the present invention is represented by the following formula (I).
  • A is a C1-6 alkyl group or a phenyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • the method for producing a crosslinked protein or peptide according to the second aspect of the present invention is a method in which at least two thiol groups of a single or separated protein or peptide represented by the following formula are bonded.
  • a method for producing a cross-linked protein or peptide is a method in which at least two thiol groups of a single or separated protein or peptide represented by the following formula are bonded.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different
  • the protein or peptide is reacted with the cross-linking agent according to the first aspect of the present invention to form the two thiol groups as the following groups.
  • A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • the protein or peptide is a protein or peptide in which all or part of the at least two thiol groups form a disulfide bond, and the reduction of the disulfide bond gives rise to two thiol groups. It may be that.
  • the method for improving the alkali resistance of a protein or peptide according to the third aspect of the present invention is a protein or a protein crosslinked by binding a thiol group in the protein or peptide by the method according to the second aspect of the present invention. Includes obtaining peptides.
  • the method for producing a protein or peptide according to the fourth aspect of the present invention is a method for producing a protein or peptide to which a reactive functional group represented by the following formula is bound.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group.
  • the method for producing a protein or peptide according to the fifth aspect of the present invention is a method for producing a protein or peptide to which a reactive functional group represented by the following formula is bound.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group.
  • the method for producing a complex of a protein or peptide and a drug according to the sixth aspect of the present invention is a method for producing a complex of a protein or peptide represented by the following formula and a drug.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents the linker and D represents the drug.
  • a drug having a functional group capable of reacting with the reactive functional group By reacting the obtained protein or peptide to which the reactive functional group is bound with a drug having a functional group capable of reacting with the reactive functional group, the drug is applied to the crosslinked portion of the crosslinked protein or peptide. Including binding.
  • the method for producing a complex of a protein or peptide and a drug according to the seventh aspect of the present invention is a method for producing a complex of a protein or peptide represented by the following formula and a drug.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents the linker and D represents the drug.
  • a protein or peptide to which a reactive functional group represented by the following formula is bound [In the formula, A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom, and Protein / Peptide represents a protein or peptide, and Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group].
  • L represents a linker and Z represents a reactive functional group.
  • the linker may contain a portion that can be cleaved by a proteolytic enzyme.
  • the protein or peptide may be a peptide of 5 to 50 amino acids.
  • the thiol group to be crosslinked may be a thiol group present in a single protein or peptide.
  • the protein or peptide may be an Fc-binding peptide.
  • the crosslinked thiol group may be a thiol group present in the separated protein or peptide.
  • crosslinked protein or peptide according to the eighth aspect of the present invention is represented by the following formula.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different
  • the protein or peptide to which the reactive functional group according to the ninth aspect of the present invention is bound is represented by the following formula.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents the linker and D represents the drug
  • the linker may contain a portion that can be cleaved by a proteolytic enzyme.
  • the protein or peptide may be a peptide of 5 to 50 amino acids.
  • the thiol group to be crosslinked is a thiol group present in a single protein or peptide. Yes, it may be.
  • the protein or peptide may be an Fc-binding peptide.
  • the amino group of the lysine residue, cysteine residue, aspartic acid residue, glutamic acid residue, 2-aminosveric acid, diaminopropionic acid, arginine residue or the amino acid at the 1-position is DSG (discussin).
  • Imidylglutarate DSS (discussin imidazole svelate), DMA (dimethyldihydrochloride acid), DMP (dimethyldihydrochloride pimelimide), DMS (dimethyldihydrochloride subberimide), DTBP (3, It may be modified with 3'-dithiobispropionimide acid dimethyl dihydrochloride) or DSP (dithiobis (succinimidylpropionic acid)).
  • the thiol group to be crosslinked is a thiol group present in the separated protein or peptide. , May be.
  • the method for producing a molecule having an IgG Fc region to which a crosslinked Fc-binding peptide according to the eleventh aspect of the present invention is bound comprises a molecule having an IgG Fc region and the eighth or ninth aspect of the present invention. It comprises contacting with a protein, peptide according to a viewpoint or a complex according to a tenth aspect of the present invention.
  • a method for producing a molecule having an Fc region of IgG to which a crosslinked Fc-binding peptide according to the twelfth aspect of the present invention is bound is described.
  • the Fc-binding peptide bound to the molecule having the Fc region of IgG is reacted with the cross-linking agent according to the first aspect of the present invention to form the following groups of two thiol groups in the Fc-binding peptide.
  • A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • A is a C1 to 6 alkyl group, a phenyl group and a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • L represents a linker
  • Z is a reactive functional group.
  • the method for producing a molecule having an Fc region of IgG to which the molecule is bound is Cross-linked Fc-binding peptide represented by the following formula [In the formula, A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom.
  • the cross-linked Fc-binding peptide was reacted by reacting a molecule having an Fc region of IgG to which the Fc was bound with NH2 -L-Z (L represents a linker and Z represents a reactive functional group). Includes the introduction of reactive functional groups into the crosslinked moiety in.
  • the method for producing a molecule having an Fc region of IgG to which the molecule is bound is Fc-binding peptide to which the reactive functional group Z represented by the following formula is bound [In the formula, A is a C1 to 6 alkyl group, a phenyl group and a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom, L represents a linker, and Z is a reactive functional group.
  • the method according to the eleventh to fourteenth aspects of the present invention may further include covalently binding the Fc-binding peptide to a molecule having an Fc region of IgG.
  • the molecule having the Fc region of IgG according to the fifteenth aspect of the present invention is bound to the protein or peptide according to the eighth and ninth aspects of the present invention, or the complex according to the tenth aspect of the present invention. There is.
  • the molecule according to the fifteenth aspect of the present invention may be a covalent bond between the Fc-binding peptide and the molecule having an Fc region of IgG.
  • the carrier according to the 16th aspect of the present invention is bound to the peptide according to the 8th or 9th aspect of the present invention.
  • the method for purifying a molecule having an Fc region of IgG according to the seventeenth aspect of the present invention is as follows. Bringing a liquid containing a molecule having an Fc region of IgG into contact with the carrier according to the sixteenth aspect of the present invention. This includes washing and removing the components that are not bound to the carrier, and eluting the components that are bound to the carrier to recover the molecule having the Fc region of the IgG.
  • the method for producing a crosslinked protein or peptide according to the eighteenth aspect of the present invention comprises a crosslinked protein or peptide to which at least two thiol groups of the protein or peptide represented by the following formula are bonded. It ’s a manufacturing method, [In the formula, A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom, and Protein / Peptide represents a protein or peptide].
  • the protein or peptide is synthesized by the Fmoc method, and in the synthesis, instead of at least one cysteine residue, the following formula is used. Includes the use of compounds represented by.
  • the method for producing a protein or peptide according to the 19th aspect of the present invention is a method for producing a protein or peptide to which a reactive functional group represented by the following formula is bound.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • L is.
  • Linker, Z for reactive functional group, D for drug The protein or peptide is synthesized by the Fmoc method, and in the synthesis, instead of at least one cysteine residue, the following formula is used. Includes the use of compounds represented by.
  • the bonds between SHs in the amino acids obtained by the method of the present invention can form a crosslinked structure with high yield. Moreover, since it has excellent alkali resistance, the bond is not broken even when exposed to alkaline conditions. Therefore, it is possible to provide proteins and peptides having strong alkali resistance, and it can be used for proteins and peptides used or washed under alkaline conditions. Furthermore, the crosslinked structure maintains the structure of the original protein and peptide, and its function is not impaired, so that it can be used for stabilizing various peptides and proteins.
  • FIG. 1 It is a schematic diagram of an intramolecular SS bond.
  • A indicates SS bonds in natural proteins and peptides.
  • B indicates the cross-linking in the prior art.
  • C indicates the crosslinked bond of the present invention.
  • It is a graph which shows the result of the evaluation of DBC using the 1,1-dichloroacetone cross-linked peptide derivative immobilization column.
  • the vertical axis represents the absorbance at 280 nm, and the horizontal axis represents the elution time.
  • the dashed line indicates the 10% breakthrough point.
  • the solid line shows the result from 1 to 5 times of alkaline washing (the dark line shows the number of washings many times), and the dotted line shows the result from 10 times to 30 times of alkaline washing (the dark line shows the number of washings many times).
  • LC-MS liquid chromatography mass spectrometer
  • a and B are diagrams showing elution chromatograms of the oxytocin SS oxidation type before the cross-linking reaction and the reaction product after the cross-linking reaction, respectively. It is a figure which shows the result of the analysis by LC-MS which concerns on the cross-linking reaction of vasopressin before and after the cross-linking reaction in Example 12.
  • a and B are diagrams showing elution chromatograms of the vasopressin SS oxidized form before the cross-linking reaction and the reaction product after the cross-linking reaction, respectively.
  • Cross-linking agent for protein or peptide cross-linking in one embodiment is represented by the following formula (I).
  • H represents a halogen atom
  • the two halogen atoms may be the same or different
  • A may be a hydrogen atom, a phenyl group or a C1-6 alkyl group substituted with a halogen atom. , Or a phenyl group
  • alkyl group means a linear, branched, or cyclic saturated hydrocarbon group.
  • C represents the number of carbon atoms
  • the C1 to 6 alkyl group means an alkyl group having 1 to 6 carbon atoms.
  • the alkyl group include methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec-butyl group, t-butyl group, isobutyl group, pentyl group, isopentyl group, 2,3. -Includes dimethylpropyl group, hexyl group, cyclohexyl group and the like.
  • the number of substituents of the C1 to 6 alkyl groups in the group A is 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 3 and 2 It can be one and / or one, for example, trifluoromethane can be raised.
  • 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, and more preferably a fluorine atom or a chlorine atom. ..
  • the two Hals are the same halogen atom, for example, both may be a fluorine atom, both may be a chlorine atom, or both may be a bromine atom.
  • cross-linking agent examples include 1,1-dichloroacetone, 1,1-dichloropinacolin, and 2,2-dichloroacetophenone.
  • the method for producing a crosslinked protein or peptide is a crosslinked protein or peptide to which at least two thiol groups of a single or separated protein or peptide represented by the following formula are linked.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different
  • the protein or peptide is reacted with the cross-linking agent according to the present embodiment, and the two thiol groups are the following groups.
  • A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • the method for producing a crosslinked protein can be expressed, for example, by the following reaction formula.
  • TCEP / HCl dissolved in a buffer solution such as PBS is added, and the mixture is stirred at room temperature for 30 minutes to 1 day to reduce the disulfide bond.
  • a cross-linking agent dissolved in acetonitrile can be added to a protein or peptide solution that has been cleaved to generate SH groups, and the mixture can be stirred at room temperature for 30 minutes to 1 day.
  • the reaction solution can be purified by HPLC or the like, if necessary.
  • the raw material protein or peptide may be protected with Fmoc, if necessary, in which case a base such as piperidine is added to deprotect the Fmoc protecting group of the protein or peptide after the reaction. do.
  • the reaction solution can be further purified by HPLC (C18 reverse phase column), if necessary. Whether or not a crosslinked protein or peptide is produced can be confirmed by, for example, confirming the molecular weight by LC-MS analysis.
  • the cross-linking agent according to the present embodiment may cross-link SH groups existing in the two separated proteins or peptides, and in this case, the two proteins or peptides are reacted with the cross-linking agent to bind them (A). ).
  • the two molecules may be the same type of molecule or different types of molecules.
  • two SH groups existing in a single molecule may be cross-linked, in which case one protein or peptide is reacted with a cross-linking agent to cross-link (C).
  • the cross-linking agent of the present specification cross-links two thiol groups.
  • the "thiol group” or “SH group” is usually derived from a cysteine residue constituting a protein or peptide, but may be an artificially introduced group such as by using an artificial amino acid.
  • the two thiol groups may or may not form a disulfide bond in their natural or pre-crosslinked state.
  • a step of reducing the disulfide bond to form a thiol group may be included before carrying out the above-mentioned cross-linking method (. B and D).
  • the reduction of disulfide bonds is commercially available, for example, tris (2-carboethoxy) phosphine (TCEP), its hydrochlorides, dithioethanol (DTT), 2-mercaptoethanol, and cysteine hydrochloride (Cys-HCl). This can be done using a reducing agent for protein disulfide.
  • the two thiol groups may be thiol groups existing in a cysteine residue contained in a single protein or peptide, or a separated protein or peptide (protein-to-protein, peptide-to-protein, combination of protein and peptide). Included) may be a thiol group.
  • a thiol group When cross-linking a thiol group that forms a disulfide bond within a single protein or peptide, it is desirable that the cross-linked structure give the same three-dimensional structure as the disulfide bond.
  • Each protein or peptide to be crosslinked has at least two SH groups in the case of intramolecular cross-linking and at least one SH group in the case of intermolecular cross-linking.
  • Protein or peptide For example, one or more intramolecular or intermolecular (eg, 1-10, 2-8, 4-6, 1, 2, 3, 4, 5, or 6). ) May have a disulfide bond.
  • 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.
  • such proteins include enzymes, glycoproteins (such as erythropoetin), cytokines, toxins, adjuvants, structural proteins, antibodies, Fc fusion proteins, antibody fragments (F (ab') 2 , Fab', Fab, Fab 3 , Single Chain Fv (scFv), (Tandem) Bispecific Single Chain Fv (sc (Fv) 2 ), Single Chain Triple Body, Nanobody, Diverent VHH, Pentavalent VHH, Minibody, (Double Chain) Diabody , Tandem Diabody, Bispecific Tribody, Bispecific Bibody, Dual Affinity Retargeting Mole (DART), Triabody (or Tribody), Tetrabody (or [sc (Fv) 2 ] 2 ), or (scFv-SA) ) 4 ) Disulfide-bonded Fv, compact IgG, heavy chain antibody, or a polymer thereof, etc.) may be used.
  • glycoproteins such as erythropoetin
  • the peptide may be a peptide having 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 such peptides include vaccines, microantibodies, antibacterial peptides and the like.
  • proteins and peptides containing the Fc region of an antibody are referred to as "antibodies and the like".
  • the above-mentioned cross-linking method may form a single or the same cross-linking structure, or may form a plurality of cross-linking structures.
  • cross-linking between a plurality of proteins or peptides the same or different three or more proteins and / or peptides may be cross-linked.
  • the intramolecular crosslink and the intermolecular crosslink may be combined and crosslinked.
  • the intramolecular crosslink in the Fc-binding peptide described later and the intermolecular crosslink between the peptide and the Fc region of the antibody may be used. Two types of cross-linking may be performed.
  • the cross-linking method is a method for producing a cross-linked protein or peptide in which at least two thiol groups of the protein or peptide represented by the following formula are bonded.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide.
  • the following formula is used to synthesize a protein or peptide by the Fmoc method, and in the synthesis, instead of at least one cystein residue.
  • a peptide containing two Cys to be crosslinked is synthesized from the C-terminal by the Fmoc method on a carrier such as a peptide synthesis resin (resin).
  • the first Cys binding is performed using normal Cys (SH group may be protected if necessary), and the amino acid immediately preceding the remaining Cys is synthesized by the normal Fmoc method. do.
  • the method for producing a protein or peptide in one embodiment is a method for producing a protein or peptide to which a reactive functional group represented by the following formula is bound.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group.
  • the method for producing a protein or peptide to which a reactive functional group is bound can be carried out using an already crosslinked substance.
  • another embodiment is a method of producing a protein or peptide to which a reactive functional group is attached.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group.
  • the method for producing a protein or peptide in another embodiment is a method for producing a protein or peptide to which a reactive functional group represented by the following formula is bound.
  • A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • L is.
  • Linker, Z for reactive functional group, D for drug The above protein or peptide is synthesized by the Fmoc method, and in the synthesis, the following formula is used instead of at least one cystein residue. Includes the use of compounds represented by. This method can be carried out 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.
  • the reactive functional group is obtained by reacting a protein or peptide crosslinked with the cross-linking agent according to the present embodiment with an amino compound ( NH2 -LZ) having the reactive functional group. It can be introduced by forming an oxime (oxym method).
  • the amino compound having a reactive functional group to be used can be NH 2 -X-L'-Z (where X is O, NH, or N (C1-4 alkyl)).
  • Linker As used herein, L and L'represent a linker. As used herein, the term "linker” is particularly limited as long as it has a length at which a reactive functional group that binds to the linker or a protein or peptide cross-linked with a drug can be bound and has a structure that does not affect the cross-linking reaction. It is not a thing and may not exist (may show a bond). As an example, the linker may contain one or more alkylenes, alkenylene, and alkynylene. The alkylene, alkenylene, and alkynylene may be linear, 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 number of carbon atoms in alkenylene and alkynylene. Can be 2 to 20, 2 to 10, 2 to 6, 2 to 4, 2 to 3, or two.
  • the linker may be 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 may be included.
  • the linker may contain one or more amino acid moieties, eg 1-100, 1-50, 1-25, 1-20, 1-15, 1-10, 1-5, 1-. It may contain 4, 1 to 3, 1 to 2, 2, or one amino acid moiety.
  • the hydrogen atom bonded to the carbon atom may be substituted with a halogen atom.
  • Linkers are preferably specific in vivo, such as those that bind to molecules with Fc regions (eg, antibody-drug complexes (ADCs)) and are intended to release the drug locally in the target.
  • Conditions preferably, include a portion that can be cut according to the conditions characteristic of the local area. Such conditions include, for example, the presence of an enzyme, pH, and the like.
  • the linker may comprise a peptide bond that is cleaved by a protease or esterase (see, eg, Marcin Poreva, The FEBS Journal (2020) 287: 1936-1969, such as GGFG cleaved by a lysosomal enzyme) or an ester. ..
  • the linker may contain an amino acid moiety other than the peptide bond cleaved by the protease.
  • alkylene represents a divalent group obtained by removing one hydrogen atom from the alkyl group.
  • alkylene group examples include C1 to 20 linear or branched alkylene and 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 may be substituted with a hydroxyl group, a halogen atom, an amino group, or the like, if necessary.
  • the number of substituents of the alkylene is 1 to 10, 1 to 8, and 1 to 6. It can be 1 to 5, 1 to 4, 1 to 3, 3, 2, and / or 1.
  • Alkenylene is a divalent product obtained by removing two hydrogen atoms from any carbon atom of a linear, branched, or cyclic unsaturated hydrocarbon having one or more carbon-carbon double bonds. Means the basis of.
  • alkenylene include C2 to 20 alkenylene, and examples thereof include vinylene, propenylene, isopropenilen, butenylene, pentenylene, hexenylene, heptenylene, octenilen, nonenylene, and desenylene.
  • Alkinylene means a divalent group obtained by removing two hydrogen atoms from any carbon atom of a linear or branched unsaturated hydrocarbon having one or more carbon-carbon triple double bonds.
  • alkynylene examples include C2-20 alkynylene, and examples thereof include ethynylene, propynylene, butynylene, pentinylene, hexynylene, and phenylethynylene.
  • Heteroallylene and “heterocyclene” are 5-14 members, each containing at least one aromatic and non-aromatic heteroatom of one or more selected from a nitrogen atom, an oxygen atom, and a sulfur atom. It means a monocyclic or fused heterocyclic group of valence.
  • 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- to 6-membered ring.
  • the fused heterocyclic group is preferably an 8- to 10-membered ring.
  • heteroarylene and heterocyclene examples include piperidylene, piperazylene, morpholilen, quinuclidilene, pyrrolidylene, azetidylene, oxetylene, aziridinylene, tropanilen, furylene, tetrahydrofrilen, thienylene, pyrrolylene, pyrrolinylene, pyrrolidinylene, dioxolarene, Ren, thiazolilen, thiazolinylene, isothiazolylen, imidazolylene, imidazolinylene, imidazolidilenene, oxazolidinylene, thiazolidinylene, pyrazolylene, pyrazolinylene, pyrazolidinylene, oxadiazolilen, pyrazizolinylene, oxadiazolilen, pyrazizolinylene , Pyrazineylene, piperaziniren, diox
  • a group represented by a “reactive functional group” or “Z” can react and bind to a peptide, protein, nucleic acid, small molecule drug or the like under relatively mild conditions.
  • Reactive functional groups include maleimide, thiol or protected thiol, alcohols, acrylates, acrylamides, amines or protected amines, carboxylic acids or protected carboxylic acids, azides, alkins containing cycloalkynes, cyclopentadiene and furans 1,3-.
  • alpha-halocarbonyl N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, nitrophenyl ester, carbonate, dibenzocyclooctine (DBCO), tetrazine, methyltetrazine (MTZ), transcyclooctene ( TCO), azide, carboxy, tosyl, amino, epoxy, acyl, isothiocyanate, isocyanate, acyl azide, NHS ester, acidified, aldehyde, glyoxal, epoxide, oxylane, carbonate, arylating agent, imide ester, carbodiimide, acid anhydride Substances, haloacetyls, alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, diazoalkanes, diazoacetyl compounds, carbonyls, ketones, carbodiimides, e
  • N-Hydroxysuccinimidyl chlorogitate alkyl halides, isocyanates, hydrazines, Schiff bases, reductive amination products, Mannich condensation products, diazonium derivatives, Mannich condensation products, iodization reaction products, aryl azide , Halylated aryl azide, benzophonone, diazo compound, diazirin derivative and the like.
  • the reactive functional group may be a group that brings about 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 as a bond form with a drug.
  • N-succinimidyl ester or N-sulfosuccinimidyl ester is suitable for the reaction with the primary amine
  • p-nitrophenyl ester and dinitrophenyl ester are suitable for the reaction with the amino group.
  • pentafluorophenyl ester there is a pentafluorophenyl ester, and those suitable for the reaction with the mercapto group include a maleimide group, a carboxylated product, a pyridyldithio group, a nitropyridyldithio group, a haloalkyl group, and a haloacetyl group, which are suitable for the reaction with a hydroxy group. Suitable ones are isocynates (Greg t. Hermanson, Bioconjugate Technologies Second Edition, p234-345).
  • the reactive functional group is a reaction in which an oxime bond is formed by a reaction with an alkoxyamine, and a Cu (I) -catalyzed reaction with an alkyne or azide to form a 1,3-dipolar cyclization of Husgen.
  • a reaction in which an oxime bond is formed by a reaction with an alkoxyamine, and a Cu (I) -catalyzed reaction with an alkyne or azide to form a 1,3-dipolar cyclization of Husgen.
  • lick addition reactions
  • Diel's-Alder reactions reverse electron-required Diel's-Alder reactions
  • Michael reactions metasessis reactions
  • transition metal-catalyzed cross-coupling reactions radical polymerization reactions, oxidation coupling reactions, acyl transition reactions or photoclick reactions.
  • It also contains a reactive functional group involved (that reaction is possible) (Kim CH et al, Curr Opin Chem Biol. 2013 Jun; 17 (3):
  • the introduction of a reactive functional group is carried out by introducing the above-mentioned protein or the above-mentioned peptide or a fusion thereof bound to each other via a cross-linking agent and a hydroxyamine derivative in a buffer solution such as PBS (pH 7.4) from 4 ° C. It can be carried out by introducing a reactive functional group such as an alkyne group by reacting in any environment at room temperature for 30 minutes to overnight.
  • the cross-linking agent according to the present embodiment can be used to introduce a drug (payload) into a protein or peptide or a fusion thereof. Specifically, by reacting and binding the reactive functional group of the protein or peptide into which the above-mentioned reactive functional group has been introduced or the reactive functional group thereof and the drug (loadage), the drug (the drug or the peptide thereof or the fusion thereof) Peptides) can be introduced.
  • the 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 represented by the following formula and a drug.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents the linker and D represents the drug.
  • the method for producing a complex of the above protein or peptide and a drug can be produced using a protein and / or peptide in which a reactive functional group has already been introduced. Therefore, another 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 represented by the following formula and a drug.
  • A is a C1-6 alkyl group, a phenyl group, or a C1-6 alkyl group that may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • Protein / Peptide represents a protein or peptide
  • Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents the linker and D represents the drug
  • a protein or peptide to which a reactive functional group represented by the following formula is bound [In the formula, A is a C1-6 alkyl group, a phenyl group, a C1-6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom, and Protein / Peptide represents a protein or peptide, and Protein / Peptide A and Protein / Peptide B may be the same or different, where L represents a linker and Z represents a reactive functional group]. And, by reacting the reactive functional group with a drug having a reactive functional group, the agent is bound to the crosslinked portion of the crosslinked protein or peptide.
  • the “drug” is not particularly limited as long as it is a drug introduced into a protein or peptide and used, but for example, a therapeutic agent, a preventive agent, a targeting agent, a labeling agent, or a diagnostic agent.
  • therapeutic or prophylactic agents include monomethyloristatin, auristatin, meitansine, emtansine, doxorubicin, bleomycin, ozogamycin, vedotin, pastodox, deruxtecan, maytansinol calikea sewing machine, exatecan, pyrobenzodiazepine dimer, duocarmycin.
  • Anti-cancer agents such as mycin, elibrin, SN-38, PNU-159682, emtansine (DM1), mertansine or derivatives thereof; radioisotopes such as 90Y ; bind to receptors on the blood-brain barrier to the central nervous system Drugs that enable migration; Targeting agents such as drugs that bind to cancer cells and enable the transfer of antibodies into cells; Radiolabels, enzymes, fluorescent labels, bioluminescent labels, and chemical luminescent labels. Detectable signs such as metal can be mentioned.
  • a protein or peptide in which a maleimide group has been introduced as a reactive functional group or a fusion thereof by an oxime reaction in advance, and a product in which an azido group has been introduced into the SH group of mertansine, which is an anticancer agent, as a drug are PBS (pH 7.4).
  • the drug can be introduced by reacting in a buffer solution such as 4 ° C. to room temperature for 30 minutes to overnight.
  • a protein and / or peptide crosslinked with the above cross-linking agent is provided.
  • a fusion protein or fusion peptide having the following structure is provided in which two thiol groups present in the separated protein and / or peptide are bonded to each other via the cross-linking agent.
  • Protein / Peptide A and Protein / Peptide B may be different substances from each other or may be the same substance.
  • a protein or peptide having the following structure in which two thiol groups existing in a single protein or peptide are bonded to each other via the cross-linking agent of the present invention.
  • the cross-linked protein and peptide may be a protein or peptide to which a reactive functional group is bound via a cross-linking agent, or a protein or peptide to which a drug is bound via a cross-linking agent, and is a molecule represented by the following. including.
  • L represents a linker
  • Z represents a reactive functional group
  • D represents a drug
  • the linker can bind to a plurality of functional groups Z or drug D by having a branched structure.
  • the number of functional groups Z or drug D bonded to the cross-linking agent according to the present embodiment is 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, and 1 to 5. It can be 1 to 4, 1 to 3, 1 to 2, or 1.
  • the structure can be as follows (L1 and L2 represent a linker; the linker is the same as the above definition).
  • the cross-linking agent according to the present embodiment can be used for the purpose of enhancing the binding stability of a desired protein, peptide or a complex thereof.
  • the crosslinked protein or peptide may have a group A as a hydrogen atom and contains a molecule represented by the following.
  • cross-linked molecules are molecules with improved alkali resistance and stabilization compared to disulfide bonds.
  • the number of bonds via the above-mentioned cross-linking agent may be one or two or more. If more than one crosslink is present, it may be a crosslink within a single protein or peptide, a crosslink within a separated protein and / or peptide, or a crosslink within a single protein or peptide. May be combined with cross-linking within the separated protein and / or peptide.
  • crosslinks there are two or more crosslinks within the separated protein or peptide, the same two proteins and / or peptides may be crosslinked, or multiple proteins and / or peptides may be crosslinked to three or more proteins. And / or the peptide may be bound by cross-linking.
  • the peptide cross-linked herein may be an Fc-binding peptide. Therefore, in another embodiment, the Fc-binding peptide cross-linked with the above-mentioned cross-linking agent or a method for producing the same, which is represented by the following formula (in the formula, A, L, Z, and D are the same as the above definitions). Is provided.
  • the term "Fc-binding peptide” means a peptide that specifically binds to the Fc region of IgG.
  • the "Fc region of IgG” typically means a C-terminal fragment obtained as a papain-treated product of the proteolytic enzyme papain of IgG.
  • the Fc-binding peptide is preferably a site selected from Lys248, Lys246, Lys338, Lys288, Lys290, Lys360, Lys414, and Lys439 according to Eu numbering in Fc and / or a region close thereto, preferably Lys248 and / or its proximity.
  • the Fc-binding peptide may be a partial peptide of protein A having Fc-binding ability or a variant thereof.
  • Specific examples of such peptides are International Publication No. 2008/0504030, International Publication No. 2013/027796, International Publication No. 2016/186206, International Publication No. 2018/230257, and Kyohei Muguruma et al., ACS Omega ( 2019); 4 (11): 14390-14397. And they can be appropriately prepared according to the methods described in their respective literatures.
  • two cysteine residues preferably the cysteine closest to the C-terminal and the cysteine closest to the N-terminal, are cross-linked by the cross-linking agent herein.
  • IgG refers to mammals such as primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, domestic animals such as pigs, cows, horses, sheep, and goats, and dogs. And IgG of pet animals such as cats, preferably human IgG (IgG1, IgG2, IgG3 or IgG4).
  • the IgG in the present specification is preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.
  • the Fc-binding peptide may be a peptide selected from the following (i) to (iv): (I) Peptide represented by the following formula (I): NH 2- (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, where 1 to 3 X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , and 1 to 3 X 8 are.
  • Each X 1 , X 2 , X 3 and each X 8 represent any amino acid residue other than C that is independent of each other and is the same or different.
  • X 4 is H, R, S, or D.
  • X 5 is K, C, D, E, R, V, F, L, 2-aminosuberic acid, Dpr, Orn, AcOrn, AcDab, Dab, Nle, Nva, Tle, Ala (t-Bu), and Cha.
  • One amino acid residue selected from X 6 is E, N, R, or D, X 7 is I or V];
  • X 11 and X 12 are independently selected from the group consisting of R, H, D, E, S, T, N, Q, Y, and C];
  • Each X 1 , X 2 , X 3 and each X 8 represent any amino acid residue other than C that is independent of each other and is the same or different.
  • X 4 is H, R, S, or D.
  • X 5 is K, C, D, E, R, V, F, L, 2-aminosuberic acid, Dpr, Orn, AcOrn, AcDab, Dab, Nle, Nva, Tle, Ala (t-Bu), and Cha.
  • One amino acid residue selected from X 6 is E, N, R, or D, X 7 is I or V] (Iv)
  • X 9 1-2 NMQCQX 14 RFYEALHDPNLNEEQRNAX 11 IX 12 SIRDDC- (Linker2) -NH 2 (SEQ ID NO: 3) ...
  • X 11 and X 12 are independently selected from the group consisting of R, H, D, E, S, T, N, Q, Y, C, and K (Z).
  • X 14 is R, C, K (Z), Z is a reactive functional group].
  • X m (m is an integer) represents an amino acid.
  • X m n means that n amino acids X m are bound, and "X m " in which n is not described means that one amino acid X m is present.
  • n 2 or more, the plurality of X m may be independently the same or different amino acids.
  • pq it means that p to q amino acids X m are present.
  • 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
  • V is a valine residue.
  • Hcy is a homocysteine
  • Dpr is a diaminopropionic acid
  • Orn is an ornithine residue
  • ⁇ Ala is a ⁇ -alanine residue
  • Dab is a 2,4-diaminobutyric acid residue
  • Nle is a norleucine. It is a residue
  • Nva is a norvaline residue
  • Tle is a tert-leucine residue
  • Ala (t-Bu) is a tert-butylalanine residue
  • Cha is a cyclohexylalanine residue.
  • amino group in the residue having an amino group in the side chain may be acetylated, if necessary.
  • Acetylated forms of natural and artificial amino acids are sometimes described herein with the Ac prefix in the amino acid designations described above, unless in particular it is inconsistent to interpret them as such. It is understood that it may include an acetylated form even if it is not described as Ac.
  • K (Z) means a functional group-bound lysine residue, preferably K (Z) is K (Azide).
  • K (Azide) represents an azide-bound lysine residue.
  • the Fc-binding peptide may be a carrier-binding peptide whose purpose is to bind the antibody to the carrier by binding to the carrier, or to bind the drug to the antibody via the peptide. It may be a peptide for drug binding for the purpose of.
  • the carrier-binding peptide the above-mentioned peptide (i) or (ii) is preferable.
  • the N-terminal amino group may be acetylated (in this case, the Lys residue is introduced at an appropriate position near the N-terminal in the Linker on the N-terminal side).
  • X 1 1-3 is (S, G, F, or none)-(D, G, A, S, P, Hcy, or none)-(S, D, T, N, E, or none). It is an amino acid sequence represented by R).
  • X 1 1-3 are D, GPD, R, GPR, SPD, GDD, GPS, SDD, RGN, G-Hcy-D, RGP, or GPD.
  • X 1 1-3 is D or GPD.
  • X 2 is A, S, or T.
  • X 2 is A or T.
  • X 2 is A.
  • X 3 is Y or W.
  • X 3 is Y.
  • X 4 is H.
  • X 5 is 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 one amino acid residue selected from Cha.
  • X 5 is K, R, AcOrn.
  • X 5 is one amino acid residue selected from V, Dab, F, R, L, Nva, Nle, Ala (t-Bu), and Cha.
  • X 5 is one amino acid residue selected from F, R, L, Nva, Nle, Ala (t-Bu), and Cha.
  • X 5 is one amino acid residue selected from L, Ala (t-Bu), and Cha.
  • X 6 is E or N.
  • X 6 is E.
  • X 7 is V.
  • 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)-(Y, F, H, M, or none). None).
  • X 8 1-3 is T, TFH, S, SFH, THH, TFY, TYH, or T-Hcy-H.
  • X 8 1-3 is T or TFH.
  • the peptide represented by the formula (I) may be any one or a combination of two or more of the above conditions, and may be, for example, a peptide satisfying the conditions described below: [ 8] and [9]; [8] and [17]; [9] and [17]; [8] and [9] and [17]; or any one of these and [10] to [14]. It is a combination of.
  • X5 is the same as described above, may have an NH 2- (Linker) -group at the N-terminus, and -NH at the C-terminus. It may have two or NH 2- (Linker) -groups) :.
  • a peptide having the following structure can be used as a carrier bond.
  • Preferred examples of the peptide having the amino acid sequence represented by the formula (II) include the following peptides.
  • X 9 is selected from the group consisting of GF, F, and K.
  • X 11 and X 12 are independently selected from the group consisting of R, H, and E, respectively.
  • X 11 and X 12 are R.
  • the following peptides can be mentioned as the peptide having the amino acid sequence represented by the above formula (II): 60) FNMQCQRRFYEARHDPNLEQRNARIRSIRDDDC (SEQ ID NO: 63), 61) GFNMQCQRRFYEARHDPNLEQRNARIRSIRDDDC (SEQ ID NO: 64), 62) KNMQCQRRFYEARHDPNLEQRNARIRSIRDDDC (SEQ ID NO: 65), 63) GFNMQCQKRFYEARHDPNLEQRNARIRSIRDDDC (SEQ ID NO: 66), 64) KNMQCQKRFYEALHDPNLEQRNARIRSIRDDDC (SEQ ID NO: 67), Or 66) GKNMQCQRRFYEARHDPNLEQRNARIRSIRDDDC (SEQ ID NO: 68).
  • a peptide having the following structure can be used as a carrier-binding peptide.
  • the peptide for carrier binding has at least one amino group (-NH 2 ) for covalent binding with the carrier.
  • an amino group is preferably an N-terminal amino group, but as long as it can be bonded to a carrier, a lysine residue (eg, located in a linker) near the N-terminal or C-terminal, cysteine. It may be a side chain amino group of a residue, an aspartic acid residue, a glutamic acid residue, 2-aminosveric acid, Dpr, and an arginine residue.
  • the peptide represented by the above formula (I') may have a functional group at the C-terminal instead of having a functional group at the N-terminal. That is, the peptide represented by the above formula (I') 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 the 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 )-(Linker 3)] is attached to any part of the structure, and (Linker 3) represents a linker, 1-3.
  • the X1, X2, X3, X4 , X5 , X6 , X7 , and 1-3 X8s respectively, independently of each other, show the same or different amino acid residues.
  • Each X 1 , X 2 , X 3 and each X 8 represent any amino acid residue other than C that is independent of each other and is the same or different.
  • X4 is H, R, S, or T
  • X 5 is K, C, D, E, R, V, F, L, 2-aminosuberic acid, Dpr, Orn, AcOrn, AcDab, Dab, Nle, Nva, Tle, Ala (t-Bu), and Cha.
  • One amino acid residue selected from X 6 is E, N, R, or D
  • X 7 is I or V.
  • Linker 3 is RRRGS, EEGGS or (PEG) 1-8 (preferably (PEG) 4 ) or is absent.
  • Examples of the peptide having the amino acid sequences represented by the formulas (I') and (I'') include Z- ( Linker3)-(X1 1-3 ) -C- (X2)- ( X3). -(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 )-(Linker 3) -Z You may.
  • the preferred amino acid sequence is the same as the preferred amino acid sequence in the peptide represented by the above formula (I).
  • the following peptides can be mentioned as the peptide for drug binding.
  • Linker2 may be SGSGSK, SRRCR, SRRK (Z) R, SRRCRRCRC, SRRK (Z) RRK (Z) RRK (Z), or (PEG) 1-8 -Lys (preferably (PEG) 1-8-Lys. ) 4 -Lys) or does not exist.
  • the Cys residue (C) contained in the linker may be bound to another functional molecule via a maleimide group, if necessary.
  • Preferred examples of the peptide having the amino acid sequence represented by the formula (II') include the following peptides.
  • [A] From the group consisting of GF, AF, ⁇ AlaF , NH2- ( PEG) n -CO (n 1-50) -F, F, K, Orn, C, Dpr, and Acetyl-K. Be selected.
  • X 11 and X 12 are independently selected from the group consisting of R, H, and E, respectively.
  • [C] X 11 is R.
  • [D] X 12 is R or K (Z) (preferably Z is an azide).
  • peptide having the amino acid sequence represented by the above formula (II') include the peptides described in the above 60) to 66) (however, the lysine residue contained therein is necessary. Functional groups may be attached accordingly): FNMQQQCRFYEARHDPNLEQRNARICSIRDDP-SRRCRRCRRC-NH 2 Acetyl-KNMQQQCRFYEALHDPNLNEEQRNARICSIRDDP-SRRCRRCRRC-NH 2 GFNMQQQCRFYEARHDPNLEQRNARICSIRDDP-SRRCRRCRRC-NH 2 FNMQCQZRFYEARHDPNLEQRNARIRSIRDDC-NH 2 Acetyl-KNMQCQZRFYEALHDPNLNEEQLNERIRSIRDDC-NH 2 GFNMQCQK (Z) RFYEALHDPNLEQRNARIRSIRDDDC-SRRK (Z) R-NH 2 FNMQCQK (Z) RFYEALHDPNLEQRNARIRSIRDDDC-S
  • the drug-binding peptide herein has a reactive functional group (preferably an azide group) bonded to the N-terminal or C-terminal (preferably the N-terminal) via a linker, if necessary. May be good.
  • a reactive functional group eg, an azide group
  • a peptide having an azide group can link the other functional molecule to the peptide by click-reacting with another functional molecule having Divenzycyclooctyne (DBCO), alkyne, TCO.
  • DBCO Divenzycyclooctyne
  • the binding between the peptide and other functional substances can also be carried out by a method known to those skilled in the art, for example, a reaction between a maleimide group and a sulfhydryl group.
  • the peptide of the present specification may be bound to other functional molecules.
  • such other molecules can also be attached via the reactive functional groups described above (eg, to amino ends, etc.), and amino acids in the peptide (eg, lysine residues) can form reactive functional groups. If so, it can be attached to the reactive functional group (eg, the azido group that the lysine residue has as a substituent), or the Cys residue in the peptide (eg, the Cys residue in Linker 2). It can also be attached to a maleimide group via a maleimide group.
  • peptides include, but are not limited to, labeling substances or medical agents, including, but not limited to, peptides, proteins, nucleic acids, or small molecule drugs. Any molecule to which the antigen specificity and other properties of the Fc molecule can be applied can be attached as another molecule. Examples of such substances include anticancer agents, small molecule drugs, radioactive labels, fluorescent labels, nucleic acid drugs, gene therapy drugs, peptide drugs, antibodies such as IgA or VHH, and the like.
  • the drug binding peptide has at least one amino group (-NH 2 ) for covalent binding with the antibody.
  • an amino group may be an amino group at the amino end, a lysine residue, a cysteine residue, an aspartic acid residue, a glutamate residue, 2-aminosveric acid, a diaminopropionic acid, and an arginine residue. It may be a side chain amino group of.
  • the cross-linking method according to the present embodiment is a method for improving the alkali resistance of a protein or peptide having two or more SH groups or a fusion thereof. can do.
  • the cross-linking method according to the present embodiment may be, for example, a method for improving the alkali resistance of a disulfide bond, a method for improving the alkali resistance of a protein or peptide having a disulfide bond in the molecule, or a method for improving stability.
  • a method for improving the alkali resistance of a protein or peptide which comprises binding two thiol groups in the protein or peptide by the above method is provided.
  • the complex according to another embodiment is a complex of an Fc-binding peptide whose intramolecularly crosslinked with the above-mentioned cross-linking agent and a molecule having an Fc region of IgG.
  • the complex is a complex in which an Fc-binding peptide crosslinked with the above-mentioned cross-linking agent containing a drug is bound to a molecule having an Fc region of IgG; and a reactive functional group is added to a molecule having an Fc region of IgG.
  • the cross-linking agent according to the present embodiment may cross-link between the Fc-binding peptide and the molecule having the Fc region of IgG.
  • the Fc-binding peptide may be bound to the 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 crosslink may be further cross-linked in the molecule.
  • the reactive functional group / drug is bound to the Fc-binding peptide by the intramolecular cross-linking.
  • the Fc-binding peptide may be further crosslinked with a molecule having an Fc region such as an antibody.
  • the complex according to another embodiment is a complex of an Fc-binding peptide crosslinked by the above-mentioned cross-linking agent and a molecule having an Fc region of IgG.
  • the complex is a complex in which a molecule having an Fc region of IgG and an Fc-binding peptide are crosslinked with the above-mentioned cross-linking agent containing a drug; and a molecule having an Fc region of IgG and an Fc-binding peptide.
  • the term "molecule having an Fc region of IgG” means a peptide, protein, or other complex containing the Fc region of IgG, which means a wild-type or artificial IgG or a variant thereof, as well as Fc.
  • IgG Fc region represented by fusion protein and other substances (active ingredient, drug, protein, low molecular weight compound, medium molecular weight compound, high molecular weight compound, matrix, lipid, liposome, nanoparticles, vehicle for DDS, nucleic acid and / Or a fusion with a peptide), and a molecule consisting only of an Fc region.
  • the Fc molecule is an Fc fusion protein
  • the protein or peptide to be fused with Fc may be a receptor, cytokine, interleukin, blood coagulation factor VIII, CTLA4, human lactoferrin, TNF receptor, or LFA-3.
  • a part thereof (preferably a target binding portion) and the like can be mentioned.
  • the complex can be produced by contacting the Fc-binding peptide cross-linked with the above-mentioned cross-linking agent with a molecule having an Fc region of IgG.
  • a method for producing a molecule having an IgG Fc region fused with a drug comprises contacting the molecule having an IgG Fc region with the Fc-binding peptide crosslinked with the cross-linking agent.
  • the complex can be produced by binding the Fc-binding peptide to a molecule having an Fc region of IgG and then cross-linking the molecule intramolecularly and / or between the molecules with the above-mentioned cross-linking agent.
  • the method for producing a molecule having an Fc region of IgG is a method for producing a molecule having an Fc region of IgG to which a crosslinked Fc-binding peptide is bound.
  • the Fc-binding peptide bound to the molecule having the Fc region of IgG is reacted with the cross-linking agent to form the following groups of two thiol groups in the Fc-binding peptide.
  • A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • the method for producing a molecule having an Fc region of IgG is a Fc-binding peptide to which a reactive functional group Z represented by the following formula is bound.
  • A is a C1 to 6 alkyl group, a phenyl group and a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom
  • L represents a linker
  • Z is a reactive functional group.
  • Is a method for producing a molecule having an Fc region of IgG to which the molecule is bound.
  • Cross-linked Fc-binding peptide represented by the following formula [In the formula, A is a C1 to 6 alkyl group, a phenyl group, or a C1 to 6 alkyl group which may be substituted with a hydrogen atom, a phenyl group or a halogen atom].
  • the cross-linked Fc-binding peptide was reacted by reacting a molecule having an Fc region of IgG to which the Fc was bound with NH2 -L-Z (L represents a linker and Z represents a reactive functional group). Includes the introduction of reactive functional groups into the crosslinked moiety in.
  • the method for binding the Fc-binding peptide to an antibody or the like can be carried out with reference to International Publication No. 2013/027796, International Publication No. 2018/092867, International Publication No. 2020/07570, and the like.
  • the amino group may be optionally modified with a portion for covalently binding to the antibody, and when the peptide is bound to the antibody or the like, the amino group may be covalently bound to the antibody or the like at the portion.
  • Fc-binding peptides modified with such moieties may be referred to as "CCAP reagents”.
  • the "part for covalently binding to an antibody” is a chemical structure for covalently linking an Fc-binding peptide and a molecule having an Fc region of IgG, and is a desired amino acid (for example,).
  • Ricin residue, cysteine residue, aspartic acid residue, glutamate residue, 2-aminosveric acid, diaminopropionic acid, arginine residue, etc. can be combined with at least one site. ..
  • DSG dissin imidazole glutarate
  • DSS disssed imidazole svelate
  • DMA dimethyldihydrochloride acid
  • DMP pilimide
  • Acid dimethyl dihydrochloride DMS (suberimide acid dimethyl dihydrochloride), DTBP (3,3'-dithiobispropionimide acid dimethyl dihydrochloride), and DSP (dithiobis (succinimidylpropionic acid)). It can be, preferably DSG, DSS, or DSP.
  • a succinimidyl group such as DSS or DSG reacts with the side chain of the lysine residue and the primary amine present at the N-terminal of the polypeptide, so that the N-terminal of the Fc-binding peptide is blocked and then reacted with DSS or DSG.
  • DSS or DSG a succinimidyl group
  • the cross-linking between the Fc-binding peptide and IgG is, for example, Lys248 or Lys246 of the amino acid residues of the above-mentioned X5, X9 , X11, X12, X14 of the Fc-binding peptide and the Fc region of IgG, preferably. Can be site-specifically generated between Lys248.
  • the binding between the Fc-binding peptide, which is a CCAP reagent, and the antibody or the like is not particularly limited as long as it is carried out under conditions that cause a cross-linking reaction.
  • the Fc-binding peptide and the antibody or the like are placed in an appropriate buffer at room temperature.
  • the reaction can be carried out by mixing at (for example, about 15 ° C to 30 ° C).
  • the mixing step may be carried out by adding an appropriate amount of a catalyst that promotes the crosslinking reaction, if necessary.
  • the mixing ratio of the Fc-binding peptide and the antibody or the like in the mixing step is, for example, the molar ratio of the Fc-binding peptide: antibody or the like is 1: 1 to 20: 1, preferably 2: 1 to 20: 1 or 5. It can be 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, if desired.
  • Fc region such as IgG usually forms a pair of symmetrical heavy chain constant regions, there may be two sites to which the Fc-binding peptide binds. Therefore, one or two Fc-binding peptides can be attached to one molecule having an Fc region of IgG, preferably one.
  • the Fc-binding peptide can be used for purification of an antibody or the like by binding to a carrier such as a column. Since the cross-linking method of the present invention improves resistance to alkali, the carrier can be reused after being washed with alkali once or multiple times by using the Fc-binding peptide cross-linked with the cross-linking agent of the present invention. Is. Therefore, in one embodiment, a carrier to which an Fc-binding peptide intramolecularly crosslinked by the above-mentioned cross-linking agent is bound is provided.
  • the binding of the peptide to the carrier can be carried out, for example, by reacting the peptide with a carrier having a functional group capable of reacting with an amino group.
  • the reaction is carried out under conditions where the two are sufficiently bound, and can be carried out, for example, by contacting them in a buffer solution at room temperature for 1 to 5 hours (preferably 2.5 to 3.5 hours).
  • the carrier examples include shapes such as gel (for example, column gel), particles, beads, nanoparticles, fine particles, macrobeads, membranes, microplates, and arrays, and the materials thereof include magnetic substances, latexs, and agaroses. , Glass, cellulose, sepharose, nitrocellulose, polystyrene, and other polymeric materials.
  • the carrier is a column gel (column chromatography).
  • HiTrap NHS-activated HP GE Healthcare
  • the method for purifying an antibody or the like in one embodiment is to bring an antibody or the like-containing solution into contact with the carrier to bind the antibody or the like to the carrier, to wash and remove components that do not bind to the carrier, and to remove the components bound to the carrier. Includes elution and recovery.
  • the contact between the antibody-containing liquid and the carrier is performed under conditions where both can be sufficiently contacted.
  • the carrier is a column
  • it is carried out by injecting a solution containing an antibody or the like into the column.
  • the component that did not bind to the carrier can be removed by a conventional method, for example, by washing the carrier to which an antibody or the like is bound with a buffer solution (pH about 7.0). ..
  • the antibody or the like bound to the carrier can be recovered at pH 2.5 or higher, but it is desirable that the acidity is weak in order to prevent denaturation of the antibody or the like, preferably pH 3.6 or higher, for example, pH 3.6. It can be set to pH 4.3.
  • the antibody or the like can be recovered by contacting the carrier with the antibody or the like, then collecting the beads by centrifugation or the like and resuspending them in the eluate.
  • composition in another embodiment, a medical treatment containing, as an active ingredient, a peptide and / or a protein crosslinked by the above-mentioned cross-linking agent, or a molecule having an Fc region of IgG to which an Fc-binding peptide cross-linked by the cross-linking agent of the present invention is bound.
  • Compositions for use, in particular therapeutic, prophylactic, or diagnostic agents are provided.
  • a 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.
  • the cross-linked peptide and / or protein, or a drug thereof when the peptide and / or protein or the drug D bound via the cross-linking agent is a drug having a function as a therapeutic drug, a prophylactic drug, or a vaccine, the cross-linked peptide and / or protein, or a drug thereof.
  • the antibody or the like bound to the above can be used for therapeutic or prophylactic purposes.
  • the peptide and / or protein or the drug D bound via the cross-linking agent is a drug having a function as a label
  • the cross-linked peptide and / or protein, or the antibody to which the cross-linking agent is bound etc. It can be used for diagnosis or detection.
  • the drug is a therapeutic or prophylactic agent
  • the drug is a labeling substance.
  • the target disease of the medical composition can be appropriately set by selecting a peptide, protein, antibody or the like to be used or a binding agent, and for example, cancer, inflammatory disease, infectious disease, and neurodegenerative disease. Can be mentioned.
  • the medical composition can be used as an injection, and includes dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection.
  • dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection.
  • injections can be prepared according to known methods, for example, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily solution normally used for injections.
  • the prepared injection solution is usually filled in a suitable ampoule, vial or syringe.
  • a lyophilized preparation can be prepared and dissolved in water for injection, physiological saline or the like at the time of use to obtain an injection solution.
  • oral administration of proteins such as antibodies is difficult because they are decomposed by the digestive tract, but oral administration may be possible due to the ingenuity of antibody fragments, modified antibody fragments and dosage forms.
  • the orally administered preparation include capsules, tablets, syrups, granules and the like.
  • the medical composition is prepared in a dosage form of a dosage unit suitable for the dose of the active ingredient.
  • dosage unit dosage forms include injections (ampoules, vials, prefilled syringes), which usually contain 5 to 500 mg, 5 to 100 mg, 10 to 250 mg of active ingredient or drug per dosage unit dosage form. You may have.
  • the administration route of the medical composition may be local or systemic.
  • the administration method is not particularly limited, and is administered parenterally or orally as described above.
  • Examples of the parenteral route of administration include subcutaneous, intraperitoneal, blood (intravenous or intraarterial) or spinal fluid injection or infusion, and administration to blood is preferable.
  • the medical or diagnostic composition may be administered transiently, or may be administered continuously or intermittently. For example, administration can be continued for 1 minute to 2 weeks.
  • the administration regimen of the medical composition is not particularly limited as long as it is a dose and administration time at which a desired therapeutic effect or preventive effect can be obtained, and can be appropriately determined depending on symptoms, gender, age and the like.
  • a single dose of the active ingredient is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, for the above-mentioned diseases. It is convenient to administer by intravenous injection about 1 to 10 times a month, preferably about 1 to 5 times a month before and / or after the occurrence of clinical symptoms. In the case of other parenteral administration and oral administration, an equivalent amount can be administered.
  • Example 1 Preparation of 1,1-dichloroacetone crosslinked cyclic peptide
  • the raw material peptide [Sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1): IgGBP-longGS or IgGBP-LGS] is a solid phase peptide synthesis method (SEQ ID NO: 1). It was synthesized by consignment using the Fmoc method) (Eurofins).
  • Example 2 Preparation of 1,1-dichloropinacolin cross-linked cyclic peptide
  • the raw material peptide [Sequence: Fmoc-HN-GSGGS-GPDCAYHRGELVWCTFH (SEQ ID NO: 1)] is synthesized by consignment by the solid phase peptide synthesis method (Fmoc method). (Eurofins). 10 mg (3.9 ⁇ mol) of the above raw material peptide was dissolved in 1500 ⁇ L of DMF, and TCEP / HCl (2.24 mg, 7.8 ⁇ mol, 2 equal volume mol) previously dissolved in 2 ml of PBS (pH 7.4) was added thereto. The reaction was carried out at room temperature with stirring for 30 minutes.
  • 1,1-dichloropinacolin (1.32 mg, 7.8 ⁇ mol, 2 equal volumes) dissolved in 120 ⁇ L of acetonitrile was added, and the mixture was stirred at room temperature. After 1 hour, the completion of the reaction was confirmed by LC-MS analysis (manufactured by Shimadzu Corporation, LC-MS8030), and the reaction solution was purified by HPLC (C18 reverse phase column) to obtain an Fmoc cyclized peptide (7 mg, 2). .7 ⁇ mol) was obtained.
  • Example 4 Measurement of binding affinity of crosslinked cyclic peptide Affinity analysis was performed by the following method. First, 0.4M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.1M sulfo-N-hydroxysuccinimide mixed in equal amounts on a CM5 sensor chip set in BIAcoreT200 (GE healthcare). The sensor chip was activated by injecting the (sulfo-NHS) solution into the sensor chip at a flow rate of 10 ⁇ l / ml. Then, IgG was immobilized on the sensor chip under the condition of pH 5.5 (10 mM Na acetate).
  • pH 5.5 10 mM Na acetate
  • HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, 3 mM EDTA, pH 7.4) was used, and the flow rate was 50 ⁇ l / ml at 15.625, 31.2, 62.
  • the binding reaction was monitored by injecting 5, 125, 250, 500 nM peptides for 180 seconds.
  • the dissociation reaction only the buffer solution was injected for 600 seconds. Analysis of interaction parameters was performed using BIA evolution T100 software.
  • Figure 1 shows the IgG-binding peptide derivatives that were comparatively evaluated.
  • the results of evaluating the affinity of these peptides with human IgG1 are shown in Table 1.
  • the affinity of the original IgG-binding peptide having a disulfide bond was 8.2 nM in Kd value, but the affinity of the 1,3-dichloroacetone crosslinked cyclic peptide was 4.9 ⁇ M, which was about 500 times lower.
  • the Kd value of the 1,1-dichloroacetone crosslinked cyclic peptide was 45.6 nM, which was about 5 times lower than that of the original peptide
  • the Kd value of the 1,1-dichloropinacolin crosslinked cyclic peptide was 112 nM, which was the original value.
  • the Kd value was 6.4 nM, which was about the same as the original peptide.
  • Example 5 Preparation of peptide-immobilized column and purification of IgG 5 mL of 1 mM hydrochloric acid was sent to a 1 mL volume of NHS-activated prepack column, and the isopropanol solution in the column was removed. Next, the 10.0 mg / mL peptide solution (dissolved in 100 ⁇ L of DMSO) is diluted 10-fold with a coupling solution (20 mM carbonate buffer, 50 mM sodium chloride, pH 8.3), and 1 mL of the diluted solution is sent to room temperature. It was fixed for 4 hours. Then, unreacted NHS was blocked at room temperature for 1 hour with 5 mL of 1M Tris (pH 8.0).
  • the prepared IgG-binding peptide-immobilized column was connected to a BioLogic LP (Bio-Rad) liquid chromatography system and equilibrated with PBS.
  • a BioLogic LP Bio-Rad liquid chromatography system
  • 1 mg / mL human serum-derived IgG (manufactured by Sigma-Aldrich) dissolved in PBS was sent at a flow rate of 1 mL / min for 1 min.
  • the column was washed with PBS and an elution solution (100 mM glycine buffer, pH 2.8) was sent to elute IgG, which is an adsorbed component. Elution of IgG from the column was detected with an absorbance of 280 nm.
  • Example 6 Measurement of dynamic binding capacity (DBC) of peptide-immobilized column and evaluation of alkali resistance 1
  • a column having a peptide immobilization amount of 1 mg was prepared by the same method as described above. After equilibrating the prepared column with PBS, 1 mg / mL human serum-derived IgG (manufactured by Sigma-Aldrich) dissolved in PBS was sent at a flow rate of 1 mL / min (residence time 1 min). DBC shows what was determined from the amount of added protein at the time when 10% leakage of 280 nm absorbance of the added sample was observed.
  • DBC dynamic binding capacity
  • the initial DBC value of each peptide column is 17.24 mg / mL-column for the original peptide and 2.3 mg / mL-column for the 1,3-dichloroacetone cross-linked peptide, while 1,1-dichloro is this time. It was 12.8 mg / mL-column for the acetone cross-linked peptide and 16.70 mg / mL-column for the 1,1-dichloropinacolin cross-linked peptide. Therefore, it was clarified that the IgG adsorption performance was higher than that of the conventional crosslinked cyclic peptide.
  • Example 7 Measurement of dynamic binding capacity (DBC) of peptide-immobilized column and evaluation of alkali resistance 2 5 mL of 0.1 M sodium hydroxide solution was sent to the prepared 1 mg peptide-immobilized 1 mL column, and then the cells were washed with 5 mL PBS. This is regarded as one cycle, and after performing 1 to 30 times of NaOH aqueous solution washing / PBS washing treatment, DBC measurement is performed at a flow rate of 1 mL / min at a specified number of times (1, 2, 3, 4, 5, 10, 20, 30 times). Was done.
  • DBC dynamic binding capacity
  • Figure 3 shows the results of comparing the rate of change in the amount of antibody binding with a value of DBC 10%.
  • DBC decreased to 50% or less by 5 times of alkaline washing
  • 1,1-dichloroacetone cross-linked peptide 90% or more by 30 times of washing was 1
  • 1-Dichloropinacolin cross-linked peptide maintained 85% or more after 10 washes (Fig. 3). From this, it was found that the 1,1-dichloroacetone cross-linked peptide and the 1,1-dichloropinacolin cross-linked peptide clearly acquired high alkali resistance.
  • the IgG-bound peptide-immobilized column having the original disulfide bond was 17.24 mg / ml
  • the 1,1-dichloroacetone cross-linked peptide column was 12.8 mg / ml and 74. %, which was about 96% in the 1,1-dichloropinacolin crosslinked peptide column, which was about 16.70 mg / ml.
  • Triethylamine (0.017 mL) in 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 eq) under an argon stream. , 0.121 mmol, 1.1 eq) and stirred at room temperature for 10 minutes. After adding water to stop the reaction, the aqueous layer was extracted with dichloromethane, the organic layer was washed with saturated brine, and dried over sodium sulfate.
  • the obtained reaction product was analyzed by LC-MS as follows. After diluting the reaction 5-fold with 0.1% formic acid, 20 ⁇ L was concatenated with a Peptide BEH-C18 column (130 ⁇ , 1.7 ⁇ m, 2.1 ⁇ 100 mm, manufactured by Waters) in an Accuracy UPLC / SQ detector system (1 0 ⁇ , 1.7 ⁇ m, 2.1 ⁇ 100 mm, manufactured by Waters). Analyzed by Waters) (flow rate: 0.2 mL / min, elution: linear gradient from 4% CH 3 CN to 70% CH 3 CN containing 0.1% formic acid, column temperature: 25 ° C.).
  • FIGS. 5A and 5B The analysis results of oxytocin-OX and the reaction product by LC-MS before the addition of TCEP hydrochloride are shown in FIGS. 5A and 5B, respectively.
  • the measured value of the peak mass of oxytocin-OX was 1006.3, which was almost the same as the theoretical value of oxytocin-OX, 1007.19.
  • the peak in the reaction product of the 1.1-dichloroacetone cross-linking reaction was eluted later than the original peak and had a mass of 1060.4. Since this mass was almost the same as the theoretical value of the oxytocin dichloroacetone crosslinked type (oxytocin-DA) shown in the formula A1: 1063.26, it was judged that the crosslinked product having the desired structure was obtained.
  • oxytocin-DA oxytocin dichloroacetone crosslinked type
  • Example 12 Crosslinking of vasopressin 1.1 Using dichloroacetone, vasopressin (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH 2 (SEQ ID NO: 78), intramolecular SS bond , Molecular weight: 1084.24). 5.5 mg (5.07 ⁇ mol) of vasopressin SS oxidized (vasopressin-OX) acetate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) represented by the following formula B was added to 1.65 mL of 0.1 M phosphate buffer (pH 8.0).
  • the obtained reaction product was analyzed by LC-MS in the same manner as in Test Example 11.
  • the analysis results of vasopressin-OX and the reaction product by LC-MS before the addition of TCEP hydrochloride are shown in FIGS. 6A and 6B, respectively.
  • the measured mass of the peak of vasopressin-OX was 1083.7, which was in good agreement with the theoretical value of vasopressin-OX: 1084.24.
  • the peak in the reaction product of the 1.1-dichloroacetone cross-linking reaction was eluted with a delay from the original peak, and was a peak with a mass of 1139.0 and a mass of 1121.6.
  • vasopressin-DA vasopressin dichloroacetone crosslinked type
  • the mass of 1121.6 is 17.4 smaller than 1139.0, which means that the vasopressin-DA represented by the formula B1 undergoes a reduction reaction by TCEP and is a vasopressin dichloroacetone crosslinked reduced product represented by the following formula B2.
  • vasopressin-DA-R vasopressin-DA-R
  • vasopressin-DA-RDH theoretical mass value: 1124.3
  • Example 13 Acquisition of resistance to protease by cross-linking oxytocin
  • ⁇ -chymotrypsin derived from bovine pancreas, MP Biomedicals
  • oxytocin cross-linked with 2,2-dichloroacetophenone was used using oxytocin cross-linked with 2,2-dichloroacetophenone.
  • the resistance to protease degradation by (manufactured by) was evaluated.
  • Cross-linking of oxytocin with 2,2-dichloroacetophenone was carried out in the same manner by replacing 1.1-dichloroacetone in Example 11 with 2,2-dichloroacetophenone, and the target product was separated by reverse phase HPLC.
  • the obtained sample was applied to an InertStain C18 column (5 ⁇ m, 14 ⁇ 250 mm, manufactured by GL Science) connected to LC-Forte (manufactured by YMC) (flow rate 5 mL / min). Elution was performed with a 4% to 70% linear gradient containing 0.1% formic acid. After the target product was separated, acetonitrile was removed under negative pressure, and then freeze-dried.
  • oxytocin-OX and the oxytocin SH reduced form represented by the following formula A3 were used for evaluation. That is, the oxytocin solution dissolved in 0.1 M phosphate buffer (pH 7.0) at 0.5 mg / mL was designated as oxytocin-OX, and the 0.1 M phosphate buffer (pH 7.0) containing 0.5 mg / mL TCEP was used. ) was added with an oxytocin solution dissolved at 0.5 mg / mL, and the substance obtained after 30 minutes was designated as oxytocin-RD.
  • 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.
  • ⁇ -chimotrypsin was added and analyzed by reverse phase HPLC.
  • Figure 9 shows the elution chromatogram of the reverse phase HPLC of the sample prepared from oxytocin-DP.
  • two peaks A and B appeared.
  • the peak A was mass 1126.7 and the peak B was mass 1106.5.
  • These are the theoretical value of 1125.34 of the mass of the oxytocin dichloroacetone phenone cross-linked type (oxytocin-DP) represented by the following formula A4, and the oxytocin-DP-DP obtained by dehydrating the reduced product (oxytocin-DP-R, the following formula A5). It was almost the same as the theoretical value of 1109.33 of the mass of RDH (formula A6 below).
  • this sample was a mixture of oxytocin-DP and oxytocin-DP-RDH.
  • reaction of adding 1/10 amount of ⁇ -chymotrypsin by weight to this sample was followed, unlike the case of oxytocin-RD, as in the case of oxytocin-OX, two bottles were added after the addition of ⁇ -chymotrypsin. No decrease in the peak of was observed.
  • FIG. 10 shows the results of plotting the relative value (%) with respect to the peak area of the blank over time in order to see the amount of change in each of these molecular species after the addition of ⁇ -chymotrypsin.
  • oxytocin-RD the peak disappeared rapidly immediately after the addition of ⁇ -chymotrypsin, so it is considered that the resistance to protease is extremely low.
  • no degradation was observed, and since it has high protease resistance, it can be seen that cross-linking by SS bond greatly contributes to the resistance.
  • the dichloroacetonephenone cross-linked type (Oxytocin-DP and Oxytocin-DP-RDH) also showed high resistance to ⁇ -chymotrypsin digestion in the presence of the reducing agent TCEP. This indicates that this cross-linking method can greatly contribute to the stability of the peptide, especially the resistance (stability) to the protease.
  • the present invention is useful for cross-linking peptides and proteins.

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KR1020237018809A KR20230107276A (ko) 2020-11-09 2021-10-22 펩타이드 가교제 및 상기 가교제를 이용하여 가교된 가교 펩타이드
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008054030A1 (fr) 2006-11-02 2008-05-08 Kagoshima University Peptide liant l'igg
WO2013027796A1 (ja) 2011-08-24 2013-02-28 大塚化学株式会社 IgG結合性ペプチド及びそれによるIgGの検出および精製方法
JP2015027957A (ja) * 2013-07-30 2015-02-12 国立大学法人東京農工大学 ジスルフィド結合を有するペプチドの合成方法
WO2016186206A1 (ja) 2015-05-20 2016-11-24 国立大学法人鹿児島大学 ＩｇＧ結合ペプチドによる抗体の特異的修飾
WO2018092867A1 (ja) 2016-11-18 2018-05-24 国立大学法人鹿児島大学 ＩｇＧ結合ペプチドを含む固相担体及びＩｇＧの分離方法
WO2018230257A1 (ja) 2017-06-16 2018-12-20 国立大学法人鹿児島大学 ＩｇＧ結合ペプチド、及び該ペプチドによる抗体の特異的修飾
WO2020075670A1 (ja) 2018-10-10 2020-04-16 国立大学法人鹿児島大学 ＩｇＧ結合性ペプチドを含む固相担体及びＩｇＧの分離方法
JP2020063254A (ja) * 2019-11-22 2020-04-23 ハンジョウ ディーエーシー バイオテック シーオー．，エルティディ．Ｈａｎｇｚｈｏｕ Ｄａｃ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． 細胞結合分子の特異的共役体
WO2020101032A1 (ja) * 2018-11-16 2020-05-22 味の素株式会社 分子内ｓ－ｓ結合を有する環化ペプチドの製造方法
JP2020186833A (ja) 2019-05-10 2020-11-19 マックス株式会社 設備機器
JP2021082739A (ja) 2019-11-20 2021-05-27 株式会社ディスコ カセット収容機構及び加工装置

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3426906A1 (de) * 1984-07-20 1986-01-30 Bayer Ag, 5090 Leverkusen Verfahren und zwischenprodukte zur synthese diastereomerer verbindungen
ES2732574T3 (es) 2012-11-30 2019-11-25 Novartis Ag Métodos para preparar conjugados a partir de proteínas que contienen disulfuro

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008054030A1 (fr) 2006-11-02 2008-05-08 Kagoshima University Peptide liant l'igg
WO2013027796A1 (ja) 2011-08-24 2013-02-28 大塚化学株式会社 IgG結合性ペプチド及びそれによるIgGの検出および精製方法
JP2015027957A (ja) * 2013-07-30 2015-02-12 国立大学法人東京農工大学 ジスルフィド結合を有するペプチドの合成方法
WO2016186206A1 (ja) 2015-05-20 2016-11-24 国立大学法人鹿児島大学 ＩｇＧ結合ペプチドによる抗体の特異的修飾
WO2018092867A1 (ja) 2016-11-18 2018-05-24 国立大学法人鹿児島大学 ＩｇＧ結合ペプチドを含む固相担体及びＩｇＧの分離方法
WO2018230257A1 (ja) 2017-06-16 2018-12-20 国立大学法人鹿児島大学 ＩｇＧ結合ペプチド、及び該ペプチドによる抗体の特異的修飾
WO2020075670A1 (ja) 2018-10-10 2020-04-16 国立大学法人鹿児島大学 ＩｇＧ結合性ペプチドを含む固相担体及びＩｇＧの分離方法
WO2020101032A1 (ja) * 2018-11-16 2020-05-22 味の素株式会社 分子内ｓ－ｓ結合を有する環化ペプチドの製造方法
JP2020186833A (ja) 2019-05-10 2020-11-19 マックス株式会社 設備機器
JP2021082739A (ja) 2019-11-20 2021-05-27 株式会社ディスコ カセット収容機構及び加工装置
JP2020063254A (ja) * 2019-11-22 2020-04-23 ハンジョウ ディーエーシー バイオテック シーオー．，エルティディ．Ｈａｎｇｚｈｏｕ Ｄａｃ Ｂｉｏｔｅｃｈ Ｃｏ．，Ｌｔｄ． 細胞結合分子の特異的共役体

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
GALLUCCI, R. RGOING, R., JOURNAL OF ORGANIC CHEMISTRY, vol. 46, 1981, pages 2532 - 2538
GREG T. HERMANSON, BIOCONJUGATE TECHINIQUES SECOND EDITION, pages 234 - 345
KIM CH ET AL., CURR OPIN CHEM BIOL, vol. 17, no. 3, June 2013 (2013-06-01), pages 412 - 9
KYOHEI MUGURUMA ET AL., ACS OMEGA, vol. 4, no. 11, 2019, pages 14390 - 14397
MARCIN POREBA, THE FEBS JOURNAL, vol. 287, 2020, pages 1936 - 1969
See also references of EP4242220A4
SHAO-LIN ZHANGZHENG YANGXIAOHUI HUKIN YIP TAM, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 28, 15 November 2018 (2018-11-15), pages 3441 - 3445

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