WO2023277574A1 - Procédé de production d'un conjugué urate oxydase-albumine utilisant un lieur bcn et son utilisation - Google Patents

Procédé de production d'un conjugué urate oxydase-albumine utilisant un lieur bcn et son utilisation Download PDF

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WO2023277574A1
WO2023277574A1 PCT/KR2022/009319 KR2022009319W WO2023277574A1 WO 2023277574 A1 WO2023277574 A1 WO 2023277574A1 KR 2022009319 W KR2022009319 W KR 2022009319W WO 2023277574 A1 WO2023277574 A1 WO 2023277574A1
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tyrosine
albumin
phenylalanine
uric acid
acid oxidase
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PCT/KR2022/009319
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English (en)
Korean (ko)
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조정행
김현우
김형석
김선관
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주식회사 프로앱텍
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Priority to KR1020237045220A priority Critical patent/KR20240031241A/ko
Publication of WO2023277574A1 publication Critical patent/WO2023277574A1/fr

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    • 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
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/04—Drugs for disorders of the urinary system for urolithiasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase

Definitions

  • the present specification relates to a method for producing a uric acid oxidase-albumin conjugate in which a uric acid oxidase variant containing an unnatural amino acid is bound to albumin and a use thereof.
  • the present specification relates to a method for producing a uric acid oxidase-albumin conjugate comprising linking a urate oxidase variant and albumin using a linker containing a BCN group and a use thereof.
  • therapeutic proteins have achieved clinical success in the treatment of a variety of diseases, which continues to serve as an important growth driver for the pharmaceutical sector.
  • therapeutic proteins are easily destroyed by proteolytic enzymes in the body or have an extremely short half-life in blood, so they require repeated administration rather than a single administration, resulting in side effects such as immunogenicity. Therefore, one of the important considerations in the development of therapeutic proteins is to prolong the duration of drug efficacy in order to avoid repeated administration.
  • One of the efficient methods is to use human serum albumin to increase the durability of therapeutic protein.
  • a therapeutic protein having a multi-subunit and complex tertiary structure it is not easy to obtain the effect of albumin. This is because the production yield of the therapeutic protein is lowered or the activity is lowered due to albumin binding.
  • site-specific protein linkages are being studied. The binding at a specific site shows the possibility of producing a protein-albumin conjugate for treatment without a problem in reducing protein production yield or activity, and research on a method for producing the conjugate using the same is required.
  • the present inventors studied a method for prolonging the duration of drug efficacy by increasing the production yield of the uric acid oxidase-albumin complex and the number of albumin bound to uric acid oxidase. As a result, it was confirmed that the number of albumin bound to uric acid oxidase was increased in the uric acid oxidase-albumin complex by using site-specific albumin binding and specific click chemistry.
  • albumin can be site-specifically conjugated to a uric acid oxidase variant.
  • the production yield of the uric acid oxidase-albumin conjugate can be increased.
  • the production yield of a uric acid oxidase-albumin conjugate in which one or more albumins are conjugated to a uric acid oxidase variant can be increased.
  • One embodiment of the present application provides a method for producing a urate oxidase-albumin conjugate using a linker containing a BCN group.
  • One embodiment of the present application provides a uric acid oxidase-albumin conjugate produced according to the above production method.
  • One embodiment of the present application provides a pharmaceutical composition for preventing or treating gout comprising a uric acid oxidase-albumin conjugate produced according to the above production method.
  • One embodiment of the present application provides a food composition for preventing or improving gout containing a uric acid oxidase-albumin conjugate produced according to the above production method.
  • the method for producing the uric acid oxidase-albumin conjugate disclosed herein can improve the half-life of uric acid oxidase.
  • the method for producing the uric acid oxidase-albumin conjugate disclosed herein may increase the drug durability of uric acid oxidase.
  • the uric acid oxidase-albumin conjugates disclosed herein may have reduced immunogenicity.
  • the urate oxidase-albumin conjugate disclosed herein can produce a urate oxidase-albumin conjugate having an increased number of albumin bonds.
  • Figure 1 shows the growth curves of Uox-AzF producing strains with different agitation rates and internal pressures.
  • Figure 2 shows the culture results of the Uox-AzF producing strain.
  • FIG. 3 shows the results of SDS-PAGE analysis of Uox-AzF protein expression.
  • Figure 4 shows the purity of Uox-AzF and rHSA-linker conjugation reactants.
  • 6 and 7 are results of primary cation chromatography purification purity analysis of Uox-rHSA.
  • 10 is a purification result of a conjugate produced using a linker containing a DBCO group or a BCN group.
  • 11 is a result of SEC-HPLC analysis of a conjugate produced using a linker containing a DBCO group.
  • 13 is a result of albumin conjugation using a linker containing an APN or maleimide group.
  • the present application provides a method for preparing a uric acid oxidase-albumin conjugate.
  • a method for preparing a uric acid oxidase-albumin conjugate comprising:
  • the linker includes a first click chemical functional group at one end and a thiol reactive group at the other end, and the first click chemical functional group is a bicyclononyne group,
  • an albumin-linker conjugate is prepared,
  • the uric acid oxidase variant includes at least one non-natural amino acid
  • the non-natural amino acid includes a second click chemical functional group capable of performing a click chemical reaction with the first click chemical functional group
  • a uric acid oxidase-albumin conjugate is prepared through a reaction between the first click chemical functional group at one end of the albumin-linker conjugate and the second click chemical functional group included in the non-natural amino acid.
  • the second click chemical functional group may be an azide group.
  • the unnatural amino acid may be p-azido-L-phenylalanine.
  • the uric acid oxidase variant may be in the form of a tetramer, consisting of three uric acid oxidase variant subunits and one wild-type uric acid oxidase subunit, or four uric acid oxidase variant subunits. .
  • the uric acid oxidase variant subunit is 8th tyrosine, 16th tyrosine, 30th tyrosine, 46th tyrosine, 65th tyrosine, 79th phenylalanine of the amino acid sequence of SEQ ID NO: 1, 87th phenylalanine, 91st tyrosine, 106th tryptophan, 120th phenylalanine, 159th phenylalanine, 160th tryptophan, 162nd phenylalanine, 167th tyrosine, 174th tryptophan, 186th tryptophan, 188th tryptophan, 191st phenylalanine, 204th At least one amino acid selected from the group consisting of phenylalanine, 208th tryptophan, 219th phenylalanine, 233rd tyrosine, 251st tyrosine, 258th ty
  • the urate oxidase variant subunit is an amino acid sequence in which one or more amino acids selected from tryptophan at position 160 or tryptophan at position 174 of the amino acid sequence of SEQ ID NO: 1 is substituted with p-Azido-L-phenylalanine (AzF).
  • AzF p-Azido-L-phenylalanine
  • the uric acid oxidase variant subunit is tyrosine 10, tyrosine 163, phenylalanine 17, phenylalanine 45, tyrosine 59, tyrosine 77 of the amino acid sequence of SEQ ID NO: 2.
  • the urate oxidase variant subunit is at least one selected from tyrosine at position 163, phenylalanine at position 170, tyrosine at position 200, and tryptophan at position 271 of the amino acid sequence of SEQ ID NO: 2. It may include an amino acid sequence in which an amino acid is substituted with p-Azido-L-phenylalanine (AzF).
  • the linker may have the structure of Formula 1 below:
  • F 1 is a first reactive functional group including the first click chemical functional group
  • F 2 is a second reactive functional group including the thiol-reactive group, wherein the thiol-reactive group is a maleimide group or a 3-arylpropiolonitriles group;
  • L is substituted or unsubstituted C 1-50 alkylene, substituted or unsubstituted C 1-50 heteroalkylene, substituted or unsubstituted C 2-50 alkenylene, substituted or unsubstituted C 2-50 heteroalke Nylene, substituted or unsubstituted C 2-50 alkynylene, substituted or unsubstituted C 2-50 heteroalkynylene,
  • heteroalkylene, heteroalkenylene, and heteroalkynylene each independently include one or more heteroatoms, wherein the heteroatoms are each independently selected from O, S, and N;
  • the linker may have a structure of Formula 1-2:
  • np is an integer of 1 or more and 6 or less
  • L 1 is a bond, or an unsubstituted C 1-3 alkylene or an unsubstituted C 1-3 heteroalkylene;
  • L 3 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
  • the linker may have a structure of Formula 1-3 below:
  • np is an integer of 1 or more and 6 or less.
  • the albumin may be human serum albumin or a variant thereof.
  • the albumin may include any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
  • the albumin is human serum albumin or a variant thereof, wherein, in (a), the thiol group of cysteine of albumin reacting with the thiol-reactive group may be a thiol group of cysteine 34.
  • the uric acid oxidase-albumin conjugate can include:
  • subunit-albumin conjugate 1 subunit-albumin conjugate, and 3 uric acid oxidase variant subunits; two subunits-albumin conjugate, and two urate oxidase variant subunits; 3 subunits - albumin conjugate, and 1 uric acid oxidase variant subunit; or 4 subunit-albumin conjugates, wherein the subunit-albumin conjugate is one albumin conjugated to one urate oxidase variant subunit.
  • a urate oxidase-albumin conjugate having the structure of Formula 3-2, wherein one or more albumin is conjugated to a urate oxidase variant is provided:
  • n is an integer of 1 or more and 4 or less;
  • Uoxv is a uric acid oxidase variant, wherein the uric acid oxidase variant contains one or more non-natural amino acids, wherein the non-natural amino acids contain an azide group;
  • A is albumin
  • X 1 includes the following structure formed by the reaction of the azide group and the bicyclononyne group included in the non-natural amino acid,
  • X 2 includes any one of the following structures formed by reacting a thiol group of cysteine contained in albumin with a thiol-reactive functional group;
  • S is derived from the thiol group of the cysteine of the albumin
  • np is an integer of 1 or more and 6 or less
  • L 1 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene;
  • L 3 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene;
  • heteroalkylene each independently, includes one or more heteroatoms, wherein the heteroatoms are, each independently, O, S, or N.
  • the uric acid oxidase variant may be in the form of a tetramer consisting of four uric acid oxidase variant subunits.
  • the uric acid oxidase variant subunit is 8th tyrosine, 16th tyrosine, 30th tyrosine, 46th tyrosine, 65th tyrosine, 79th phenylalanine of the amino acid sequence of SEQ ID NO: 1, 87th phenylalanine, 91st tyrosine, 106th tryptophan, 120th phenylalanine, 159th phenylalanine, 160th tryptophan, 162nd phenylalanine, 167th tyrosine, 174th tryptophan, 186th tryptophan, 188th tryptophan, 191st phenylalanine, 204th At least one amino acid selected from the group consisting of phenylalanine, 208th tryptophan, 219th phenylalanine, 233rd tyrosine, 251st tyrosine, 258th ty
  • the uric acid oxidase variant subunit is tyrosine 10, tyrosine 163, phenylalanine 17, phenylalanine 45, tyrosine 59, tyrosine 77 of the amino acid sequence of SEQ ID NO: 2.
  • the albumin may be human serum albumin or a variant thereof.
  • the albumin may include any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
  • the albumin is human serum albumin or a variant thereof, and X 2 S may be derived from a thiol group of cysteine 34 of the albumin.
  • the uric acid oxidase-albumin conjugate can consist of any of the following:
  • a complex of one subunit-albumin conjugate and three urate oxidase variant subunits a complex of two subunit-albumin conjugates and two urate oxidase variant subunits
  • a complex of three subunit-albumin conjugates and one urate oxidase variant subunit a complex of four subunit-albumin conjugates, wherein the subunit-albumin conjugate is one albumin conjugated to one urate oxidase variant subunit.
  • TLS tumor lysis syndrome
  • hyperuricemia gout
  • deposition of urate crystals in joints due to deposition of urate crystals
  • a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of acute gouty arthritis, urolithiasis, nephrolithiasis and gouty nephropathy is provided.
  • chemical structures are disclosed with corresponding chemical names. In case of dispute, the meaning should be determined by the chemical structure, prior to the chemical name.
  • Halogen or "halo” refers to a group containing fluorine, chlorine, bromine and iodine included in the halogen group of elements in the periodic table.
  • hetero refers to a compound or group containing one or more heteroatoms.
  • heteroatom refers to an atom other than carbon or hydrogen, for example, B, Si, N, P, O, S, and Se, of which preferably N, O, and S, or polyvalent elements such as F, Cl , Br, and monovalent elements such as I, and the like, but are not limited thereto.
  • Alkyl or “alkane” is a chain or branched hydrocarbon that is fully saturated.
  • a chain-like or branched alkenyl group can have 2 to about 50, 2 to 20, or 2 to 10 carbon atoms.
  • Chain and branched alkyl groups include, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, iso-butyl, pentyl, hexyl, heptyl (heptyl), octyl, nonyl, and decyl.
  • Alkyl groups may include cyclic cyclic structures.
  • C xy for example when used with an alkyl group, is intended to include moieties containing from x to y carbons in the chain or ring.
  • C xy alkyl includes substituted or unsubstituted, chain-like alkyl groups, branched alkyl groups, or alkyl groups containing a cyclic structure that contain from x to y carbons in the chain; , exemplarily including haloalkyl groups such as difluoromethyl and 2,2,2-trifluoroethyl, and the like.
  • C 0 Alkyl means hydrogen.
  • C 1-4 alkyl examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, iso-butyl, difluoromethyl, and 2,2,2-trifluoro roethyl and the like, but are not limited thereto.
  • heteroalkyl refers to an alkyl containing one or more heteroatoms.
  • Alkenyl or “alkene” is a chain or branched non-aromatic hydrocarbon containing one or more double bonds.
  • a chain-like or branched alkenyl group can have 2 to about 50, 2 to 20, or 2 to 10 carbon atoms.
  • Alkenyl groups can include cyclic structures.
  • heteroalkene refers to an alkene containing one or more heteroatoms.
  • Alkynyl or “alkyne” is a chain or branched non-aromatic hydrocarbon containing one or more triple bonds.
  • a chain or branched alkynyl group can have 2 to about 50, 2 to 20, or 2 to 10 carbon atoms.
  • An alkynyl group may contain one or more double bonds in addition to one or more triple bonds.
  • Alkynyl groups can include cyclic structures.
  • heteroalkyn means an alkyne containing one or more heteroatoms.
  • alkylene when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from an alkyl.
  • alkylene may be used with the terms “substituted” or “unsubstituted”, as appropriate.
  • alkylene is intended to include both substituted and unsubstituted alkylenes.
  • Alkylene may be exemplified by -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, and -CH 2 CH 2 CH 2 CH 2 -, but is not limited thereto.
  • alkylene can be written as C 2 alkylene, which refers to an alkylene group containing two carbon atoms in the main chain.
  • C xy alkylene is used to mean an alkylene including both substituted or unsubstituted alkylenes having X to Y number of carbon atoms in the main chain.
  • heteroalkylene when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkyl.
  • heteroalkylene may be used with the terms “substituted” or “unsubstituted” as appropriate.
  • heteroalkylene when the term “heteroalkylene” is not used in conjunction with the terms “substituted” or “unsubstituted”, the term “heteroalkylene” includes both substituted and unsubstituted heteroalkylenes.
  • heteroalkylene groups include, but are not limited to —CH 2 —CH 2 —O—CH 2 —CH 2 —, and —CH 2 —O—CH 2 —CH 2 —NH—CH 2 —.
  • Heteroalkylene groups may contain one or more heteroatoms at non-terminal positions of the chain or branch, and each heteroatom may be the same or different.
  • Heteroalkylene groups may contain one or more heteroatoms at each or all ends of the chain or branch, and each heteroatom may be the same or different.
  • C xy heteroalkylene is used to include all substituted or unsubstituted heteroalkylenes having X to Y number of carbon atoms in the main chain.
  • alkenylene when used as a molecule by itself or as part of a molecule refers to a divalent radical derived from an alkene.
  • alkenylene may be used with the terms “substituted” or “unsubstituted” as appropriate.
  • the term “alkenylene” is intended to include both substituted and unsubstituted alkenylene.
  • C xy alkenylene is used to include all substituted or unsubstituted alkenylene having X to Y number of carbon atoms in the main chain.
  • heteroalkenylene when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkene.
  • heteroalkenylene may be used with the terms “substituted” or “unsubstituted” as appropriate.
  • heteroalkenylene when the term “heteroalkenylene” is not used with the terms “substituted” or “unsubstituted”, the term “heteroalkenylene” includes both substituted and unsubstituted heteroalkenylenes. It is intended to A teroalkenylene group may contain one or more heteroatoms at non-terminal positions of the chain or branch, and each heteroatom may be the same or different.
  • Heteroalkenylene groups may contain one or more heteroatoms at each or all ends of the chain or branch, and each heteroatom may be the same or different.
  • alkynylene when used as a molecule by itself or as part of a molecule refers to a divalent radical derived from an alkyne.
  • alkynylene may be used with the terms “substituted” or “unsubstituted” as appropriate.
  • the term “alkynylene” is intended to include both substituted and unsubstituted alkynylenes.
  • an alkynylene group includes, but is not limited to, -C ⁇ C-, -CH 2 C ⁇ CCH 2 -, and -C ⁇ CC ⁇ C-.
  • C xy alkynylene is used to include all substituted or unsubstituted alkynylenes having X to Y number of carbon atoms in the main chain.
  • heteroalkynylene when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkyne.
  • heteroalkynylene may be used with the terms “substituted” or “unsubstituted” as appropriate.
  • heteroalkynylene when the term “heteroalkynylene” is not used with the terms “substituted” or “unsubstituted”, the term “heteroalkynylene” includes both substituted and unsubstituted heteroalkynylene.
  • Heteroalkynylene groups may contain one or more heteroatoms at non-terminal positions of the chain or branch, and each heteroatom may be the same or different.
  • the heteroalkynylene group may contain one or more heteroatoms at each or all ends of the chain or branch, and each heteroatom may be the same or different.
  • substituted means that one or more hydrogen atoms on an atom, where the valence of the atom is normal and the substituted compound is stable, is replaced with a substituent including deuterium and hydrogen variants.
  • one substituent is a halogen (eg, Cl, F, Br, and I, etc.)
  • one hydrogen atom has been replaced with a halogen.
  • substituents present in the group may be the same or different. Unless otherwise specified, the type and number of substituents can be arbitrary, as long as they are chemically achievable.
  • substituted C 10-20 alkylene may mean that one or more hydrogen atoms linked to the main chain are substituted with substituents, and each substituent may be independently selected.
  • Compounds of the present application may have certain geometric or stereoisomeric forms. Where a compound is disclosed in this application unless otherwise specified, cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, portions of said compound Isomers such as stereoisomers, (D)-isomers, (L)-isomers, and racemates are included within the scope of this application. That is, special indications related to isomers in the formulas or structures disclosed in this application (eg, *, , , and etc.), the formula or structure disclosed is meant to include all possible isomers.
  • the term "about” means approximately as close to any quantity, 30, 25, 20 relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. , 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term "natural amino acid” or "standard amino acid” refers to 20 types of amino acids synthesized through gene transcription and translation in the body of an organism. it means. Specifically, the standard amino acids are Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys) , C), glutamic acid (Glu, E), glutamine (Gln, Q), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).
  • the standard amino acids are Alanine (Ala
  • Each of the above standard amino acids has a corresponding DNA codon, and can be represented by a general amino acid one-letter or three-letter notation.
  • the subject referred to by the term standard amino acid should be appropriately interpreted according to the context, and includes all other meanings that can be recognized by those skilled in the art.
  • nonnatural amino acid refers to an amino acid that is not synthesized in the body but artificially synthesized.
  • the non-natural amino acids include, for example, p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), LHomopropargylglycine (HPG), O-propargyl-L-tyrosine (oPa), ppropargyloxyphenylalanine (pPa), 2 -amino-3-(4-azidophenyl)propanoic acid, 2-amino-4-(4-azidophenyl)butanoic acid, and 4-(1,2,4,5-tetrazin-3-yl) phenylalanine (frTet), etc.
  • non-natural amino acid does not have a corresponding DNA codon and cannot be expressed in a general amino acid one-letter or three-letter notation, it is indicated using other characters and additionally supplemented.
  • the subject referred to by the term non-natural amino acid should be appropriately interpreted according to the context, and includes all other meanings that can be recognized by those skilled in the art.
  • amino acid sequence in this specification it is written in the direction from the N-terminal to the C-terminal using the one-letter notation of amino acids or the three-letter notation.
  • RNVP when expressed as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected from the N-terminal to the C-terminal.
  • Thr-Leu-Lys when expressed as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected from the N-terminal to the C-terminal.
  • amino acids that cannot be expressed by the one-letter notation or the three-letter notation other characters are used and additionally supplemented descriptions are provided.
  • treatment refers to an approach for obtaining beneficial or desirable clinical results.
  • beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction of extent of disease, stabilization of disease state (i.e., not worsening), delay or reduction in rate of disease progression, prevention of disease. , amelioration or palliation and alleviation (partial or total) of the disease state, detectable or undetectable.
  • the treatment refers to both therapeutic treatment and prophylactic or prophylactic methods.
  • Subject refers to an animal in need of treatment that can be achieved by a molecule of the invention.
  • Animals that can be treated according to the present invention include vertebrates, with mammals such as murine, bovine, canine, equine, feline, ovine, porcine and primates (including humans and non-human primates) being particularly preferred examples. .
  • the term "uric acid oxidase-albumin conjugate” refers to a complex in which a variant of uric acid oxidase and albumin are bound.
  • the term conjugate can be used interchangeably with complex.
  • the uric acid oxidase-albumin complex or the uric acid oxidase-albumin conjugate is a complex in which one albumin is bound to a tetrameric uric acid oxidase variant, a complex in which two albumin are bound, and three albumin. It includes bound complexes and complexes in which four albumins are bound.
  • the bond may be a chemical bond through a linker.
  • click-chemistry as used herein is defined by K. Barry Sharpless of the Scripps Research Institute to describe complementary chemical groups and chemical reactions designed to quickly and stably form a covalent bond between two molecules. It is a chemical concept introduced for Click chemistry in the present specification does not mean a specific reaction, but means a concept of a fast and stable reaction. In one embodiment, in order to form bonds between molecules by click chemistry, several conditions must be satisfied. The conditions are high yield, excellent selectivity for the reaction site, organic molecular bonding by operating in a modular manner, and rapid and accurate product production by proceeding in a thermodynamically stabilized direction.
  • the click chemistry of the present specification is a click chemical functional group (eg, terminal alkyne, azide, strained alkyne, diene, dienophile, trans cyclo Among trans-cyclooctene, alkene, thiol, tetrazine, dibenzocyclooctyne (DBCO) and bicyclononyne (including bicyclo[6.1.0]non-4-yne) This includes reacting pairs that are reactive with each other.
  • click chemistry herein includes the reaction of bicyclononine and azide.
  • a urate oxidase-albumin conjugate can be prepared by reacting a urate oxidase variant, albumin, and a linker.
  • Uric acid oxidase is an enzyme that has the function of decomposing uric acid. Since the human body does not produce uric acid oxidase, if the decomposition of uric acid is not smooth, uric acid is deposited in the body and can cause various diseases. Thus, the uric acid oxidase can be used to treat diseases caused by high levels of uric acid, including, for example, gout.
  • the uric acid oxidase may exist in the form of a tetramer or an octamer in which four monomers having the same structure are combined. That is, the uric acid oxidase is a tetramer or octamer formed by oligomerization of four uric acid oxidase subunits.
  • the uric acid oxidase mutant used in the preparation of the uric acid oxidase-albumin conjugate disclosed herein is characterized in that a part of the sequence of wild-type uric acid oxidase derived from a microorganism is modified.
  • the uric acid oxidase variant contains one or more non-natural amino acids and can be site-specifically conjugated to albumin via each non-natural amino acid residue.
  • the uric acid oxidase variant may contain one, two, three, or four, or more unnatural amino acids.
  • the uric acid oxidase variant is a tetramer formed by oligomerization of four uric acid oxidase variant subunits, and each uric acid oxidase variant subunit has at least one amino acid in its sequence compared to a wild-type urate oxidase subunit. It is characterized in that it is substituted with a natural amino acid.
  • Wild-type uric acid oxidase which is the prototype of the uric acid oxidase variant provided herein, is derived from a microorganism.
  • the wild-type uric acid oxidase may be uric acid oxidase derived from a microorganism selected from Aspergillus Flavus, Arthrobacter globiformis, and Candidas Utilis.
  • the wild-type uric acid oxidase is a tetrameric protein in which four identical wild-type uric acid oxidase subunits are oligomerized.
  • the peptide sequence of the subunit is from the N-terminus to the C-terminus
  • the peptide sequence of the subunit is from the N-terminus to the C-terminus
  • the peptide sequence of the subunit is from the N-terminus to the C-terminus
  • the uric acid oxidase used herein includes microbial uric acid oxidase and mammalian uric acid oxidase.
  • the mutant uric acid oxidase is also a tetrameric protein comprising 4 subunits.
  • the uric acid oxidase variant includes 1 to 4 uric acid oxidase variant subunits, wherein the uric acid oxidase variant subunit is a nocturnal uric acid oxidase subunit having one or more amino acids substituted with a non-natural amino acid.
  • the uric acid oxidase variant may include one uric acid oxidase variant subunit and three wild-type uric acid oxidase subunits.
  • the uric acid oxidase variant may include two uric acid oxidase variant subunits and one wild-type uric acid oxidase subunit. In another embodiment, the uric acid oxidase variant may include three uric acid oxidase variant subunits and one wild-type uric acid oxidase subunit. In another embodiment, the uric acid oxidase variant may include four uric acid oxidase variant subunits.
  • Uric acid oxidase variant subunits include one or more unnatural amino acids.
  • the non-natural amino acid includes a first click chemical functional group and a second click chemical functional group capable of performing a click chemical reaction.
  • the second click chemical functional group is a terminal alkyne, azide, strained alkyne, diene, dienophile, trans-cyclooctene ), an alkene, a thiol, a tetrazine, a dibenzocyclooctyne (DBCO), and a bicyclononyne, but is not limited thereto.
  • the non-natural amino acids are each independently p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), LHomopropargylglycine (HPG), O-propargyl-L-tyrosine (oPa) ppropargyloxyphenylalanine (pPa), and 4-(1,2,4,5-tetrazin-3-yl) phenylalanine (frTet).
  • the unnatural amino acid may be AzF.
  • p-Azido-L-phenylalanine (AzF) may have a structure of Formula 6 or 6-1 below.
  • Amino acids that play an important role in the activity and structure of uric acid oxidase since the structure and function of the original uric acid oxidase should not be affected as much as possible when creating a uric acid oxidase mutant by inserting a non-natural amino acid into wild-type uric acid oxidase. cannot be substituted with an unnatural amino acid.
  • the non-natural amino acid needs to bind to a linker during the preparation of the uric acid oxidase-albumin conjugate, it is advantageous to substitute an amino acid at a position with relatively high solvent accessibility in the three-dimensional structure of uric acid oxidase.
  • Various methods can be used to select sites with high solvent accessibility while minimizing the effect on the structure and function of wild-type uric acid oxidase. For example, through molecular modeling calculations, it is possible to select candidate sites that are similar in atomic energy to wild-type uric acid oxidase and have high solvent accessibility.
  • the uric acid oxidase variant subunit may be obtained by replacing one or more amino acids of the amino acid sequence of SEQ ID NO: 1 with a non-natural amino acid.
  • the uric acid oxidase variant subunit is 8th tyrosine, 16th tyrosine, 30th tyrosine, 46th tyrosine, 65th tyrosine, 79th phenylalanine, 87th tyrosine of the amino acid sequence of SEQ ID NO: 1 Phenylalanine, position 91 tyrosine, position 106 tryptophan, position 120 phenylalanine, position 159 phenylalanine, position 160 tryptophan, position 162 phenylalanine, position 167 tyrosine, position 174 tryptophan, position 186 tryptophan, position 188 tryptophan, position 191 phenylalanine, position 204 phenylalanine, At least one residue selected from the
  • the uric acid oxidase variant subunit may be one or more amino acids of the amino acid sequence of SEQ ID NO: 2 substituted with a non-natural amino acid.
  • the uric acid oxidase variant subunit is 10th tyrosine, 163rd tyrosine, 17th phenylalanine, 45th phenylalanine, 59th tyrosine, 77th tryptophan of the amino acid sequence of SEQ ID NO: 2 (tryptophan), position 82 phenylalanine, position 90 phenylalanine, position 94 tyrosine, position 109 tryptophan, position 112 tyrosine, position 123 phenylalanine, position 136 tyrosine, position 137 tyrosine, position 143 tyrosine, 162 position phenylalanine, position 163 tyrosine, position 165 tyrosine, position 170 phenylalanine, position
  • At least one residue selected from tyrosine at position 163, phenylalanine at position 170, tyrosine at position 200, and tryptophan at position 271 of the amino acid sequence of SEQ ID NO: 2 is substituted with a non-natural amino acid. It could be
  • the uric acid oxidase variant subunit may be obtained by replacing one or more amino acids of the amino acid sequence of SEQ ID NO: 3 with a non-natural amino acid.
  • the uric acid oxidase variant subunit is 20th tyrosine, 52nd phenylalanine, 75th tyrosine, 77th phenylalanine, 82nd phenylalanine, 88th phenylalanine, 96th phenylalanine, 100th phenylalanine, Tryptophan at position 108, phenylalanine at position 113, phenylalanine position 114, tryptophan position 115, phenylalanine position 125, phenylalanine position 163, phenylalanine position 166, tyrosine position 171, tryptophan position 190, tyrosine position 192, phenylalanine position 199, phenylalanine position 203
  • One or more amino acids e
  • albumin is used in the preparation of uric acid oxidase-albumin conjugates.
  • Albumin is a simple protein that is widely distributed in body fluids, and serves as a transport protein that binds and transports various molecules.
  • a representative example of albumin is serum albumin.
  • the albumin of the present application may be mammalian albumin, such as serum albumin.
  • serum albumin may be any one selected from human serum albumin (HSA), bovine serum albumin (BSA), ovalbumin, other vertebrate albumin, and variants thereof. They may be wild-type or recombinant forms.
  • the albumin can be wild type or recombinant human serum albumin.
  • the human serum albumin has a long half-life of 2 weeks or more. This is because 1) it is not easily filtered in the glomerulus due to the electrostatic repulsion of the albumin molecule, and 2) it is degraded in the body due to the recycling action mediated by the neonatal Fc receptor (FcRn) of the endothelium. because it is long
  • albumin may be human serum albumin, and in this case, human serum albumin may include the following amino acid sequence.
  • the albumin may be human serum albumin or a variant thereof, and the human serum albumin or variant thereof may include a sequence selected from any one of the following sequences:
  • a thiol residue of cysteine included in human serum albumin or a variant thereof may react with a second reactive functional group at one end of the linker. More specifically, a thiol residue of cysteine included in human serum albumin or a variant thereof may react with a thiol-reactive group at one end of the linker. At this time, the cysteine reacting with one end of the linker may be cysteine 34 (Cys 34).
  • the linker means a structure that reacts with uric acid oxidase variants and albumin and serves to connect uric acid oxidase variants and albumin.
  • the linker may react with albumin to form an albumin-linker conjugate, and the resulting albumin-linker conjugate may react with a urate oxidase variant to form a urate oxidase-albumin conjugate.
  • the linker may react with the uric acid oxidase variant to form a linker-uric acid oxidase complex, and the resulting linker-uric acid oxidase complex may react with albumin to form a uric acid oxidase-albumin conjugate.
  • a uric acid oxidase-albumin conjugate may be formed by reaction of a linker, a uric acid oxidase variant, and albumin. That is, one end of the linker is configured to react with a urate oxidase variant containing an unnatural amino acid, and the other end is configured to react with albumin.
  • the linker may include a structure that reacts with a urate oxidase variant, a structure that reacts with albumin, and/or a linking structure between the two structures.
  • the linker may react with the non-natural amino acid of the uric acid oxidase variant.
  • This reaction is a reaction between the first reactive functional group of the linker and the non-natural amino acid of the urate oxidase variant.
  • the first reaction functional group includes a first click chemistry functional group.
  • the first reactive functional group may include a first click chemical functional group, wherein the first click chemical functional group is selected from among dibenzocyclooctyne, azide, tetrazine, transcyclooctyne, and bicyclononine. can be chosen
  • linkers can react with cysteine residues of albumin. This reaction is a reaction between the second reactive functional group of the linker and the thiol group of the cysteine of albumin.
  • the second reactive functional group includes a functional group having reactivity to a thiol of a cysteine residue.
  • the second reactive functional group may include a thiol reactive group.
  • the second reactive functional group is maleimide (MAL), 3-arylpropiolonitriles (APN)), haloacetal, pyridyl disulfide, and functional groups capable of reacting with other known thiols.
  • a linker is used to prepare the uric acid oxidase-albumin conjugate.
  • the linker of the present application may have a structure of Formula 1 below.
  • F 1 is a first reactive functional group
  • F 2 is a second reactive functional group
  • L is a linker moiety
  • F 1 that is, the first reactive functional group may include a first click chemical functional group.
  • the first click chemical functional group may be any one selected from a dibenzocyclooctyne group, an azide group, a tetrazine group, a transcyclooctyne group, and a bicyclononine group, but is not limited thereto. It may be a group commonly used in the industry as a click chemical functional group. In certain embodiments, the first click chemofunctional group may be a bicyclononine.
  • the first click chemistry functional group can be represented by any one of the following structures.
  • F 2 that is, the second reactive functional group may include a functional group having reactivity to a thiol group.
  • the second reactive functional group may include a thiol reactive group.
  • the thiol-reactive group may be any one selected from a maleimide (MAL) group, a 3-arylpropiolonitriles group, a haloacetal group, and a pyridyl disulfide group.
  • MAL maleimide
  • the second reactive functional group may include a functional group having reactivity to an amine group.
  • the second reactive functional group may include N-hydroxysuccinimide ester (NHS) and imidoester, but is not limited thereto, and is typically a functional group capable of reacting with an amine group.
  • NHS N-hydroxysuccinimide ester
  • thiol-reactive groups can be represented by the structure:
  • L ie the linker moiety
  • L can include an alkylene, alkenylene, alkynylene, aralkylene, arylalkylene or (C 2 H 4 O) np , where np is greater than 1 and less than or equal to 6 may be an integer of
  • L is a substituted or unsubstituted C 1-50 alkylene, a substituted or unsubstituted C 1-50 heteroalkylene, a substituted or unsubstituted C 2-50 alkenylene, a substituted or unsubstituted C 2-50 heteroalkenylene, substituted or unsubstituted C 2-50 alkynylene, or substituted or unsubstituted C 2-50 heteroalkynylene.
  • the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms.
  • the heteroatoms can each independently be selected from O, S, and N.
  • L is a substituted or unsubstituted C 10-30 alkylene, a substituted or unsubstituted C 10-30 heteroalkylene, a substituted or unsubstituted C 10-30 alkenylene, or a substituted or unsubstituted C 10-30 heteroalkylene.
  • L is substituted or unsubstituted C 10-30 alkylene, substituted or unsubstituted C 10-30 heteroalkylene, substituted or unsubstituted C 10-30 alkylene, including (C 2 H 4 O) np .
  • L is a substituted or unsubstituted C 12-20 alkylene, a substituted or unsubstituted C 12-20 heteroalkylene, a substituted or unsubstituted C 12-20 alkenylene, or a substituted or unsubstituted C 12-20 heteroalkylene.
  • L is substituted or unsubstituted C 12-20 alkylene, substituted or unsubstituted C 12-20 heteroalkylene, substituted or unsubstituted C 12-20 alkylene, including (C 2 H 4 O) np .
  • the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms.
  • the heteroatoms can each independently be selected from O, S, and N.
  • linker moiety (L) can be represented by any of the following structures:
  • L 1 is a bond, substituted or unsubstituted C 1-6 alkylene, substituted or unsubstituted C 1-6 heteroalkylene, or substituted or unsubstituted C 2-6 alkenylene. , substituted or unsubstituted C 2-6 heteroalkenylene, substituted or unsubstituted C 2-6 alkynylene, or substituted or unsubstituted C 2-6 heteroalkynylene.
  • L 1 can be -O-, -NH- or -S-.
  • L 1 can be a bond, unsubstituted C 1-3 alkylene, or unsubstituted C 1-3 heteroalkylene.
  • L 2 may include alkylene, alkenylene, alkynylene, aralkylene, arylalkylene, or (C 2 H 4 O) np , where np may be an integer of 1 or more and 6 or less.
  • L 2 is substituted or unsubstituted C 1-30 alkylene, substituted or unsubstituted C 1-30 heteroalkylene, substituted or unsubstituted C 2-30 alkenylene, substituted or unsubstituted C 1-30 C 2-30 heteroalkenylene, substituted or unsubstituted C 2-30 alkynylene, or substituted or unsubstituted C 2-30 heteroalkynylene.
  • L 2 is substituted or unsubstituted C 1-30 alkylene, substituted or unsubstituted C 1-30 heteroalkylene, substituted or unsubstituted, including (C 2 H 4 O) np .
  • L 2 is substituted or unsubstituted C 10-20 alkylene, substituted or unsubstituted C 10-20 heteroalkylene, substituted or unsubstituted C 10-20 alkenylene, or substituted or unsubstituted C 10-20 heteroalkylene.
  • L 2 is substituted or unsubstituted C 10-20 alkylene, substituted or unsubstituted C 10-20 heteroalkylene, substituted or unsubstituted, including (C 2 H 4 O) np .
  • C 10-20 alkenylene substituted or unsubstituted C 10-20 heteroalkenylene, substituted or unsubstituted C 10-20 alkynylene, or substituted or unsubstituted C 10-20 heteroalkynylene.
  • L 3 is a bond, substituted or unsubstituted C 1-6 alkylene, substituted or unsubstituted C 1-6 heteroalkylene, or substituted or unsubstituted C 2-6 alkenylene. , substituted or unsubstituted C 2-6 heteroalkenylene, substituted or unsubstituted C 2-6 alkynylene, or substituted or unsubstituted C 2-6 heteroalkynylene.
  • L 3 may be -O-, -NH-, or -S-.
  • L 3 can be a bond, unsubstituted C 1-3 alkylene, or unsubstituted C 1-3 heteroalkylene.
  • Formula 1 may be represented by Formula 1-1, and the linker may have the structure of Formula 1-1:
  • each of F 1 , L 1 , L 2 , L 3 , and F 2 is as described above.
  • Formula 1-1 may be represented by Formula 1-2, and the linker may have a structure of Formula 1-2:
  • each of F 1 , L 1 , L 2 , and F 2 is as described above, and np may be an integer of 1 or more and 6 or less.
  • Formula 1-2 may be represented by Formula 1-3, and the linker may have a structure of Formula 1-3:
  • np is an integer of 1 or more and 6 or less.
  • a linker can have the structure of Formulas 1-4:
  • np is an integer of 1 or more and 6 or less.
  • the linker may have a structure of any one of the following formulas:
  • a uric acid oxidase-albumin conjugate site-specifically bound to albumin In order to produce a uric acid oxidase-albumin conjugate site-specifically bound to albumin, a uric acid oxidase variant containing an unnatural amino acid is produced.
  • one or more residues in the amino acid sequence of wild type uric acid oxidase can be changed to an unnatural amino acid.
  • the position may be selected from phenylalanine, tryptophan, and tyrosine.
  • it may be selected from positions having high solvent accessibility in order to achieve efficient binding with the linker.
  • the change to the non-natural amino acid may be a substitution of one or more residues of the amino acid sequence of SEQ ID NO: 1.
  • SEQ ID NO: 1 is Ser Ala Val Lys Ala Ala Arg Tyr Gly Lys Asp Asn Val Arg Val Tyr Lys Val His Lys Asp Glu Lys Thr Gly Val Gln Thr Val Tyr Glu Met Thr Val Cys Val Leu Leu Glu Gly Glu Ile Glu Thr Ser Tyr Thr Lys Ala Asp Asn Ser Val Ile Val Ala Thr Asp Ser Ile Lys Asn Thr Ile Tyr Ile Thr Ala Lys Gln Asn Pro Val Thr Pro Pro Glu Leu Phe Gly Ser Ile Leu Gly Thr His Phe Ile Glu Lys Tyr Asn His Ile His Ala Ala His Val Asn Ile Val Cys His Arg Trp Thr Arg Met Asp Ile Asp Gly Lys Pro His Pro His Ser Phe Ile Arg Asp Arg Asp G
  • one or more residues of the amino acid sequence of SEQ ID NO: 2 may be substituted with a non-natural amino acid.
  • SEQ ID NO: 2 Met Ser Thr Thr Leu Ser Ser Ser Thr Tyr Gly Lys Asp Asn Val Lys Phe Leu Lys Val Lys Lys Asp Pro Gln Asn Pro Lys Lys Gln Glu Val Met Glu Ala Thr Val Thr Cys Leu Leu Glu Gly Gly Phe Asp Thr Ser Tyr Thr Glu Ala Asp Asn Ser Ser Ile Val Pro Thr Asp Thr Val Lys Asn Thr Ile Leu Val Leu Ala Lys Thr Thr Glu Ile Trp Pro Ile Glu Arg Phe Ala Ala Lys Leu Ala Thr His Phe Val Glu Lys Tyr Ser His Val Ser Gly Val Ser Val Lys Ile Val Gln Asp Arg Trp Val Lys Tyr Ala Val Asp Gly Lys Pro His Asp His Ser Phe Ile Ile
  • At least one residue selected from tyrosine at position 163, phenylalanine at position 170, tyrosine at position 200, and tryptophan at position 271 of the amino acid sequence of SEQ ID NO: 2 is a non-natural amino acid may be substituted with
  • the non-natural amino acid refers to an amino acid that is not one of the 20 common amino acids, pyrrolysine and selenocysteine.
  • the non-natural amino acid includes a structure capable of reacting with the first reactive functional group of the linker.
  • the first reactive functional group of the linker includes a first click chemistry functional group.
  • the non-natural amino acids are p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), LHomopropargylglycine (HPG), O-propargyl-L-tyrosine (oPa), ppropargyloxyphenylalanine (pPa), and It may be one or more selected from 4-(1,2,4,5-tetrazin-3-yl) phenylalanine (frTet).
  • AzF p-Azido-L-phenylalanine
  • pEthF p-ethynyl-phenylalanine
  • HPG LHomopropargylglycine
  • oPa O-propargyl-L-tyrosine
  • pPa ppropargyloxyphenylalanine
  • It may be one or more selected from 4-(1,2,4,5-tetrazin-3-yl)
  • pQE80-Uox can be produced by obtaining the coding sequence of Uox by PCR and then cloning it into pQE80. Next, site-directed mutagenic PCR can be performed using the pQE80-Uox as a template to replace amino acid residues at specific positions of uric acid oxidase with amber codons.
  • pEVOL-pAzF plasmid (Plasmid ID: 31186) containing an AzF-specific engineered pair consisting of tyrosyl-tRNA synthetase and amber suppressor tRNA derived from Methanococcus jannaschii can be used.
  • the production strain in order to express a variant of uric acid oxidase containing an unnatural amino acid, is simultaneously transformed with pQE80-Uox.Xamb and pEVOL-pAzF in which an amber codon is introduced at a specific position, for example, the X position. can make it
  • the method disclosed in the present application includes effectively culturing a strain producing uric acid oxidase for mass production of variants of uric acid oxidase containing non-natural amino acids.
  • the production strain includes bacteria.
  • the bacteria are Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus, Pseudomonas ), Leptospira, Salmonella and Brevibacterium, Hyphomonas, Chromobactorium, Norcardia or Fungi. (fungi), or yeast (yeast).
  • a fed-batch process may be performed while supplying a carbon source and a nitrogen source at a constant rate.
  • the culture may include seed culture and main culture.
  • the medium for the culture may include soy peptone, yeast extract, KH 2 PO 4 , and K 2 HPO 4 , and may additionally include antibiotics such as kanamycin and chloramphenicol.
  • Carbon and nitrogen sources may be supplied during the culturing process.
  • glucose and MgSO 4 may be supplied as carbon sources
  • yeast extract and (NH 4 ) 2 SO 4 may be supplied as nitrogen sources.
  • the culturing is performed in a fermentor, and the agitation speed of the fermentor may be changed to increase the production yield of the urate oxidase variants containing unnatural amino acids.
  • the stirring speed may be set to about 300 rpm, 400 rpm, 500 rpm, 600 rpm, or 700 rpm. In a specific embodiment, the stirring speed may be set to about 600 rpm for 50L mass culture.
  • the pressure conditions of the fermentor may be changed.
  • the internal pressure may be set to about 100 mbar, 200 mbar, 300 mbar, 400 mbar, or 500 mbar.
  • the internal pressure condition may be set to about 400 mbar for 50L mass culture.
  • Air supply conditions may be changed to increase the production yield of the uric acid oxidase variants containing non-natural amino acids.
  • the air supply condition may be about 1 vvm, 1.5 vvm, or 2 vvm. In a specific embodiment, air supply conditions may be set to about 1.5vvm for 50L mass culture.
  • the air may mean having a composition of 78% nitrogen, 21% oxygen, and the like, in one embodiment, 1%.
  • the method may include disrupting the strain to obtain a urate oxidase variant containing an unnatural amino acid.
  • Appropriate disruption conditions may be set to increase the yield and enzymatic activity of uric acid oxidase variants.
  • the crushing pressure may be varied for appropriate crushing conditions.
  • the cell disruption pressure may be set to about 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 psi.
  • the volume of the disruption buffer may be changed for appropriate disruption conditions.
  • about 5 ml, 10 ml, and 15 ml of buffer based on 1 g of wet-cell may be added.
  • the number of crushing cycles can be varied for suitable crushing conditions.
  • the crushing cycle may be set to 1 time, 2 times, 3 times, 4 times, and 5 times.
  • the method may further include performing chromatography to separate and purify the variant uric acid oxidase containing the non-natural amino acid.
  • two or more steps of chromatography may be performed for the separation and purification.
  • the chromatography may include cation chromatography, anion chromatography, and size exclusion chromatography.
  • primary anion chromatography and secondary cation chromatography may be performed for the separation and purification.
  • the conditions of the elution buffer may be changed to optimize the separation and purification of uric acid oxidase variants.
  • condition of the elution buffer in the primary anion chromatography may be 20mM sodium phosphate pH 6.0 + 0.1M NaCl. In one embodiment, the condition of the elution buffer in secondary cation chromatography may be 20mM sodium phosphate pH 8.5.
  • the conjugate preparation method of the present application includes performing a process of linking albumin and a linker in order to bind a urate oxidase variant and albumin.
  • the production of the albumin-linker conjugate is performed before 3. binding of the uric acid oxidase variant containing a non-natural amino acid to the albumin-linker conjugate.
  • Production of the albumin-linker conjugate may be performed before, during, or after the production of a uric acid oxidase variant containing a non-natural amino acid.
  • the linker may be connected through a specific amino acid residue of albumin.
  • albumin may be linked to albumin through a cysteine residue, and the linkage may be formed by a reaction between the second reactive functional group of the linker and the cysteine residue of albumin.
  • albumin is linked with a linker including a second reactive functional group having reactivity to a thiol of a cysteine residue of albumin.
  • albumin is maleimide (MAL), 3-arylpropiolonitriles, haloacetal, pyridyl disulfide, or other known thiols. It is linked with a linker comprising a reactive group capable of reacting with a group.
  • albumin is linked with a linker including Maleimide (MAL) or 3-Arylpropiolonitrile.
  • an albumin-linker conjugate may be prepared by reacting a thiol group of cysteine contained in human serum albumin (HSA) with a second reactive functional group of a linker.
  • an albumin-linker conjugate may be prepared by reacting a thiol group of cysteine 34 (Cys 34) of human serum albumin (HSA) with a thiol reactive group included in the second reactive functional group of the linker.
  • an albumin-linker conjugate may be prepared by reacting a maleimide group included in the second functional group of the linker with a thiol group of Cys 34 of human serum albumin or a variant thereof.
  • a linker containing 3-arylpropiolonitriles when connected with a linker containing 3-arylpropiolonitriles, it may have an effect of increasing stability in the body.
  • it is not limited thereto, and includes all those connected by commonly used coupling reactions.
  • an albumin-linker conjugate resulting from the reaction of a linker with albumin may have the structure of Formula 2 below:
  • F 1 is a first reactive functional group
  • L is a linker moiety
  • X 2 is a structure formed by the reaction of the second reactive functional group with albumin
  • A is albumin.
  • F 1 and L are derived from a linker and are as described in the relevant paragraphs.
  • Formula 2 may be represented by Formula 2-1, and the albumin-linker conjugate may have the structure of Formula 2-1:
  • np is an integer of 1 or more and 6 or less.
  • the X 2 is a structure formed by reacting albumin with a reactive functional group for albumin. That is, the X 2 may be a structure formed by reacting the second reactive functional group of the linker with albumin.
  • the second reactive functional group may be, for example, a reactive functional group having reactivity to a thiol of a cysteine residue of albumin.
  • the second reactive functional group includes maleimide (MAL), 3-arylpropiolonitriles, haloacetal, or pyridyl disulfide, and is limited thereto. and may contain a functional group that can react with a thiol group.
  • the second reactive functional group may have reactivity to the lysine residue of albumin.
  • the second reactive functional group includes N-hydroxysuccinimide ester (NHS) and imidoester, but is not limited thereto, and typically includes a functional group capable of reacting with an amine group. can
  • the X 2 may have a structure formed by reacting a thiol reactive group included in the second reactive functional group with a thiol residue of Cys 34 of human serum albumin (HSA).
  • HSA human serum albumin
  • X 2 can be represented by the structure:
  • S is derived from the thiol group of cysteine of albumin.
  • S may be derived from Cys 34 of human serum albumin.
  • albumin and the linker may be reacted at a constant ratio. For example, it may be reacted at a ratio of 1:1, 1:2, 1;3, 1:4, 1:5, 1:6, 1:7 or 1:8. In one embodiment, it may be reacted with a linker including HSA and MAL at a molar ratio of 1:4.
  • a process of removing the unreacted linker may be additionally performed.
  • a process of desalting with PD-10 may be performed to remove unreacted linkers.
  • desalting may occur at pH 5 to 8 using 10 to 30 mM sodium phosphate. More specifically, desalting can occur at pH 6 using 20 mM sodium phosphate.
  • the conjugate preparation method of the present application includes a step of linking a linker bound to albumin with a uric acid oxidase variant in order to bind the urate oxidase variant and albumin.
  • uric acid oxidase-albumin conjugates can be prepared by conjugated uric acid oxidase variants with 1 to 4 albumin-linkers.
  • a urate oxidase-albumin conjugate can be prepared by a method comprising: preparing a subunit-albumin conjugate wherein one albumin is conjugated to a urate oxidase variant subunit; and 4 of the subunit-albumin conjugates constitute a tetramer or the subunit-albumin conjugate and the uric acid oxidase variant subunit constitute a tetramer to prepare a uric acid oxidase-albumin conjugate.
  • the uric acid oxidase-albumin conjugate comprises one to four subunit-albumin conjugates.
  • the linker linked to albumin at one end is conjugated with a urate oxidase variant using a reactive functional group at the other end.
  • the linker may be connected to the uric acid oxidase variant through the unnatural amino acid of the uric acid oxidase variant, and in this case, the linker is involved in the reaction between the first reactive functional group of the linker and the non-natural amino acid of the uric acid oxidase variant.
  • the non-natural amino acid of the uric acid oxidase variant and the albumin-linker conjugate may be linked through a click chemistry reaction.
  • the second click chemical functional group of the non-natural amino acid contained in the uric acid oxidase variant and the first click chemical functional group contained in the first reactive functional group of the albumin-linker conjugate are combined with the click chemical functional group.
  • the second click chemical functional group may be a group capable of performing a click chemical reaction with the first click chemical functional group.
  • the first click chemical functional group may be a bicyclononine
  • the second click chemical functional group may be an azide, but is not limited thereto.
  • the albumin-linker conjugate includes a first click chemofunctional group.
  • the first click chemical functional group is a terminal alkyne, azide, strained alkyne, diene, dienophile, trans-cyclooctene ), an alkene, a thiol, a tetrazine, a dibenzocyclooctyne (DBCO), and a bicyclononyne, but is not limited thereto.
  • the non-natural amino acid of the uric acid oxidase variant includes a second click chemical functional group.
  • the second click chemical functional group is a terminal alkyne, azide, strained alkyne, diene, dienophile, trans-cyclooctene ), an alkene, a thiol, a tetrazine, a dibenzocyclooctyne (DBCO), and a bicyclononyne, but is not limited thereto.
  • the first click chemistry functional group may be a functional group capable of performing a click chemistry reaction with the second click chemistry functional group.
  • the second click chemistry functional group may be a functional group capable of performing a click chemistry reaction with the first click chemistry functional group.
  • the second click chemical functional group may be an azide, and the first click chemical functional group may be a bicyclononine.
  • the azide contained in the non-natural amino acid of the uric acid oxidase variant and the bicyclononine contained in the albumin-linker conjugate may be linked through a click chemistry reaction.
  • Combining a urate oxidase variant containing an unnatural amino acid with an albumin-linker conjugate by a click chemical reaction using the bicyclononine may have the advantage of increasing the number of bound albumins (see Table 1).
  • the uric acid oxidase variant and the albumin-linker conjugate can be reacted at a constant ratio for efficient linkage.
  • the uric acid oxidase variant and the albumin-linker conjugate are in a molar ratio of 1:1, 1:2, 1;3, 1:4, 1:5, 1:6, 1:7 or 1:8. can be combined with.
  • the uric acid oxidase variant and the albumin-linker conjugate may be combined at a molar ratio of 1:4 or 1:8.
  • chromatography may be additionally performed to isolate and purify the conjugate in which the uric acid oxidase variant and albumin are bound.
  • two or more steps of chromatography may be performed for the separation and purification.
  • the chromatography may include cation chromatography, anion chromatography, and size exclusion chromatography.
  • primary cation chromatography and secondary anion chromatography may be performed to isolate and purify the uric acid oxidase-albumin conjugate.
  • a flow rate of 100 cm/h, binding buffer 20 mM sodium phosphate pH6.0, and elution buffer 20 mM sodium phosphate pH6.0 +0.5 M NaCl may be used.
  • a flow rate of 100 cm/h, binding buffer 20 mM bis-tris pH 6.5, and elution buffer 20 mM bis-tris pH 6.5 + 0.3 M NaCl may be used.
  • size exclusion chromatography may be additionally performed.
  • the size exclusion chromatography refers to a technique for separating mixtures based on the rate (permeability) of solutes of various sizes passing through a porous matrix.
  • a sample to be analyzed is passed through a column filled with a porous stationary phase such as gel, matrix, or beads, large molecules that cannot pass through the pores of the column cannot enter the pores and pass through the surrounding empty space. It uses the principle that small molecules come out through the pores of the column and move out of the column relatively slowly while leaving the column quickly. This method is generally used for desalting for buffer exchange, separation for purification, or molecular weight determination based on solute size.
  • the most widely used gels for size exclusion chromatography are Sepharose (GE Healthcare), Superose (GE Healthcare), Sephadex (Pharmacia), Bio-Gel P (Bio-Rad), Superdex ® (superdex' GE Healthcare) and TSKgel® (silicabased; Sigma).
  • Superdex 200 increase 10/300 GL may be used.
  • the method for producing a uric acid oxidase-albumin conjugate using bicyclononine (BCN) disclosed herein has a yield of multi-HSA compared to the method for producing a uric acid oxidase-albumin conjugate using dibenzocyclooctyne (DBCO). is high That is, when the uric acid oxidase-albumin conjugate is produced using BCN, the production yield of the uric acid oxidase-albumin conjugate to which two or more albumins are bound increases.
  • BCN bicyclononine
  • Table 1 shows the results of comparing the conjugates produced according to the method for producing the uric acid oxidase-albumin conjugate using DBCO and the method for producing the uric acid oxidase-albumin conjugate using BCN.
  • BCN when BCN is used, a large number of uric acid oxidase-albumin conjugates in which two or more albumins are bound are produced.
  • BCN has a yield 1.5 times higher than that of DBCO, and in the case of di-, it has a yield 2.2 times higher.
  • the method of preparing an albumin-linker conjugate by contacting a linker with albumin, and preparing a uric acid oxidase-albumin conjugate by contacting the prepared albumin-linker conjugate with a urate oxidase variant has been described above.
  • the uric acid oxidase-albumin conjugate can be prepared by a method comprising the following steps:
  • the uric acid oxidase-linker conjugate may have the structure of Formula 5:
  • Uoxv is a uric acid oxidase variant
  • X 1 is a structure formed by the reaction of the first reactive functional group with the non-natural amino acid of the uric acid oxidase variant
  • L is a linker moiety
  • F 2 is a second reactive functional group.
  • F 2 and L are derived from the linker and are as described in the relevant paragraphs.
  • the uric acid oxidase-albumin conjugate may refer to a complex in which a variant of uric acid oxidase and albumin are bound.
  • the uric acid oxidase-albumin conjugate may contain 1 to 8 albumins.
  • a uric acid oxidase-albumin conjugate may contain 1 to 4 albumins.
  • the uric acid oxidase-albumin conjugate may be in the form of a tetrameric uric acid oxidase coupled to one or more albumins. At this time, 0 or 1 albumin may be bound to one subunit.
  • the uric acid oxidase-albumin conjugate includes four albumins
  • one albumin may be bound to each of the four subunits.
  • the uric acid oxidase-albumin conjugate contains three albumins
  • one albumin is bound to each of three subunits among the four subunits, and albumin is not bound to the other subunit.
  • uric acid oxidase and each albumin are bonded through a linker.
  • the uric acid oxidase-albumin conjugate provided by the present application is disclosed.
  • the present application provides a uric acid oxidase-albumin conjugate having the structure of Formula 3 below:
  • n is an integer greater than or equal to 1. In one embodiment, n may be an integer greater than or equal to 1 and less than or equal to 8. In certain embodiments, n may be an integer greater than or equal to 1 and less than or equal to 4.
  • Uoxv means a uric acid oxidase variant.
  • the uric acid oxidase variant may be a tetrameric protein comprising four subunits.
  • the uric acid oxidase variant includes 1 to 4 uric acid oxidase variant subunits, wherein the uric acid oxidase variant subunit is a nocturnal uric acid oxidase subunit having one or more amino acids substituted with a non-natural amino acid.
  • the uric acid oxidase variant may include one uric acid oxidase variant subunit and three wild-type uric acid oxidase subunits.
  • the uric acid oxidase variant may include two uric acid oxidase variant subunits and one wild-type uric acid oxidase subunit. In another embodiment, the uric acid oxidase variant may include three uric acid oxidase variant subunits and one wild-type uric acid oxidase subunit. In another embodiment, the uric acid oxidase variant may include four uric acid oxidase variant subunits. Uric acid oxidase variant subunits include one or more non-natural amino acids, as described above in the relevant paragraphs. In certain embodiments, a uric acid oxidase variant subunit may include AzF.
  • the uric acid oxidase variant subunit may be obtained by replacing one or more amino acids of the amino acid sequence of SEQ ID NO: 1 with a non-natural amino acid.
  • the uric acid oxidase variant subunit is 8th tyrosine, 16th tyrosine, 30th tyrosine, 46th tyrosine, 65th tyrosine, 79th phenylalanine, 87th tyrosine of the amino acid sequence of SEQ ID NO: 1 Phenylalanine, position 91 tyrosine, position 106 tryptophan, position 120 phenylalanine, position 159 phenylalanine, position 160 tryptophan, position 162 phenylalanine, position 167 tyrosine, position 174 tryptophan, position 186 tryptophan, position 188 tryptophan, position 191 phenylalanine, position 204 phenylalanine, At least one residue selected from the
  • the uric acid oxidase variant subunit may be one or more amino acids of the amino acid sequence of SEQ ID NO: 2 substituted with a non-natural amino acid.
  • the uric acid oxidase variant subunit is 10th tyrosine, 163rd tyrosine, 17th phenylalanine, 45th phenylalanine, 59th tyrosine, 77th tryptophan of the amino acid sequence of SEQ ID NO: 2 (tryptophan), position 82 phenylalanine, position 90 phenylalanine, position 94 tyrosine, position 109 tryptophan, position 112 tyrosine, position 123 phenylalanine, position 136 tyrosine, position 137 tyrosine, position 143 tyrosine, 162 position phenylalanine, position 163 tyrosine, position 165 tyrosine, position 170 phenylalanine, position
  • At least one residue selected from tyrosine at position 163, phenylalanine at position 170, tyrosine at position 200, and tryptophan at position 271 of the amino acid sequence of SEQ ID NO: 2 is substituted with a non-natural amino acid. It could be
  • the uric acid oxidase variant subunit may be obtained by replacing one or more amino acids of the amino acid sequence of SEQ ID NO: 3 with a non-natural amino acid.
  • the uric acid oxidase variant subunit is 20th tyrosine, 52nd phenylalanine, 75th tyrosine, 77th phenylalanine, 82nd phenylalanine, 88th phenylalanine, 96th phenylalanine, 100th phenylalanine, Tryptophan at position 108, phenylalanine at position 113, phenylalanine position 114, tryptophan position 115, phenylalanine position 125, phenylalanine position 163, phenylalanine position 166, tyrosine position 171, tryptophan position 190, tyrosine position 192, phenylalanine position 199, phenylalanine position 203
  • One or more amino acids e
  • the X 1 is a structure formed by a click chemical reaction between a second click chemical functional group included in the non-natural amino acid of the uric acid oxidase variant and a first click chemical functional group included in the first reactive functional group of the linker or albumin-linker conjugate.
  • a second click chemical functional group included in the non-natural amino acid of the uric acid oxidase variant and a first click chemical functional group included in the first reactive functional group of the linker or albumin-linker conjugate.
  • X 1 may include a structure formed by the reaction of an azide group included in a non-natural amino acid with a bicyclononine group included in a linker or a first reactive functional group of an albumin-linker conjugate.
  • X 1 may be represented by any one structure selected from the following structures:
  • X 1 may include a structure formed by a click chemical reaction between azide and BCN.
  • the L is a linker moiety and is derived from a linker.
  • L is a structure connecting X 1 and X 2 .
  • L may include alkylene, alkenylene, alkynylene, aralkylene, arylalkylene, or (C 2 H 4 O) np . In this case, np may be selected from 1 to 6.
  • L is a substituted or unsubstituted C 1-50 alkylene, a substituted or unsubstituted C 1-50 heteroalkylene, a substituted or unsubstituted C 2-50 alkenylene, a substituted or unsubstituted C 2-50 heteroalkenylene, substituted or unsubstituted C 2-50 alkynylene, or substituted or unsubstituted C 2-50 heteroalkynylene.
  • the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms.
  • the heteroatoms can each independently be selected from O, S, and N.
  • L is a substituted or unsubstituted C 10-30 alkylene, a substituted or unsubstituted C 10-30 heteroalkylene, a substituted or unsubstituted C 10-30 alkenylene, or a substituted or unsubstituted C 10-30 heteroalkylene.
  • L is a substituted or unsubstituted C 12-20 alkylene, a substituted or unsubstituted C 12-20 heteroalkylene, a substituted or unsubstituted C 12-20 alkenylene, or a substituted or unsubstituted C 12-20 heteroalkylene.
  • the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms.
  • the heteroatoms can each independently be selected from O, S, and N.
  • linker moiety (L) can be represented by any of the following structures:
  • L 1 is a bond, substituted or unsubstituted C 1-6 alkylene, substituted or unsubstituted C 1-6 heteroalkylene, or substituted or unsubstituted C 2-6 alkenylene. , substituted or unsubstituted C 2-6 heteroalkenylene, substituted or unsubstituted C 2-6 alkynylene, or substituted or unsubstituted C 2-6 heteroalkynylene.
  • L 1 can be -O-, -NH- or -S-.
  • L 1 can be unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
  • L 2 may include alkylene, alkenylene, alkynylene, aralkylene, arylalkylene, or (C 2 H 4 O) np , where np may be an integer of 1 or more and 6 or less.
  • L 2 is substituted or unsubstituted C 1-30 alkylene, substituted or unsubstituted C 1-30 heteroalkylene, substituted or unsubstituted C 2-30 alkenylene, substituted or unsubstituted C 1-30 C 2-30 heteroalkenylene, substituted or unsubstituted C 2-30 alkynylene, or substituted or unsubstituted C 2-30 heteroalkynylene.
  • L 2 is substituted or unsubstituted C 1-30 alkylene, substituted or unsubstituted C 1-30 heteroalkylene, substituted or unsubstituted, including (C 2 H 4 O) np .
  • L 2 is substituted or unsubstituted C 10-20 alkylene, substituted or unsubstituted C 10-20 heteroalkylene, substituted or unsubstituted C 10-20 alkenylene, or substituted or unsubstituted C 10-20 heteroalkylene.
  • L 2 is substituted or unsubstituted C 10-20 alkylene, substituted or unsubstituted C 10-20 heteroalkylene, substituted or unsubstituted, including (C 2 H 4 O) np .
  • C 10-20 alkenylene substituted or unsubstituted C 10-20 heteroalkenylene, substituted or unsubstituted C 10-20 alkynylene, or substituted or unsubstituted C 10-20 heteroalkynylene.
  • L 3 is a bond, substituted or unsubstituted C 1-6 alkylene, substituted or unsubstituted C 1-6 heteroalkylene, or substituted or unsubstituted C 2-6 alkenylene. , substituted or unsubstituted C 2-6 heteroalkenylene, substituted or unsubstituted C 2-6 alkynylene, or substituted or unsubstituted C 2-6 heteroalkynylene.
  • L 3 may be -O-, -NH- or -S-.
  • L 3 can be unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
  • linker moiety (L) can be represented by the following structure:
  • the X 2 is a structure formed by reacting albumin with a reactive functional group for albumin. That is, the X 2 may be a structure formed by reacting the second reactive functional group of the linker with albumin.
  • the second reactive functional group may be, for example, a reactive functional group having reactivity to a thiol of a cysteine residue of albumin.
  • the second reactive functional group includes maleimide (MAL), 3-arylpropiolonitriles, haloacetal, or pyridyl disulfide, and is limited thereto. and may contain a functional group that can react with a thiol group.
  • the second reactive functional group may have reactivity to the lysine residue of albumin.
  • the second reactive functional group includes N-hydroxysuccinimide ester (NHS) and imidoester, but is not limited thereto, and typically includes a functional group capable of reacting with an amine group. can
  • the X 2 may have a structure formed by reacting a thiol reactive group included in the second reactive functional group with a thiol residue of Cys 34 of human serum albumin (HSA).
  • HSA human serum albumin
  • X 2 can be represented by any of the following structures:
  • S is derived from the thiol group of cysteine of albumin.
  • S may be derived from Cys 34 of human serum albumin or variants thereof.
  • Formula A refers to albumin, as described above in the relevant paragraph.
  • the albumin can be human serum albumin.
  • the albumin may be a variant of human serum albumin.
  • albumin may include any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
  • X 2- is formed through a reaction between a thiol residue of cysteine contained in albumin and a thiol-reactive group at one end of the linker.
  • the cysteine included in albumin that reacts with the thiol-reactive group may be Cys 34.
  • Formula 3 may be represented by Formula 3-1 below, and the uric acid oxidase-albumin conjugate may have a structure of Formula 3-1:
  • Formula 3-1 may be represented by Formula 3-2 below, and the uric acid oxidase-albumin conjugate may have a structure of Formula 3-2:
  • each of Uoxv, A, X 1 , X 2 , L 1 , L 3 , n, and np is as described above in the related paragraph.
  • Formula 3-2 may be represented by Formula 3-3 below, and the uric acid oxidase-albumin conjugate may have a structure of Formula 3-3 below:
  • Formula 3 may be represented by Formula 3-4 below, and the uric acid oxidase-albumin conjugate may have a structure of Formula 3-4:
  • Formula 3-4 may be represented by Formula 3-5 below, and the uric acid oxidase-albumin conjugate may have a structure of Formula 3-5:
  • the uric acid oxidase-albumin conjugate may include one or more subunit-albumin conjugates.
  • the subunit-albumin conjugate is one in which albumin is conjugated to a urate oxidase mutant subunit.
  • the urate oxidase-albumin conjugate may include 1, 2, 3, 4, 5, 6, 7, or 8 subunit-albumin conjugates.
  • the subunit-albumin conjugate may have a structure represented by Chemical Formula 4 below.
  • Uoxv' is a uric acid oxidase variant subunit, and the uric acid oxidase variant subunit is as described above in the relevant paragraph.
  • a multi-albumin conjugate in which two or more albumins are bonded is present in a high proportion.
  • the conjugate produced by the method of the present specification has a high ratio of uric acid oxidase-albumin conjugate to which two or more albumins are bonded, the efficacy is improved compared to the conjugate produced by the conventional method using DBCO.
  • the present application relates to tumor lysis syndrome (TLS), hyperuricemia, gout, deposition of urate crystals in joints, and urate crystals containing the urate oxidase-albumin conjugate produced by the above method as an active ingredient.
  • TLS tumor lysis syndrome
  • a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of acute gouty arthritis, urolithiasis, nephrolithiasis and gouty nephropathy due to deposition is provided.
  • tumor lysis syndrome occurs when intracellular uric acid, potassium, and phosphorus are released into the bloodstream due to rapid destruction of tumor cells after administration of anticancer drugs.
  • Significantly increased uric acid excretion results in the precipitation of uric acid crystals in the urinary tubules, which can lead to urinary tubule obstruction and renal failure.
  • patients with tumor lysis syndrome may rapidly deteriorate due to metabolic abnormalities.
  • hyperuricemia is known as the most common target abnormality in tumor lysis syndrome.
  • hyperuricemia is a disease in which the concentration of uric acid in the blood is increased. This occurs when the concentration of monosodium urate in serum exceeds the limited solubility limit.
  • the uric acid saturation of plasma at 37°C is about 7 mg/dl. Therefore, when this concentration is exceeded, it becomes supersaturated physically and chemically.
  • the serum uric acid concentration is relatively higher when it exceeds +2 standard deviation than the average serum uric acid concentration of normal subjects.
  • the upper limit is 7 mg/dl for men and 6 mg/dl for women. For this reason, the practical upper limit for hyperuricemia is defined as 7.0 mg/dl or higher.
  • the hyperuricemia and hyperuricemia-related metabolic disorders are diseases or diseases caused by excess uric acid remaining in the blood and increasing blood uric acid levels when uric acid is higher than normal and the ability of the kidneys to excrete uric acid is lowered say the disease
  • the hyperuricemia-related metabolic disorders include gout, uric acid crystals, intra-articular urate crystal deposition, acute gouty arthritis due to urate crystal deposition, monoarticular arthritis, pain attacks of inflammatory arthritis, urolithiasis, nephrolithiasis and gouty nephropathy. Long-term nephrolithiasis and gouty nephropathy are known to increase the risk of kidney damage and renal failure.
  • Gout is a medical condition usually characterized by recurrent attacks of acute inflammatory arthritis and commonly occurs in the metatarsophalangeal joint at the base of the big toe. Also, gout is caused by blood uric acid crystallized and deposited in joints, tendons and surrounding tissues, and may exist as gouty nodules, kidney stones or urate nephropathy.
  • prevention refers to all activities that suppress or delay the onset of gout, hyperuricemia, or hyperuricemia-related metabolic disorders by administration of the pharmaceutical composition according to the present application.
  • treatment refers to all activities that improve or beneficially change symptoms caused by gout, hyperuricemia, or hyperuricemia-related metabolic disorders by administration of the pharmaceutical composition according to the present application.
  • composition containing the uric acid oxidase-albumin conjugate of the present application may contain one or more active ingredients exhibiting the same or similar functions in addition to the above ingredients.
  • the pharmaceutical composition of the present application may further include a pharmaceutically acceptable carrier in addition to containing the uric acid oxidase-albumin conjugate as an active ingredient.
  • the type of carrier that can be used in the present application is not particularly limited, and any carrier commonly used in the art may be used.
  • Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. can These may be used alone or in combination of two or more.
  • the pharmaceutical composition of the present application may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , a binder or a lubricant may be additionally added and used.
  • other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , a binder or a lubricant may be additionally added and used.
  • the uric acid oxidase-albumin conjugate may be included in an amount of 0.00001% to 99.99% by weight, preferably 0.1% to 90% by weight, based on the total weight of the pharmaceutical composition. , More preferably 0.1% by weight to 70% by weight, more preferably 0.1% to 50% by weight, but may be included, but is not limited thereto, and variously changed depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, etc. It can be. If necessary, it may be included in the entire content of the pharmaceutical composition.
  • the term "administration” means introducing the pharmaceutical composition of the present application to a patient by any suitable method, and the route of administration of the composition of the present application can be oral or parenteral as long as it can reach the target tissue. It can be administered via any route.
  • composition of the present application may be formulated and used in various dosage forms suitable for oral or parenteral administration.
  • Non-limiting examples of formulations for oral administration using the pharmaceutical composition of the present application include troches, lozenges, tablets, aqueous suspensions, oily suspensions, powdered preparations, granules, emulsions, hard capsules, and soft capsules, syrups or elixirs; and the like.
  • a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate and the like; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be used, and sweeteners, aromatics, syrups, and the like may also be used.
  • a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.
  • Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present application include injection solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, and the like.
  • aqueous solutions In order to formulate the pharmaceutical composition of the present application for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, external preparations, etc. may be used, and the non-aqueous solvents and suspensions include propylene glycol, polyethylene Glycols, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used.
  • the pharmaceutical composition of the present application is formulated as an injection solution
  • the pharmaceutical composition of the present application is mixed in water together with a stabilizer or buffer to prepare a solution or suspension, which is used for unit administration in an ampoule or vial can be formulated.
  • a propellant or the like may be blended with additives so that the water-dispersed concentrate or wet powder is dispersed.
  • composition of the present application When the pharmaceutical composition of the present application is formulated into an ointment, cream, powder for application, oil, external skin preparation, etc., animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite , silica, talc, zinc oxide, etc. may be formulated using a carrier.
  • the pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present application may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type of response to be achieved by administration of the pharmaceutical composition. and degree, type of subject to be administered, age, weight, general health condition, symptom or severity of disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, and other components of the composition, etc. It can be varied according to factors and similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.
  • the daily dose of the pharmaceutical composition of the present application is 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.
  • Administration of the pharmaceutical composition of the present application may be administered once a day, or may be divided and administered several times.
  • the pharmaceutical composition of the present application may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it can be administered in an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the administration route and administration method of the pharmaceutical composition of the present application may be independent, and may follow any route and administration method without particular limitation as long as the pharmaceutical composition can reach the target site.
  • the pharmaceutical composition may be administered orally or parenterally.
  • intravenous administration intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, etc.
  • a method of applying, spraying, or inhaling the composition to the diseased area It can also be used, but is not limited thereto.
  • the pharmaceutical composition of the present application may be additionally used in combination with various methods such as hormone therapy and drug therapy to prevent or treat gout, hyperuricemia, or hyperuricemia-related metabolic disorders.
  • the present application relates to hyperuricemia, gout, intra-articular urate crystal deposition, acute gouty arthritis due to urate crystal deposition, urolithiasis, renal It provides a food composition for preventing or improving one or more diseases selected from the group consisting of calculus and gouty nephropathy.
  • the term “improvement” refers to all activities that improve or beneficially change a disease by administering the composition of the present application.
  • the food composition of the present application can be used as a health functional food.
  • health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Health Functional Food Act No. 6727, and “functional” refers to the structure of the human body. And it refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients for functions or physiological functions.
  • the food composition of the present application may include additional ingredients that are commonly used and can improve smell, taste, and vision.
  • vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included.
  • minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included.
  • amino acids such as lysine, tryptophan, cysteine, and valine may be included.
  • preservatives potassium sorbate, sodium benzoate, salicylic acid, sodium dihydroacetate, etc.
  • disinfectants bleaching powder, high bleaching powder, sodium hypochlorite, etc.
  • antioxidants butylhydroxyanisole (BHA), butylhydroxytoluene (BHT) ), etc.
  • coloring agents tar color, etc.
  • coloring agents sodium nitrite, sodium nitrite, etc.
  • bleaching agents sodium sulfite, etc.
  • seasonings MSG sodium glutamate, etc.
  • sweeteners dulcin, cyclemate, saccharin, sodium, etc.
  • Food additives such as flavoring (vanillin, lactones, etc.), leavening agent (alum, D-potassium hydrogentartrate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc.
  • the additive may be selected according to the type of food
  • the food composition of the present application When using the food composition of the present application as a food additive, it may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.
  • the content of the uric acid oxidase-albumin conjugate is not particularly limited, and may be variously changed depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, and the like. If necessary, it may also be included in the total content of food.
  • p-Azido-L-phenylalanine was purchased from Chem-Impex International (Wood Dale, IL), and was dissolved in 0.2 M NaOH to make a 100 mM stock solution.
  • the pQE80 plasmid was purchased from Qiagen (Valencia, CA).
  • the pEVOL-pAzF plasmid (Plasmid ID: 31186) containing an AzF-specific engineered pair consisting of tyrosyl-tRNA synthetase and amber suppressor tRNA derived from Methanococcus jannaschii was purchased from Addgene (Cambridge, MA) and used without further modification.
  • Addgene Click-through, MA
  • a bacterial expression vector for expressing recombinant urea oxidase (Uox variant) derived from Aspergillus flavus the coding sequence (SEQ ID NO: 1) of the Uox subunit was synthesized and cloned into pQE80 to obtain pQE80-Uox. made.
  • Site-directed mutagenic PCR was performed using the pQE80-Uox as a template to replace tryptophan at position 174 of Uox with an amber codon (UAG).
  • UAG amber codon
  • 5-CACTGAAGGAGACTTAGGATAGAATCCTG-3 SEQ ID NO: 16
  • 5-CAGGATTCTATCCTAAGTCTCCTTCAGTG-3 SEQ ID NO: 17
  • 150mL of medium for primary seed culture was prepared with the following composition and sterilized.
  • Soy peptone (TATUA, Cat no. HSP-349): 12g/L, Yeast extract (Procelys, Cat no. 0354): 24g/L, KH 2 PO 4 (Duksan, Cat no. 432): 2.3g/L ( Added after separate sterilization), K 2 HPO 4 (Duksan, Cat no. 562): 12.53 g/mL (added after separate sterilization), Kanamycin (Sigma, Cat no. 60615): 35 ⁇ g/mL (added after sterilization filtration), Chloramphenicol (WAKO, Cat no. 036-10571): 35 ⁇ g/mL (added after sterilization filtration).
  • 2L of medium for secondary seed culture was prepared with the following composition and sterilized.
  • Soy peptone 12g/L
  • Yeast extract 24g/L
  • KH 2 PO 4 2.3g/L
  • K 2 HPO 4 12.53 g/mL
  • Kanamycin 35 ⁇ g/L mL
  • Chloramphenicol 35 ⁇ g/mL (added after sterilization filtration).
  • 25mL of the primary seed culture was inoculated into 500mL secondary seed culture medium/2L baffled flask, and the secondary seed culture was performed under the following conditions.
  • a separate sterilization medium for main culture was prepared with the following composition.
  • Glucose (Daejeong, Cat no. 3020-4405): 20g/L (added after separate sterilization), MgSO 4 (Daejeong, Cat no. 5513-4405): 1.2g/L (added after separate sterilization), Kanamycin: 35 ⁇ g/L mL (added after disinfection filtration), chloramphenicol : 35 ⁇ g/mL (added after disinfection filtration), KH 2 PO 4 : 2.3g/L (added after separate sterilization), K 2 HPO 4 : 12.53g/L (added after separate sterilization) ).
  • C-source and N-source for feeding were prepared with the following composition and sterilized separately.
  • Glucose 300g/L(C-source), MgSO 4 :1.0g/L(C-source), Yeast extract : 211g/L(N-source), (NH 4 ) 2 SO 4 (Daejeong, Cat no. 1082 -1405) - : 1.5 g/L (N-source).
  • a 5N NaOH and 50% antifoam (Sigma, Cat no. A6426) solution was prepared and separately sterilized.
  • 5L of concentrated medium for main culture was prepared with the following composition, put into the sample inlet of a 50L fermentor, and sterilized with purified water according to the weight of the vessel to the initial culture volume. After sterilization of the enriched medium was completed, a separate sterilization medium prepared using a peristaltic pump was injected into the fermentor.
  • Soy peptone 12 g/L
  • Yeast extract 24 g/L
  • Thiamine-HCl Alfa Aesar, Cat no. A19560
  • Antifoam 0.1 g/L.
  • a site-specific strain producing urate oxidase containing unnatural amino acids is cultured. Some conditions were additionally changed to increase the culture yield.
  • Air 1.5vvm, pH 7.0, DO (%) 30, Temp:30°C, RPM:600, internal pressure 400mbar.
  • the final culture time was 42 hours, and the final OD was 150.8.
  • the weight of the final wet-cell was 11.8 kg, and the culture yield was 221.8 g/L (see Table 3 and FIG. 2).
  • the culture solution was taken at regular intervals, the wet cells were recovered by centrifugation, and the total protein concentration and Uox enzyme activity were measured after disrupting with an ultrasonicator. A protein concentration of more than mL was confirmed.
  • Cells were disrupted using a cell disruptor (Avestin Emulsiflex D20) under the following disruption conditions. 100 g of Uox-AzF wet cells were washed twice with 10 times the volume of 1L disruption buffer, then disrupted, and centrifuged supernatant was used to compare disruption efficiency.
  • a cell disruptor Avestin Emulsiflex D20
  • Disruption buffer 20 mM Tris-HCl (pH9.0), disruption pressure: 20,000 psi, disruption cycle: 1-4, wet cell (g) to buffer (mL) ratio: 1:10.
  • the supernatant obtained by centrifugation and sterilization filtration was purified in AKTA Pilot 600 with Sartobind Q 800mL using Anion exchange membrane column (Sartobind Q nano 3mL (8mm)).
  • Binding buffer was 20 mM sodium carbonate pH 9.5
  • elution buffer was 20 mM sodium phosphate pH 6.0 + 0.1 M NaCl.
  • a cationic membrane column (Sartobind S nano 1mL (4mm)) was used. Binding buffer used 20mM sodium carbonate pH 6.0. Elution buffer used 20mM sodium phosphate pH 8.5.
  • BCN-linker and rHSA (Albumedix, Recombumin Elite) (SEQ ID NO: 4) were mixed in the ratio shown in the table below, and the binding reaction was induced for 2 hours in a shaded incubator (23°C, 130 rpm). did
  • the structure of the BCN-linker used is as follows:
  • the purified Uox-AzF was concentrated 10 times and buffer changed (PBS pH 7.4), and the rHSA-linker of the binding reaction was concentrated about 3 times from 1,200mL to 400mL for Uox-rHSA binding, and reacted at the ratio shown in the table below. induced.
  • the total reaction volume was treated for 15 hours in a light-shielded incubator (23° C., 130 rpm) at 1,200 mL.
  • the Uox-rHSA binding reaction was centrifuged and the supernatant filtered for sterilization was subjected to SP-Sepharose high performance on an XK 50/30 column, and the eluted peak was measured.
  • the flow rate was 100 cm/h (33 mL/min)
  • the binding buffer was 20 mM sodium phosphate pH6.0
  • the elution buffer was 20 mM sodium phosphate pH 6.0 + 0.5 M NaCl.
  • Uox-rHSA was confirmed in elutions 1 to 4, and it was found that some of the unconjugated residues were removed during the purification process (see FIGS. 5 to 7).
  • Albumin and the linker (BCN-PEG3-MAL or DBCO-PEG4-MAL) were conjugated at room temperature for 2 hours at a molar ratio of 1:4.
  • the respective yields were 97.6% for DBCO-PEG4-HSA and 96.6% for BCN-PEG3-HSA.
  • Uric acid oxidase with AzF introduced at position 174 and an albumin-linker conjugate (BCN-PEG3-HSA or DBCO-PEG4-HSA) were conjugated at room temperature for 15 hours at a molar ratio of 1:8. Thereafter, separation and purification were performed using cation exchange chromatography (SP-HP), and each fraction was analyzed by SEC-HPLC using size exclusion chromatography (SEC).
  • the use of an APN reactor may have an effect of increasing stability in the body.

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Abstract

La présente demande concerne un conjugué urate oxydase-albumine et son procédé de production, le conjugué urate oxydase-albumine étant produit par liaison d'albumine avec un mutant d'urate oxydase dans lequel un site d'acide aminé non naturel a été spécifiquement introduit. Le conjugué urate oxydase-albumine de la présente demande est produit grâce à un lieur comprenant un groupe bicyclononyne.
PCT/KR2022/009319 2021-06-29 2022-06-29 Procédé de production d'un conjugué urate oxydase-albumine utilisant un lieur bcn et son utilisation WO2023277574A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5541098A (en) * 1989-07-13 1996-07-30 Sanofi Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells
US5700674A (en) * 1995-08-24 1997-12-23 Kikkoman Corporation Mutant uricase, a mutant uricase gene, a novel recombinant DNA, and a process for producing mutant uricase
KR101637010B1 (ko) * 2015-04-24 2016-07-07 광주과학기술원 위치 특이적으로 알부민이 연결된 요산 산화효소 및 단백질에 위치 특이적으로 알부민을 연결하는 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5541098A (en) * 1989-07-13 1996-07-30 Sanofi Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells
US5700674A (en) * 1995-08-24 1997-12-23 Kikkoman Corporation Mutant uricase, a mutant uricase gene, a novel recombinant DNA, and a process for producing mutant uricase
KR101637010B1 (ko) * 2015-04-24 2016-07-07 광주과학기술원 위치 특이적으로 알부민이 연결된 요산 산화효소 및 단백질에 위치 특이적으로 알부민을 연결하는 방법

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABBAS H. K. AL TEMIMI, THOMAS J. BOLTJE, DANIEL ZOLLINGER, FLORIS P.J.T. RUTJES, MARTIN C. FEITERS: "Peptide-appended permethylated β-cyclodextrins with hydrophilic and hydrophobic spacers", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, US , XP055393637, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.7b00319 *
DATABASE Protein GenPept; ANONYMOUS : "Chain A, SERUM ALBUMIN", XP093019735, retrieved from NCBI *

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