WO2023277574A1 - Method for producing urate oxidase-albumin conjugate using bcn linker and use thereof - Google Patents

Method for producing urate oxidase-albumin conjugate using bcn linker and use thereof 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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French (fr)
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/en
Publication of WO2023277574A1 publication Critical patent/WO2023277574A1/en

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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.

Abstract

The present application relates to a urate oxidase-albumin conjugate and a method for producing same, the urate oxidase-albumin conjugate being produced by binding albumin with a urate oxidase mutant having an unnatural amino acid site-specifically introduced therein. The urate oxidase-albumin conjugate of the present application is produced through a linker comprising a bicyclononyne group.

Description

BCN 링커를 이용한 요산산화효소-알부민 컨쥬게이트의 생산 방법 및 이의 용도Method for producing uric acid oxidase-albumin conjugate using BCN linker and use thereof
본 명세서는 비천연아미노산을 포함하는 요산산화효소 변이체와 알부민이 결합된 요산산화효소-알부민 컨쥬게이트의 생산 방법 및 이의 용도에 관한 것이다. 또한, 본 명세서는 BCN 그룹을 포함하는 링커를 이용하여 요산산화효소 변이체 및 알부민을 결합시키는 것을 포함하는 요산산화효소-알부민 컨쥬게이트 생산 방법 및 이의 용도에 관한 것이다.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. In addition, 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.
지난 30년 동안 치료용 단백질은 다양한 질병의 치료에서 임상적인 성공을 거두어 왔으며, 이는 계속해서 제약 분야의 중요한 성장 동기로 작용하고 있다. 하지만, 치료용 단백질은 체내 단백질 분해 효소에 의해 쉽게 파괴되거나 혈중 반감기가 극히 짧아 단 회 투여가 아닌 반복 투여를 하게 되어 면역 원성 등의 부작용이 발생하기 쉽다. 따라서, 치료용 단백질의 개발에서 중요한 고려사항 중 하나는 반복 투여를 피하기 위하여 약효의 지속시간을 연장시키는 것이다. Over the past 30 years, 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. However, 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.
치료용 단백질의 약효 지속성을 높이기 위해 인간 혈청 알부민(Human serum albumin)을 이용하는 것은 효율적인 방법 중 하나이다. 그러나, 멀티-서브유닛과 복잡한 3차 구조를 가지고 있는 치료용 단백질의 경우, 알부민에 의한 효과를 얻기가 쉽지 않다. 이는 알부민의 결합으로 인해 치료용 단백질의 생산 수율이 낮아지거나 활성이 저하되기 때문이다. 이와 같은 문제점을 해결하기 위해 위치 특이적인 단백질 연결이 연구되고 있다. 특정 위치의 결합은 단백질 생산 수율 또는 활성 저하의 문제없이 치료용 단백질-알부민 결합체를 생산할 수 있는 가능성을 보여주며, 이를 이용한 결합체 생산 방법에 대한 연구가 요구된다.One of the efficient methods is to use human serum albumin to increase the durability of therapeutic protein. However, in the case of 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. In order to solve this problem, 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.
추가적으로, 위치 특이적 알부민 결합 외에도 치료용 단백질의 약효 지속시간 연장을 위한 방법들에 대한 연구가 요구된다. Additionally, research on methods for extending the duration of drug efficacy of therapeutic proteins in addition to site-specific albumin binding is required.
이에 따라, 본 발명자들은 요산산화효소-알부민 복합체의 생산 수율 및 요산산화효소에 결합되는 알부민의 개수를 증가시킴을 통해 약효의 지속시간을 연장시기 위한 방법을 연구하였다. 그 결과, 위치 특이적 알부민 결합 및 특정 클릭화학반응을 이용하면 요산산화효소-알부민 복합체에서, 요산산화효소에 결합하는 알부민의 개수를 증가된다는 것을 확인하였다.Accordingly, 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.
본 출원에 개시된 요산산화효소-알부민 컨쥬게이트의 제조 방법을 통해, 위치 특이적으로 알부민을 요산산화효소 변이체에 컨쥬게이트시킬 수 있다. Through the method for preparing a uric acid oxidase-albumin conjugate disclosed in the present application, albumin can be site-specifically conjugated to a uric acid oxidase variant.
본 출원에 개시된 요산산화효소-알부민 컨쥬게이트의 제조 방법을 통해, 요산산화효소-알부민 컨쥬게이트의 제조 수율을 높일 수 있다. Through the method for preparing a uric acid oxidase-albumin conjugate disclosed in the present application, the production yield of the uric acid oxidase-albumin conjugate can be increased.
본 출원에 개시된 요산산화효소-알부민 컨쥬게이트의 제조 방법을 통해, 하나 또는 둘 이상의 알부민이 요산산화효소 변이체에 컨쥬게이트된 요산산화효소-알부민 컨쥬게이트의 제조 수율을 높일 수 있다. Through the method for preparing a uric acid oxidase-albumin conjugate disclosed in the present application, 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.
본 출원의 일 실시예는, BCN 그룹을 포함하는 링커를 이용하여 요산산화효소-알부민 컨쥬게이트를 생산하는 방법을 제공한다.One embodiment of the present application provides a method for producing a urate oxidase-albumin conjugate using a linker containing a BCN group.
본 출원의 일 실시예는, 상기 생산 방법에 따라 생산된 요산산화효소-알부민 컨쥬게이트를 제공한다.One embodiment of the present application provides a uric acid oxidase-albumin conjugate produced according to the above production method.
본 출원의 일 실시예는, 상기 생산 방법에 따라 생산된 요산산화효소-알부민 컨쥬게이트를 포함하는 통풍의 예방 또는 치료용 약학적 조성물을 제공한다.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.
본 출원의 일 실시예는, 상기 생산 방법에 따라 생산된 요산산화효소-알부민 컨쥬게이트를 포함하는 통풍의 예방 또는 개선용 식품 조성물을 제공한다.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.
본 명세서에서 개시하는 요산산화효소-알부민 컨쥬게이트 생산 방법은 요산산화효소의 반감기를 향상시킬 수 있다.The method for producing the uric acid oxidase-albumin conjugate disclosed herein can improve the half-life of uric acid oxidase.
본 명세서에서 개시하는 요산산화효소-알부민 컨쥬게이트 생산 방법은 요산산화효소의 약물 지속성이 증가될 수 있다. The method for producing the uric acid oxidase-albumin conjugate disclosed herein may increase the drug durability of uric acid oxidase.
본 명세서에서 개시하는 요산산화효소-알부민 컨쥬게이트는 면역원성이 감소될 수 있다. The uric acid oxidase-albumin conjugates disclosed herein may have reduced immunogenicity.
본 명세서에서 개시하는 요산산화효소-알부민 컨쥬게이트는 알부민 결합 개수가 증가된 요산산화효소-알부민 컨쥬게이트를 생산할 수 있다.The urate oxidase-albumin conjugate disclosed herein can produce a urate oxidase-albumin conjugate having an increased number of albumin bonds.
도 1은 교반속도 및 내압에 다른 Uox-AzF 생산 균주의 생장 곡선을 나타낸다.Figure 1 shows the growth curves of Uox-AzF producing strains with different agitation rates and internal pressures.
도 2는 Uox-AzF 생산 균주의 배양 결과를 나타낸다.Figure 2 shows the culture results of the Uox-AzF producing strain.
도 3은 Uox-AzF 단백질 발현 SDS-PAGE 분석 결과를 나타낸다.Figure 3 shows the results of SDS-PAGE analysis of Uox-AzF protein expression.
도 4는 Uox-AzF와 rHSA-링커 결합 반응물 순도를 나타낸다.Figure 4 shows the purity of Uox-AzF and rHSA-linker conjugation reactants.
도 5는 Uox-rHSA의 1차 양이온 크로마토그래피 정제 그래프이다.5 is a first cation chromatography purification graph of Uox-rHSA.
도 6 및 도 7은 Uox-rHSA의 1차 양이온 크로마토그래피 정제 순도 분석 결과이다.6 and 7 are results of primary cation chromatography purification purity analysis of Uox-rHSA.
도 8은 Uox-rHSA의 2차 음이온 크로마토그래피 정제 그래프이다.8 is a graph of secondary anion chromatography purification of Uox-rHSA.
도 9는 Uox-rHSA의 2차 음이온 크로마토그래피 정제 순도 분석 결과이다.9 is a result of secondary anion chromatography purification purity analysis of Uox-rHSA.
도 10은 DBCO 그룹 또는 BCN 그룹을 포함하는 링커를 이용하여 생산된 컨쥬게이트의 정제 결과이다.10 is a purification result of a conjugate produced using a linker containing a DBCO group or a BCN group.
도 11은 DBCO 그룹을 포함하는 링커를 이용하여 생산된 컨쥬게이트의 SEC-HPLC 분석 결과이다. 11 is a result of SEC-HPLC analysis of a conjugate produced using a linker containing a DBCO group.
도 12는 BCN 그룹을 포함하는 링커를 이용하여 생산된 컨쥬게이트의 SEC-HPLC 분석 결과이다.12 is a result of SEC-HPLC analysis of a conjugate produced using a linker containing a BCN group.
도 13은 APN 또는 Maleimide 그룹을 포함하는 링커를 이용한 알부민 컨쥬게이션 결과이다.13 is a result of albumin conjugation using a linker containing an APN or maleimide group.
도 14는 APN 또는 Maleimide 그룹을 포함하는 링커를 이용한 알부민 컨쥬게이션의 안정성을 확인한 결과이다.14 is a result of confirming the stability of albumin conjugation using a linker containing an APN or maleimide group.
본 출원은 요산산화효소-알부민 컨쥬게이트의 제조 방법을 제공한다. The present application provides a method for preparing a uric acid oxidase-albumin conjugate.
일 실시예로, 다음을 포함하는 요산산화효소-알부민 컨쥬게이트의 제조 방법이 제공된다:In one embodiment, a method for preparing a uric acid oxidase-albumin conjugate comprising:
(a) 알부민 및 링커를 접촉하여 알부민-링커 컨쥬게이트를 제조함, (a) contacting albumin and a linker to prepare an albumin-linker conjugate;
이때 상기 링커는 일 말단에 제1 클릭화학작용기를 포함하고 다른 말단에 티올 반응성 그룹을 포함하고, 상기 제1 클릭화학작용기는 비시클로노닌(bicyclononyne) 그룹이며, In this case, 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,
이때 상기 링커의 상기 티올 반응성 그룹과 상기 알부민의 시스테인의 티올기 간의 반응을 통해, 알부민-링커 컨쥬게이트가 제조됨, At this time, through the reaction between the thiol reactive group of the linker and the thiol group of the cysteine of the albumin, an albumin-linker conjugate is prepared,
(b) 상기 알부민-링커 컨쥬게이트와 요산산화효소 변이체를 접촉함, (b) contacting the albumin-linker conjugate with a urate oxidase variant;
이때 상기 요산산화효소 변이체는 하나 이상의 비천연아미노산을 포함하고, 상기 비천연아미노산은 상기 제1 클릭화학작용기와 클릭화학반응을 할 수 있는 제2 클릭화학작용기를 포함하고, In this case, the uric acid oxidase variant includes at least one non-natural amino acid, and 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,
이때 상기 알부민-링커 컨쥬게이트의 일 말단의 상기 제1 클릭화학작용기와 상기 비천연아미노산에 포함된 상기 제2 클릭화학작용기의 반응을 통해, 요산산화효소-알부민 컨쥬게이트가 제조됨. At this time, 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.
특정한 실시예에서, 상기 제2 클릭화학작용기는 아자이드 그룹일 수 있다. In a specific embodiment, the second click chemical functional group may be an azide group.
특정한 실시예에서, 상기 비천연아미노산은 p-azido-L-phenylalanine일 수 있다. In certain embodiments, the unnatural amino acid may be p-azido-L-phenylalanine.
특정한 실시예에서, 상기 요산산화효소 변이체는 3개의 요산산화효소 변이체 서브유닛 및 1개의 야생형 요산산화효소 서브유닛으로 구성되거나, 4개의 요산산화효소 변이체 서브유닛으로 구성되는, 사량체 형태일 수 있다. In certain embodiments, 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. .
특정한 실시예에서, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 아미노산 서열을 포함할 수 있다. In a specific embodiment, 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 tyrosine, 259th phenylalanine, 265th tryptophan and 279th phenylalanine is p-Azido-L-phenylalanine (AzF).
특정한 실시예에서, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 160번째 트립토판, 또는 174번째 트립토판 중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 아미노산 서열을 포함할 수 있다. In a specific embodiment, 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). can include
특정한 실시예에서, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 아미노산 서열을 포함할 수 있다. In a specific embodiment, 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. position 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 , position 162 phenylalanine, position 163 tyrosine, position 165 tyrosine, position 170 phenylalanine, position 189 tryptophan, position 191 tryptophan, position 200 tyrosine, position 211 phenylalanine, position 215 tyrosine, position 226 phenylalanine, position 239 phenylalanine, position 253 tyrosine, 257 tyrosine at position 264, phenylalanine at position 265, tryptophan at position 271, phenylalanine at position 281, and at least one amino acid selected from tyrosine at position 282 may include an amino acid sequence in which p-Azido-L-phenylalanine (AzF) is substituted. .
특정한 실시예에서, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 163번째 타이로신(tyrosine), 170번째 페닐알라닌 (phenylalanine), 200번째 타이로신, 및 271번째 트립토판(tryptophan) 중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 아미노산 서열을 포함할 수 있다. In a specific embodiment, 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).
특정한 실시예에서, 상기 링커는 하기의 화학식 1의 구조를 가질 수 있다:In certain embodiments, the linker may have the structure of Formula 1 below:
[화학식 1][Formula 1]
F1 - L - F2,F 1 - L - F 2 ,
이때, At this time,
F1은 상기 제1 클릭화학작용기를 포함하는 제1 반응 작용기이고,F 1 is a first reactive functional group including the first click chemical functional group;
F2는 상기 티올 반응성 그룹을 포함하는 제2 반응 작용기이고, 이때 상기 티올 반응성 그룹은 말레이미드 그룹 또는 3-아릴프로피올니트릴(3-Arylpropiolonitriles) 그룹이고, 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;
L은 치환 또는 비치환된 C1-50 알킬렌, 치환 또는 비치환된 C1-50 헤테로알킬렌, 치환 또는 비치환된 C2-50 알케닐렌, 치환 또는 비치환된 C2-50 헤테로알케닐렌, 치환 또는 비치환된 C2-50 알키닐렌, 치환 또는 비치환된 C2-50 헤테로알키닐렌이고, 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,
이때 상기 헤테로알킬렌, 헤테로알케닐렌, 및 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함하고, 이때 상기 헤테로원자는, 각각 독립적으로, O, S, 및 N 중에서 선택되고, wherein the heteroalkylene, heteroalkenylene, and heteroalkynylene each independently include one or more heteroatoms, wherein the heteroatoms are each independently selected from O, S, and N;
이때 상기 치환된 알킬렌, 치환된 헤테로알킬렌, 치환된 알케닐렌, 치환된 헤테로알케닐렌, 치환된 알키닐렌, 및 치환된 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 치환기를 포함하고, 이때 상기 치환기는, 각각 독립적으로 할로겐, C1-3알킬, -NH2, =O, =S, -OH, 및 -SH 중에서 선택됨.In this case, the substituted alkylene, substituted heteroalkylene, substituted alkenylene, substituted heteroalkenylene, substituted alkynylene, and substituted heteroalkynylene each independently include one or more substituents, wherein the Substituents are each independently selected from halogen, C 1-3 alkyl, -NH 2 , =O, =S, -OH, and -SH.
특정한 실시예에서, 상기 링커는 하기의 화학식 1-2의 구조를 가질 수 있다:In certain embodiments, the linker may have a structure of Formula 1-2:
[화학식 1-2][Formula 1-2]
Figure PCTKR2022009319-appb-I000001
,
Figure PCTKR2022009319-appb-I000001
,
이때 np는 1 이상 6 이하의 정수이고, In this case, np is an integer of 1 or more and 6 or less,
이때 L1은 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌이고, In this case, L 1 is a bond, or an unsubstituted C 1-3 alkylene or an unsubstituted C 1-3 heteroalkylene;
이때 L3는 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌임. In this case, L 3 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
일 실시예에서, 상기 링커는 하기의 화학식 1-3의 구조를 가질 수 있다:In one embodiment, the linker may have a structure of Formula 1-3 below:
[화학식 1-3][Formula 1-3]
Figure PCTKR2022009319-appb-I000002
,
Figure PCTKR2022009319-appb-I000002
,
이때 np는 1 이상 6 이하의 정수임. In this case, np is an integer of 1 or more and 6 or less.
특정한 실시예에서, 상기 알부민은 인간 혈청 알부민 또는 이의 변이체일 수 있다. In certain embodiments, the albumin may be human serum albumin or a variant thereof.
특정한 실시예에서, 상기 알부민은 서열번호 4 내지 15 중에서 선택된 어느 하나의 아미노산 서열을 포함할 수 있다. In a specific embodiment, the albumin may include any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
특정한 실시예에서, 상기 알부민은 인간 혈청 알부민 또는 이의 변이체이고, 이때 상기 (a)에서, 상기 티올 반응성 그룹과 반응하는 상기 알부민의 시스테인의 티올기는 34번 시스테인의 티올기일 수 있다. In a specific embodiment, 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.
특정한 실시예에서, 상기 요산산화효소-알부민 컨쥬게이트는 다음을 포함할 수 있다:In certain embodiments, the uric acid oxidase-albumin conjugate can include:
1개의 서브유닛-알부민 컨쥬게이트, 및 3개의 요산산화효소 변이체 서브유닛; 2개의 서브유닛-알부민 컨쥬게이트, 및 2개의 요산산화효소 변이체 서브유닛; 3개의 서브유닛-알부민 컨쥬게이트, 및 1개의 요산산화효소 변이체 서브유닛; 또는 4개의 서브유닛-알부민 컨쥬게이트, 이때 상기 서브유닛-알부민 컨쥬게이트는 하나의 알부민이 하나의 요산산화효소 변이체 서브유닛에 접합된 것임. 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.
일 실시예로, 하나 이상의 알부민이 요산산화효소 변이체에 접합된, 하기의 화학식 3-2의 구조를 갖는 요산산화효소-알부민 컨쥬게이트가 제공된다:In one embodiment, 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:
[화학식 3-2][Formula 3-2]
Figure PCTKR2022009319-appb-I000003
,
Figure PCTKR2022009319-appb-I000003
,
이때,At this time,
n은 1 이상 4 이하의 정수이고,n is an integer of 1 or more and 4 or less;
Uoxv는 요산산화효소 변이체이고, 이때 상기 요산산화효소 변이체는 하나 이상의 비천연아미노산을 포함하고, 이때 상기 비천연아미노산은 아자이드 그룹을 포함하고, 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;
A는 알부민이고,A is albumin;
X1은 상기 비천연아미노산에 포함된 상기 아자이드 그룹과 비시클로노닌(bicyclononyne) 그룹이 반응하여 형성된 하기의 구조를 포함하고, 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,
Figure PCTKR2022009319-appb-I000004
,
Figure PCTKR2022009319-appb-I000004
,
X2는 알부민에 포함된 시스테인의 티올기와 티올 반응성 작용기가 반응하여 형성된 하기의 구조 중 어느 하나를 포함하고, 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;
Figure PCTKR2022009319-appb-I000005
및
Figure PCTKR2022009319-appb-I000006
,
Figure PCTKR2022009319-appb-I000005
and
Figure PCTKR2022009319-appb-I000006
,
이때 S는 상기 알부민의 시스테인의 티올기로부터 유래되며, At this time, S is derived from the thiol group of the cysteine of the albumin,
np는 1 이상 6 이하의 정수이며, np is an integer of 1 or more and 6 or less,
L1은 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌이고, L 1 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene;
L3는 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌이며, L 3 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene;
이때 상기 헤테로알킬렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함하고, 이때 상기 헤테로원자는, 각각 독립적으로, O, S, 또는 N임. wherein the heteroalkylene, each independently, includes one or more heteroatoms, wherein the heteroatoms are, each independently, O, S, or N.
특정한 실시예에서, 상기 요산산화효소 변이체는 4개의 요산산화효소 변이체 서브유닛으로 구성되는 사량체 형태일 수 있다. In a specific embodiment, the uric acid oxidase variant may be in the form of a tetramer consisting of four uric acid oxidase variant subunits.
특정한 실시예에서, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 아미노산 서열을 포함할 수 있다. In a specific embodiment, 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 tyrosine, 259th phenylalanine, 265th tryptophan and 279th phenylalanine is p-Azido-L-phenylalanine (AzF).
특정한 실시예에서, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 아미노산 서열을 포함할 수 있다. In a specific embodiment, 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. position 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 , position 162 phenylalanine, position 163 tyrosine, position 165 tyrosine, position 170 phenylalanine, position 189 tryptophan, position 191 tryptophan, position 200 tyrosine, position 211 phenylalanine, position 215 tyrosine, position 226 phenylalanine, position 239 phenylalanine, position 253 tyrosine, 257 tyrosine at position 264, phenylalanine at position 265, tryptophan at position 271, phenylalanine at position 281, and at least one amino acid selected from tyrosine at position 282 may include an amino acid sequence in which p-Azido-L-phenylalanine (AzF) is substituted. .
특정한 실시예에서, 상기 알부민은 인간 혈청 알부민 또는 이의 변이체일 수 있다. In certain embodiments, the albumin may be human serum albumin or a variant thereof.
특정한 실시예에서, 상기 알부민은 서열번호 4 내지 15 중에서 선택된 어느 하나의 아미노산 서열을 포함할 수 있다. In a specific embodiment, the albumin may include any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
특정한 실시예에서, 상기 알부민은 인간 혈청 알부민 또는 이의 변이체이고, X2의 S는 상기 알부민의 34번 시스테인의 티올기로부터 유래된 것일 수 있다. In a specific embodiment, 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.
특정한 실시예에서, 상기 요산산화효소-알부민 컨쥬게이트는 다음 중 어느 하나로 구성될 수 있다:In certain embodiments, the uric acid oxidase-albumin conjugate can consist of any of the following:
1개의 서브유닛-알부민 컨쥬게이트 및 3개의 요산산화효소 변이체 서브유닛의 복합체; 2개의 서브유닛-알부민 컨쥬게이트 및 2개의 요산산화효소 변이체 서브유닛의 복합체; 3개의 서브유닛-알부민 컨쥬게이트 및 1개의 요산산화효소 변이체 서브유닛의 복합체; 및 4개의 서브유닛-알부민 컨쥬게이트의 복합체, 이때 상기 서브유닛-알부민 컨쥬게이트는 하나의 알부민이 하나의 요산산화효소 변이체 서브유닛에 접합된 것임. 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; and a complex of four subunit-albumin conjugates, wherein the subunit-albumin conjugate is one albumin conjugated to one urate oxidase variant subunit.
일 실시예로, 본 출원의 요산산화효소-알부민 컨쥬게이트를 포함하는, 종양용해증후군(tumor lysis syndrome, TLS), 고요산혈증, 통풍, 관절내 요산염 결정의 침착, 요산염 결정의 침착으로 인한 급성 통풍성 관절염, 요로 결석증, 신결석증 및 통풍성 신병증으로 이루어진 군으로부터 선택된 1종 이상의 질환의 예방 또는 치료용 약학적 조성물이 제공된다. In one embodiment, including the uric acid oxidase-albumin conjugate of the present application, tumor lysis syndrome (TLS), 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.
용어의 정의Definition of Terms
달리 정의되지 않는 한, 본 명세서에서 사용되는 모든 기술적 및 과학적 용어는 본 발명이 속하는 기술분야의 당업자에 의해 통상적으로 이해되는 것과 동일한 의미를 가진다. 본 명세서에서 언급된 모든 간행물, 특허 및 기타 다른 참고문헌은 전체가 참고로 포함된다. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents and other references mentioned herein are incorporated by reference in their entirety.
일부 실시예에서, 화학구조는 대응되는 화학명과 함께 개시된다. 분쟁이 발생하는 경우, 화학명보다 우선하여, 화학구조에 의하여 의미를 파악하여야 한다. In some embodiments, 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.
"할로겐" 또는 "할로"는 주기율표에서 할로겐 족 원소에 포함된 플루오르, 염소, 브롬 및 요오드를 포함하는 군을 의미한다."Halogen" or "halo" refers to a group containing fluorine, chlorine, bromine and iodine included in the halogen group of elements in the periodic table.
본 출원에서 사용되는 용어 "헤테로"는 하나 이상의 헤테로원자를 포함하는 화합물 또는 그룹을 지칭한다. 용어 "헤테로원자"는 탄소나 수소가 아닌 원자로서, 예를 들어 B, Si, N, P, O, S, 및 Se, 이중 바람직 하게는 N, O, 및 S와 같은 다가 원소나 F, Cl, Br, 및 I와 같은 일가 원소 등을 포함하나 이에 한정되는 것은 아니다.As used herein, the term “hetero” refers to a compound or group containing one or more heteroatoms. The term "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.
"알킬" 또는 "알칸"은 사슬 모양, 또는 가지 모양의 탄화수소로서 완전히 포화된 것이다. 예를 들어, 사슬 모양 또는 가지 모양 알케닐 기는, 2 내지 약 50 개, 2 내지 20개, 또는 2 내지 10개의 탄소 원자를 가질 수 있다. 사슬 모양 및 가지 모양 알킬기는 예를 들어, 메틸, 에틸, n-프로필, iso-프로필, n-부틸, sec-부틸, tert-부틸, iso-부틸, 펜틸(pentyl), 헥실(hexyl), 헵틸(heptyl), 옥틸(octyl), 노닐(nonyl), 및 데실(decyl) 등일 수 있다. 알킬기는 고리형 고리형 구조를 포함할 수 있다. "Cx-y"라는 용어는, 예를 들어, 알킬 그룹과 함께 사용되는 경우 사슬 또는 고리에 x 내지 y 개의 탄소를 포함하는 잔기를 포함하도록 의도된다. 예를 들어, "Cx-y알킬"이라는 용어는 치환된 또는 비치환된, 사슬 모양 알킬기, 가지 모양 알킬 기, 또는 고리형 구조를 포함하는 알킬기로서 사슬에 x 내지 y 개의 탄소를 포함하는 것을 포함하고, 예시적으로 디플루오로메틸 및 2,2,2-트리플루오로에틸, 등과 같은 할로알킬 기를 포함하는 것을 의미한다. C0 알킬은 수소를 의미한다. C1-4 알킬의 예에는 메틸, 에틸, n-프로필, iso-프로필, n-부틸, sec-부틸, tert-부틸, iso-부틸, 디플루오로메틸, 및 2,2,2-트리플루오로에틸 등이 포함되나 이에 제한되지 않는다."Alkyl" or "alkane" is a chain or branched hydrocarbon that is fully saturated. For example, 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. The term "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. For example, the term "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. Examples of C 1-4 alkyl 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.
본 출원에서 사용되는 용어 "헤테로알킬"은 하나 이상의 헤테로원자를 포함하는 알킬을 의미한다. As used herein, the term "heteroalkyl" refers to an alkyl containing one or more heteroatoms.
"알케닐" 또는 "알켄"은 사슬 모양 또는 가지 모양의 비방향족 탄화수소로서 하나 이상의 이중 결합을 포함한다. 예를 들어, 사슬 모양 또는 가지 모양 알케닐 기는, 2 내지 약 50 개, 2 내지 20개, 또는 2 내지 10개의 탄소 원자를 가질 수 있다. 알케닐 기는 고리형 구조를 포함할 수 있다. "Alkenyl" or "alkene" is a chain or branched non-aromatic hydrocarbon containing one or more double bonds. For example, 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.
본 출원에서 사용되는 용어 "헤테로알켄"은 하나 이상의 헤테로원자를 포함하는 알켄을 의미한다. As used herein, the term “heteroalkene” refers to an alkene containing one or more heteroatoms.
"알키닐" 또는 "알킨"은 사슬 모양 또는 가지 모양의 비방향족 탄화수소로서, 하나 이상의 삼중 결합을 포함한다. 예를 들어, 사슬 모양 또는 가지 모양 알키닐 기는, 2 내지 약 50 개, 2 내지 20개, 또는 2 내지 10개의 탄소 원자를 가질 수 있다. 알키닐 기는 하나 이상의 삼중 결합 외에도 하나 이상의 이중 결합을 포함할 수 있다. 알키닐 기는 고리형 구조를 포함할 수 있다. "Alkynyl" or "alkyne" is a chain or branched non-aromatic hydrocarbon containing one or more triple bonds. For example, 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.
본 출원에서 사용되는 용어 "헤테로알킨"은 하나 이상의 헤테로원자를 포함하는 알킨을 의미한다. As used in this application, the term "heteroalkyn" means an alkyne containing one or more heteroatoms.
그 자체인 분자로 쓰이거나 또는 분자의 일부로 사용되는 용어 "알킬렌"은 알킬로부터 유도된 2가 라디칼(divalent radical)을 의미한다. 용어 "알킬렌"은, 필요에 따라, "치환된" 또는 "비치환된"의 용어와 함께 사용될 수 있다. 용어 "알킬렌"이 "치환된" 또는 "비치환된"의 용어와 함께 사용되지 않는 경우에는, 용어 "알킬렌"은 치환된 알킬렌 및 비치환된 알킬렌을 모두 포함하는 것으로 의도된다. 알킬렌은 -CH2-, -CH2CH2-, -CH2CH2CH2-, 및 -CH2CH2CH2CH2- 으로 예시될 수 있으나, 이에 제한되지 않는다. 예를 들어, 알킬렌은 C2 알킬렌과 같이 쓰일 수 있으며, 이는 주사슬에 두개의 탄소 원자를 포함하는 알킬렌 그룹을 의미한다. 예시적으로, 본 출원에서 "Cx-y 알킬렌" 은 주사슬에 X 내지 Y 수의 탄소 원자를 갖는, 치환된 또는 비치환된 알킬렌을 모두 포함하는 알킬렌을 의미하는 것으로 사용된다. The term "alkylene" when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from an alkyl. The term "alkylene" may be used with the terms "substituted" or "unsubstituted", as appropriate. When the term "alkylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "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. For example, alkylene can be written as C 2 alkylene, which refers to an alkylene group containing two carbon atoms in the main chain. Illustratively, in this application, "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.
그 자체인 분자로 쓰이거나 또는 분자의 일부로 사용되는 용어 "헤테로알킬렌"은 헤테로알킬로부터 유도된 2가 라디칼을 의미한다. 용어 "헤테로알킬렌"은 필요에 따라, "치환된" 또는 "비치환된"의 용어와 함께 사용될 수 있다. 용어 "헤테로알킬렌"이 "치환된" 또는 "비치환된"의 용어와 함께 사용되지 않는 경우에는, 용어 "헤테로알킬렌"은 치환된 헤테로알킬렌 또는 비치환된 헤테로알킬렌을 모두 포함하는 것으로 의도된다. 예시적으로 헤테로알킬렌 그룹은 ―CH2―CH2―O―CH2―CH2―, 및 ―CH2―O―CH2―CH2―NH―CH2― 를 포함하나 이에 제한되지 않는다. 헤테로알킬렌 그룹은 사슬 또는 가지의 말단이 아닌 위치에 하나 이상의 헤테로원자를 포함할 수 있고, 각각의 헤테로원자는 같거나 다를 수 있다. 헤테로알킬렌 그룹은 사슬 또는 가지의 말단 각각, 또는 모든 말단에 하나 이상의 헤테로원자를 포함할 수 있고, 각각의 헤테로원자는 같거나 다를 수 있다. 예시적으로, 본 출원에서 "Cx-y 헤테로알킬렌" 은 주사슬에 X 내지 Y 수의 탄소 원자를 갖는, 치환 또는 비치환된 헤테로알킬렌을 모두 포함하는 것으로 사용된다.The term “heteroalkylene” when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkyl. The term “heteroalkylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. When the term "heteroalkylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "heteroalkylene" includes both substituted and unsubstituted heteroalkylenes. it is intended to Exemplary 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. Illustratively, in this application, “C xy heteroalkylene” is used to include all substituted or unsubstituted heteroalkylenes having X to Y number of carbon atoms in the main chain.
그 자체인 분자로 쓰이거나 또는 분자의 일부로 사용되는 용어 "알케닐렌"은 알켄으로부터 유도된 2가 라디칼을 의미한다. 용어 “알케닐렌”은 필요에 따라, “치환된” 또는 "비치환된"의 용어와 함께 사용될 수 있다. 용어 "알케닐렌"이 "치환된" 또는 "비치환된"의 용어와 함께 사용되지 않는 경우에는, 용어 "알케닐렌"은 치환된 알케닐렌 또는 비치환된 알케닐렌을 모두 포함하는 것으로 의도된다. 예시적으로 알케닐렌 그룹은 -CH=CH-, -CH2CH=CHCH2-, 및 -CH=CH-CH=CH- 을 포함하나 이에 한정되는 것은 아니다. 예시적으로, 본 출원에서 "Cx-y 알케닐렌" 은 주사슬에 X 내지 Y 수의 탄소 원자를 갖는, 치환 또는 비치환된 알케닐렌을 모두 포함하는 것으로 사용된다.The term "alkenylene" when used as a molecule by itself or as part of a molecule refers to a divalent radical derived from an alkene. The term “alkenylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. When the term "alkenylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "alkenylene" is intended to include both substituted and unsubstituted alkenylene. Illustratively, the alkenylene group includes, but is not limited to, -CH=CH-, -CH 2 CH=CHCH 2 -, and -CH=CH-CH=CH-. Illustratively, in this application, "C xy alkenylene" is used to include all substituted or unsubstituted alkenylene having X to Y number of carbon atoms in the main chain.
그 자체인 분자로 쓰이거나 또는 분자의 일부로 사용되는 용어 "헤테로알케닐렌"은 헤테로알켄으로부터 유도된 2가 라디칼을 의미한다. 용어 “헤테로알케닐렌”은 필요에 따라, “치환된” 또는 "비치환된"의 용어와 함께 사용될 수 있다. 용어 "헤테로알케닐렌"이 "치환된" 또는 "비치환된"의 용어와 함께 사용되지 않는 경우에는, 용어 "헤테로알케닐렌"은 치환된 헤테로알케닐렌 또는 비치환된 헤테로알케닐렌을 모두 포함하는 것으로 의도된다. 테로알케닐렌 그룹은 사슬 또는 가지의 말단이 아닌 위치에 하나 이상의 헤테로원자를 포함할 수 있고, 각각의 헤테로원자는 같거나 다를 수 있다. 헤테로알케닐렌 그룹은 사슬 또는 가지의 말단 각각, 또는 모든 말단에 하나 이상의 헤테로원자를 포함할 수 있고, 각각의 헤테로원자는 같거나 다를 수 있다. The term "heteroalkenylene" when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkene. The term “heteroalkenylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. 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.
그 자체인 분자로 쓰이거나 또는 분자의 일부로 사용되는 용어 "알키닐렌"은 알킨으로부터 유도된 2가 라디칼을 의미한다. 용어 “알키닐렌”은 필요에 따라, “치환된” 또는 "비치환된"의 용어와 함께 사용될 수 있다. 용어 "알키닐렌"이 "치환된" 또는 "비치환된"의 용어와 함께 사용되지 않는 경우에는, 용어 "알키닐렌"은 치환된 알키닐렌 또는 비치환된 알키닐렌을 모두 포함하는 것으로 의도된다. 예컨데, 알키닐렌 그룹은 -C≡C-, -CH2C≡CCH2-, 및 -C≡C-C≡C-를 포함하나 이에 한정되는 것은 아니다. 예시적으로, 본 출원에서 "Cx-y 알키닐렌" 은 주사슬에 X 내지 Y 수의 탄소 원자를 갖는, 치환 또는 비치환된 알키닐렌을 모두 포함하는 것으로 사용된다.The term "alkynylene" when used as a molecule by itself or as part of a molecule refers to a divalent radical derived from an alkyne. The term “alkynylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. In cases where the term "alkynylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "alkynylene" is intended to include both substituted and unsubstituted alkynylenes. For example, an alkynylene group includes, but is not limited to, -C≡C-, -CH 2 C≡CCH 2 -, and -C≡CC≡C-. Illustratively, in this application, "C xy alkynylene" is used to include all substituted or unsubstituted alkynylenes having X to Y number of carbon atoms in the main chain.
그 자체인 분자로 쓰이거나 또는 분자의 일부로 사용되는 용어 "헤테로알키닐렌"은 헤테로알킨으로부터 유도된 2가 라디칼을 의미한다. 용어 “헤테로알키닐렌”은 필요에 따라, “치환된” 또는 "비치환된"의 용어와 함께 사용될 수 있다. 용어 "헤테로알키닐렌"이 "치환된" 또는 "비치환된"의 용어와 함께 사용되지 않는 경우에는, 용어 "헤테로알키닐렌"은 치환된 헤테로알키닐렌 또는 비치환된 헤테로알키닐렌을 모두 포함하는 것으로 의도된다. 헤테로알키닐렌 그룹은 사슬 또는 가지의 말단이 아닌 위치에 하나 이상의 헤테로원자를 포함할 수 있고, 각각의 헤테로원자는 같거나 다를 수 있다. 헤테로알키닐렌 그룹은 사슬 또는 가지의 말단 각각, 또는 모든 말단에 하나 이상의 헤테로원자를 포함할 수 있고, 각각의 헤테로원자는 같거나 다를 수 있다.The term "heteroalkynylene" when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkyne. The term “heteroalkynylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. When the term "heteroalkynylene" is not used with the terms "substituted" or "unsubstituted", the term "heteroalkynylene" includes both substituted and unsubstituted heteroalkynylene. it is intended to 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.
본 출원에서 사용되는 용어 "치환된"은 원자의 원자가(valence)가 정상이고 치환된 화합물이 안정적인, 원자상의 하나 이상의 수소 원자가 중수소 및 수소 변이체(variants)를 포함하는 치환기로 치환됨을 의미한다. 치환기가 산소(즉, =O)인 경우, 이는 두개의 수소 원자가 치환되었음을 의미한다. 하나의 치환기가 할로겐(예를 들어, Cl, F, Br, 및 I 등)인 경우, 이는 하나의 수소원자가 할로겐으로 치환되었음을 의미한다. 치환기가 하나의 그룹에 두개 이상 존재할 경우, 상기 그룹에 존재하는 치환기는 같거나 다를 수 있다. 달리 명시되지 않는 한, 치환기의 유형 및 수는 화학적으로 달성 가능한 한, 임의적일 수 있다. 예시적으로, 치환기는 할로겐, C1-3알킬, -NH2, =O, =S, -OH, 및 -SH 중에서 선택될 수 있으나 이에 제한되지 않는다. 예를 들어, 치환된 C10-20 알킬렌은 주사슬에 연결된 하나 이상의 수소원자가 치환기로 치환된 것을 의미할 수 있으며, 이때 각각의 치환기는 독립적으로 선택될 수 있다. As used herein, the term "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. When the substituent is oxygen (i.e. =O), it means that two hydrogen atoms are substituted. When one substituent is a halogen (eg, Cl, F, Br, and I, etc.), it means that one hydrogen atom has been replaced with a halogen. When two or more substituents are present in one group, the 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. Illustratively, the substituent may be selected from halogen, C 1-3 alkyl, -NH 2 , =O, =S, -OH, and -SH, but is not limited thereto. For example, 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.
본 출원의 화합물은 특정 기하(geometric) 또는 입체 이성질체 형태를 가질 수 있다. 달리 명시되지 않고 본 출원에 화합물이 개시되는 경우, 상기 화합물의, 시스 및 트랜스 이성질체, (-)- 및 (+)- 거울상 이성질체(enantiomers), (R)- 및 (S)- 거울상 이성질체, 부분입체이성질체, (D)- 이성질체, (L)-이성질체, 및 라세미체 등의 이성질체는 본 출원의 범위에 포함된다. 즉, 본 출원에 개시된 화학식 또는 구조에 이성질체와 관련된 특별한 표시(예를 들어, *,
Figure PCTKR2022009319-appb-I000007
,
Figure PCTKR2022009319-appb-I000008
, 및
Figure PCTKR2022009319-appb-I000009
등)가 없는 경우, 개시된 화학식 또는 구조는 가능한 모든 이성질체를 포함함을 의미한다. 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, *,
Figure PCTKR2022009319-appb-I000007
,
Figure PCTKR2022009319-appb-I000008
, and
Figure PCTKR2022009319-appb-I000009
etc.), the formula or structure disclosed is meant to include all possible isomers.
본 명세서에서 사용되는 "약"이라는 용어는 어떤 수량에 거의 가까운 정도를 의미하며, 참조 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이에 대해 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 또는 1% 정도로 변하는 양, 수준, 값, 수, 빈도, 퍼센트, 치수, 크기, 양, 중량 또는 길이를 의미한다.As used herein, 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.
본 명세서에서 사용되는 "천연 아미노산(natural amino acid)" 또는 "표준 아미노산(standard amino acid)"이라는 용어는 유기체(organism)의 체내에서 유전자의 전사 및 번역 과정을 통해 합성되는 20종의 아미노산을 통틀어 의미한다. 구체적으로, 상기 표준 아미노산은 알라닌(Alanine; Ala, A), 아르기닌(Arginine; Arg, R), 아스파라긴(Asparagine; Asn, N), 아스파르트산(Aspartic acid; Asp, D), 시스테인(Cysteine; Cys, C), 글루탐산(Glutamic acid; Glu, E), 글루타민(Glutamine; Gln, Q), 글리신(Glycine; Gly, G), 히스티딘(Histidine; His, H), 이소류신(Isoleucine; Ile, I), 류신(Leucine; Leu, L), 리신(Lysine; Lys K), 메티오닌(Methionine; Met, M), 페닐알라닌(Phenylalanine; Phe, F), 프롤린(Proline; Pro, P), 세린(Serine; Ser, S), 트레오닌(Threonine; Thr, T), 트립토판(Tryptophan; Trp, W), 티로신(Tyrosine; Tyr, Y), 및 발린(Valine; Val, V)을 포함한다. 상기 표준 아미노산 각각은 모두 대응하는 DNA 코돈이 존재하며, 일반적인 아미노산 일문자 또는 세문자 표기법으로 나타낼 수 있다. 상기 표준 아미노산이라는 용어가 지칭하는 대상은 문맥에 따라 적절하게 해석되어야 하며, 그 외 통상의 기술자가 인식할 수 있는 의미를 모두 포함한다.As used herein, 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). 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.
본 명세서에서 사용되는 “비천연 아미노산(nonnatural amino acid)”이라는 용어는, 체내에서 합성되지 않고 인공적으로 합성한 아미노산을 지칭한다. 상기 비천연 아미노산은 예들 들어, 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, 및 4-(1,2,4,5-tetrazin-3-yl) phenylalanine (frTet), 등이 있다. 상기 비천연 아미노산은 이에 대응하는 DNA 코돈이 존재하지 않으며, 일반적인 아미노산 일문자 또는 세문자 표기법으로 나타낼 수 없으므로, 다른 문자를 사용하여 표기하며, 추가적으로 보충하여 설명한다. 상기 비천연 아미노산이라는 용어가 지칭하는 대상은 문맥에 따라 적절하게 해석되어야 하며, 그 외 통상의 기술자가 인식할 수 있는 의미를 모두 포함한다.The term “nonnatural amino acid” as used herein 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. Since the 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.
달리 서술하지 않는 한, 본 명세서에서 아미노산 서열을 기재할 때는 아미노산 일문자 표기법, 또는 세문자 표기법을 사용하여, N-터미널에서 C-터미널 방향으로 기재한다. 예를 들어, RNVP로 표기하는 경우, N-터미널에서 C-터미널 방향으로 아르기닌(arginine), 아스파라긴(asparagine), 발린(valine), 및 프롤린(proline)이 차례로 연결된 펩타이드를 의미한다. 또 다른 예를 들어, Thr-Leu-Lys로 표기하는 경우, N-터미널에서 C-터미널 방향으로 트레오닌(Threonine), 류신(Leucine), 및 리신(Lysine)이 차례로 연결된 펩타이드를 의미한다. 상기 일문자 표기법, 또는 세문자 표기법으로 나타낼 수 없는 아미노산의 경우, 다른 문자를 사용하여 표기하며, 추가적으로 보충하여 설명한다.Unless otherwise stated, when describing an 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. For example, 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. For another example, 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. In the case of 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.
용어 "치료"는 이롭거나 바람직한 임상적 결과를 수득하기 위한 접근을 의미한다. 본 발명의 목적을 위해서, 이롭거나 바람직한 임상적 결과는 제한적으로, 증상의 완화, 질병 범위의 감소, 질병 상태의 안정화 (즉, 악화되지 않음), 질병 진행의 지연 또는 속도의 감소, 질병의 예방, 질병 상태의 개선 또는 일시적 완화 및 경감 (부분적이거나 전체적으로), 검출 가능하거나 또는 검출되지 않거나의 여부를 포함한다. 상기 치료는 치료학적 치료 및 예방적 또는 예방조치 방법 모두를 가리킨다. The term "treatment" refers to an approach for obtaining beneficial or desirable clinical results. For purposes of this invention, 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.
"대상"은 본 발명의 분자에 의해 달성될 수 있는 처치를 필요로 하는 동물을 지칭한다. 본 발명에 따라 처치될 수 있는 동물에는 척추동물이 포함되고, 포유동물 예컨대 쥐과, 소과, 개과, 말과, 고양이과, 양과, 돼지과 및 영장류 (인간 및 비-인간 영장류 포함) 동물이 특히 바람직한 예이다."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. .
본 명세서에서 사용되는 용어 “요산산화효소-알부민 컨쥬게이트”는 요산산화효소 변이체와 알부민이 결합된 복합체를 의미한다. 상기 컨쥬게이트 용어는 복합체와 상호교환적으로 사용될 수 있다. 본 명세서에서 사용되는 요산산화효소-알부민 복합체 또는 요산산화효소-알부민 컨쥬게이트는 사량체 형태의 요산산화효소 변이체에 알부민이 1개 결합된 복합체, 알부민이 2개 결합된 복합체, 및 알부민이 3개 결합된 복합체 및 알부민이 4개 결합된 복합체를 포함한다. 일 구체예에서 상기 결합은 링커를 통한 화학적 결합일 수 있다.As used herein, 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. As used herein, 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. In one embodiment, the bond may be a chemical bond through a linker.
본 명세서에서 사용되는 용어 “클릭화학(click-chemistry)”은 Scripps Research Institute의 K. Barry Sharpless에 의하여, 두 개의 분자가 빠르고 안정적으로 공유 결합을 형성하도록 설계된 상보적인 화학작용기 및 화학 반응을 설명하기 위해 도입된 화학적 개념이다. 본 명세서의 클릭화학은 특정한 반응을 의미하는 것이 아닌, 빠르고 안정적인 반응의 개념을 의미한다. 일 실시예로, 클릭화학으로 분자들 사이의 결합을 형성하기 위해서는 몇 가지 조건을 만족해야 한다. 상기 조건은 높은 수득률, 반응 자리에 대한 뛰어난 선택성, 모듈식으로 작동하여 유기적으로 분자가 결합되는 것 및 열역학적으로 안정화된 방향으로 진행되어 빠르고 정확한 생성물을 만드는 것이다. 본 명세서의 클릭화학은 클릭화학작용기(예를 들어, 말단 알킨 (terminal alkyne), 아자이드 (azide), 스트레인된 알킨(strained alkyne), 다이엔 (diene), 친다이엔체 (dienophile), 트랜스 시클로옥틴(trans-cyclooctene), 알켄 (alkene), 티올 (thiol), 테트라진 (tetrazine), DBCO(dibenzocyclooctyne) 및 비시클로노닌(bicyclononyne, bicyclo[6.1.0]non-4-yne)을 포함)중에서 서로 반응성을 갖는 쌍이 반응하는 것을 포함한다. 일 예시로, 본 명세서의 클릭화학은 비시클로노닌과 아자이드의 반응을 포함한다.The term “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. As an example, click chemistry herein includes the reaction of bicyclononine and azide.
이하, 발명의 구체적인 내용을 개시한다.Hereinafter, specific details of the invention are disclosed.
Ⅰ. 요산산화효소-알부민 컨쥬게이트 생산 방법I. Method for producing uric acid oxidase-albumin conjugate
요산산화효소-알부민 컨쥬게이트는 요산산화효소 변이체, 알부민, 및 링커가 반응하여 제조될 수 있다. A urate oxidase-albumin conjugate can be prepared by reacting a urate oxidase variant, albumin, and a linker.
요산산화효소-알부민 컨쥬게이트의 생산 방법을 기술하기에 앞서, 요산산화효소-알부민 컨쥬게이트의 제조에 사용되는 물질에 대하여 개시한다. Prior to describing the production method of the uric acid oxidase-albumin conjugate, materials used in the production of the uric acid oxidase-albumin conjugate are disclosed.
요산산화효소 변이체Uric acid oxidase variants
요산산화효소는 요산을 분해하는 기능을 갖는 효소이다. 인체는 요산산화효소를 생산하지 않기 때문에 요산의 분해가 원활하지 않은 경우 체내에 요산이 침착되어 다양한 질병을 유발할 수 있다. 따라서, 상기 요산산화효소는 요산의 높은 수치로 인해 발생하는 질병, 예를 들어 통풍을 포함하는 질병을 치료하는데 사용될 수 있다. 상기 요산산화효소는 4개의 동일한 구조의 단량체가 결합한 형태를 갖는 사량체(tetramer) 또는 팔량체(octamer) 형태로 존재할 수 있다. 즉, 상기 요산산화효소는 요산산화효소 서브유닛 4개가 올리고머화(oligomerization)되어 형성된 사합체(tetramer) 또는 팔량체(octamer)이다. 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.
본 명세서에서 개시하는 요산산화효소-알부민 컨쥬게이트의 제조에 사용되는 요산산화효소 변이체는 미생물 유래의 야생형 요산산화효소의 서열 일부가 변형된 것을 특징으로 한다. 상기 요산산화효소 변이체는 1개 이상의 비천연아미노산을 포함하며, 각각의 비천연아미노산 잔기를 통해 위치-특이적으로 알부민과 접합될 수 있다. 예를 들어, 상기 요산산화효소 변이체는 1개, 2개, 3개, 또는 4개, 또는 그 이상의 비천연아미노산을 포함할 수 있다. 구체적으로, 상기 요산산화효소 변이체는 요산산화효소 변이체 서브유닛 4개가 올리고머화 되어 형성된 사합체이며, 각각의 요산산화효소 변이체 서브유닛은 야생형 요산산화효소 서브유닛과 비교해 그 서열 내 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것을 특징으로 한다. 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. For example, the uric acid oxidase variant may contain one, two, three, or four, or more unnatural amino acids. Specifically, 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.
본 명세서에서 제공하는 상기 요산산화효소 변이체의 원형이 되는 야생형 요산산화효소는 미생물에서 유래된 것이다. 일 구현예로, 상기 야생형 요산산화효소는 Aspergillus Flavus, Arthrobacter globiformis, 및 Candidas Utilis에서 선택된 미생물 유래 요산산화효소일 수 있다.Wild-type uric acid oxidase, which is the prototype of the uric acid oxidase variant provided herein, is derived from a microorganism. In one embodiment, the wild-type uric acid oxidase may be uric acid oxidase derived from a microorganism selected from Aspergillus Flavus, Arthrobacter globiformis, and Candidas Utilis.
일 실시예에서, 상기 야생형 요산산화효소는 동일한 4개의 야생형 요산산화효소 서브유닛이 올리고머화된 사량체(tetramer) 단백질이다.In one embodiment, the wild-type uric acid oxidase is a tetrameric protein in which four identical wild-type uric acid oxidase subunits are oligomerized.
일 실시예로, 상기 야생형 요산산화효소가 Aspergillus Flavus 유래 요산산화효소인 경우, 그 서브유닛의 펩타이드 서열은 N말단에서 C말단 방향으로,In one embodiment, when the wild-type uric acid oxidase is Aspergillus Flavus-derived uric acid oxidase, the peptide sequence of the subunit is from the N-terminus to the C-terminus,
SAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTVLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL (서열번호 1)일 수 있다. SAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLTVLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL (서열번호 1)일 수 있다.
일 실시예로, 상기 야생형 요산산화효소가 Candida Utilis 유래 요산산화효소인 경우, 그 서브유닛의 펩타이드 서열은 N말단에서 C말단 방향으로,In one embodiment, when the wild-type uric acid oxidase is Candida Utilis-derived uric acid oxidase, the peptide sequence of the subunit is from the N-terminus to the C-terminus,
MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGYNKCDFTTLQPTTDRILSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL(서열번호 2) 일 수 있다.MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGYNKCDFTTLQPTTDRILSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL(서열번호 2) 일 수 있다.
일 실시예로, 상기 야생형 요산산화효소가 Arthrobacter globiformis 유래 요산산화효소인 경우, 그 서브유닛의 펩타이드 서열은 N말단에서 C말단 방향으로, In one embodiment, when the wild-type uric acid oxidase is Arthrobacter globiformis-derived uric acid oxidase, the peptide sequence of the subunit is from the N-terminus to the C-terminus,
MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC (서열번호 3)일 수 있다.MTATAETSTGTKVVLGQNQYGKAEVRLVKVTRNTARHEIQDLNVTSQLRGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRADHPIWSNIAGFC (서열번호 3)일 수 있다.
본 명세서에서 사용되는 요산산화효소는 미생물 유래 요산산화효소 및 포유류 유래 요산산화효소를 포함한다. The uric acid oxidase used herein includes microbial uric acid oxidase and mammalian uric acid oxidase.
일 실시예에서, 상기 야생형 요산산화효소와 마찬가지로, 상기 요산산화효소 변이체도 4개의 서브유닛를 포함하는 사량체 단백질이다. 상기 요산산화효소 변이체는 1개 내지 4개의 요산산화효소 변이체 서브유닛을 포함하며, 상기 요산산화효소 변이체 서브유닛은 그 원형이 되는 야행성 요산산화효소 서브유닛에서 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것을 특징으로 한다. 일 실시예로, 상기 요산산화효소 변이체는 1개의 요산산화효소 변이체 서브유닛, 및 3개의 야생형 요산산화효소 서브유닛을 포함할 수 있다. 일 실시예로, 상기 요산산화효소 변이체는 2개의 요산산화효소 변이체 서브유닛, 및 1개의 야생형 요산산화효소 서브유닛을 포함할 수 있다. 다른 실시예로, 상기 요산산화효소 변이체는 3개의 요산산화효소 변이체 서브유닛, 및 1개의 야생형 요산산화효소 서브유닛을 포함할 수 있다. 또 다른 실시예로, 상기 요산산화효소 변이체는 4개의 요산산화효소 변이체 서브유닛을 포함할 수 있다. In one embodiment, like the wild-type 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. characterized by In one embodiment, the uric acid oxidase variant may include one uric acid oxidase variant subunit and three wild-type uric acid oxidase subunits. In one embodiment, 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.
요산산화효소 변이체 서브유닛Uric acid oxidase variant subunit
요산산화효소 변이체 서브유닛은 하나 이상의 비천연아미노산을 포함한다. 비천연아미노산은 제1 클릭화학작용기와 클릭화학반응을 할 수 있는 제2 클릭화학작용기를 포함한다. 이때, 상기 제2 클릭화학작용기는 말단 알킨 (terminal alkyne), 아자이드 (azide), 스트레인된 알킨(strained alkyne), 다이엔 (diene), 친다이엔체 (dienophile), 트랜스 시클로옥틴(trans-cyclooctene), 알켄 (alkene), 티올 (thiol), 테트라진 (tetrazine), DBCO(dibenzocyclooctyne) 및 비시클로노닌(bicyclononyne) 중에서 선택되는 어느 하나일 수 있으나, 이에 제한되지 않는다. 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. At this time, 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.
일 실시예로, 상기 비천연아미노산은 각각 독립적으로, p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), LHomopropargylglycine(HPG), O-propargyl-L-tyrosine (oPa) ppropargyloxyphenylalanine(pPa), 및 4-(1,2,4,5-tetrazin-3-yl) phenylalanine (frTet) 중에서 선택될 수 있다. 특정한 실시예에서, 비천연아미노산은 AzF일 수 있다. 예를 들어, p-Azido-L-phenylalanine (AzF)은 하기의 화학식 6 또는 6-1의 구조를 가질 수 있다. In one embodiment, 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). In certain embodiments, the unnatural amino acid may be AzF. For example, p-Azido-L-phenylalanine (AzF) may have a structure of Formula 6 or 6-1 below.
[화학식 6][Formula 6]
Figure PCTKR2022009319-appb-I000010
,
Figure PCTKR2022009319-appb-I000010
,
[화학식 6-1][Formula 6-1]
Figure PCTKR2022009319-appb-I000011
.
Figure PCTKR2022009319-appb-I000011
.
야생형 요산산화효소에 비천연 아미노산을 삽입해 요산산화효소 변이체를 만들 때, 본래 요산산화효소의 구조와 기능에는 가능한 한 영향을 미치지 않아야 하므로, 요산산화효소의 활성 및 구조에 있어 중요한 역할을 하는 아미노산을 비천연 아미노산으로 치환할 수는 없다. 또한, 상기 비천연 아미노산은 요산산화효소-알부민 접합체 제조 과정에서 링커와 결합해야 하기 때문에, 요산산화효소의 입체 구조 상 상대적으로 용매 접근성이 높은 위치의 아미노산을 치환하는 것이 유리하다. 야생형 요산산화효소의 구조와 기능에 미치는 영향은 최소화하면서, 용매접근성이 높은 자리를 선별하기 위해 다양한 방법을 사용할 수 있다. 예를 들어, 분자 모델링 계산을 통해 야생형 요산산화효소 고유의 원자 에너지와 유사하면서도, 용매 접근성은 높은 후보 위치를 선별할 수 있다.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. In addition, since 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.
비천연아미노산의 치환 위치Substitution positions for non-natural amino acids
일 실시예에서, 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. 구체적으로, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환된 것일 수 있다. 보다 더 구체적으로, 서열번호 1의 아미노산 서열의 160번째 트립토판, 또는 174번째 트립토판 중 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환된 것일 수 있다.In one embodiment, 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. Specifically, 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 group consisting of tryptophan at position 208, phenylalanine at position 219, tyrosine at position 233, tyrosine at position 251, tyrosine at position 258, phenylalanine at position 259, tryptophan at position 265 and phenylalanine at position 279 may be substituted with an unnatural amino acid. . More specifically, one or more residues selected from tryptophan at position 160 or tryptophan at position 174 of the amino acid sequence of SEQ ID NO: 1 may be substituted with a non-natural amino acid.
다른 실시예에서, 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. 구체적으로, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신 중 선택되는 하나 이상의 잔기가 비천연아미노산으로 치환된 것일 수 있다. 보다 더 구체적으로, 서열번호 2의 아미노산 서열의 163번째 타이로신(tyrosine), 170번째 페닐알라닌 (phenylalanine), 200번째 타이로신, 및 271번째 트립토판(tryptophan) 중 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환된 것일 수 있다.In another embodiment, 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. Specifically, 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 189 tryptophan, position 191 tryptophan, position 200 tyrosine, position 211 phenylalanine, position 215 tyrosine, position 226 phenylalanine, position 239 phenylalanine, position 253 tyrosine, position 257 tyrosine , At least one residue selected from tyrosine at position 264, phenylalanine at position 265, tryptophan at position 271, phenylalanine at position 281, and tyrosine at position 282 may be substituted with a non-natural amino acid. More specifically, 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
다른 실시예에서, 요산산화효소 변이체 서브유닛은 서열번호 3의 아미노산 서열의 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. 구체적으로, 상기 요산산화효소 변이체 서브유닛은 서열번호 3의 아미노산 서열의 20번째 타이로신, 52번째 페닐알라닌, 75번째 타이로신, 77번째 페닐알라닌, 82번째 페닐알라닌, 88번째 페닐알라닌, 96번째 페닐알라닌, 100번째 페닐알라닌, 102번째 트립토판, 108번째 트립토판, 113번째 페닐알라닌, 114번째 페닐알라닌, 115번째 트립토판, 125번째 페닐알라닌, 163번째 페닐알라닌, 166번째 페닐알라닌, 171번째 타이로신, 190번째 트립토판, 192번째 타이로신, 199번째 페닐알라닌, 203번째 타이로신, 214번째 페닐알라닌, 227번째 타이로신, 253번째 페닐알라닌, 260번째 페닐알라닌, 269번째 페닐알라닌, 270번째 타이로신, 276번째 타이로신, 295번째 트립토판, 및 301번째 페닐알라닌 중 선택된 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. In another embodiment, 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. Specifically, 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 selected from tyrosine, 214th phenylalanine, 227th tyrosine, 253rd phenylalanine, 260th phenylalanine, 269th phenylalanine, 270th tyrosine, 276th tyrosine, 295th tryptophan, and 301st phenylalanine are substituted with a non-natural amino acid. it could be
알부민albumin
전술한 바와 같이, 요산산화효소-알부민 컨쥬게이트의 제조에는 알부민이 사용된다. 알부민은 체액 중 넓게 분포되어 있는 단순 단백질로서, 다양한 분자들과 결합하여 운반시키는 수송 단백질의 역할을 한다. 알부민의 대표적인 예로는 혈청 알부민이 있다.As described above, 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.
일 실시예에서, 본 출원의 알부민은 포유류의 알부민, 예를 들어 혈청 알부민일 수 있다. 예를 들어, 인간 혈청 알부민(human serum albumin; HSA), 소혈청알부민(bovine serum albumin; BSA), 오브알부민, 기타 척추동물의 알부민, 및 이들의 변이체 중에 선택되는 어느 하나일 수 있다. 이들은 야생형 또는 재조합 형태일 수 있다.In one embodiment, the albumin of the present application may be mammalian albumin, such as serum albumin. For example, it 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.
일 실시예에서, 알부민은 야생형 또는 재조합 인간 혈청 알부민일 수 있다. 상기 인간 혈청 알부민은 2주 이상의 긴 반감기를 가진다. 이는, 1) 알부민 분자의 정전기적 반발력(electrostatic repulsion)으로 인해 사구체에서 쉽게 여과되지 않으며, 2) 내피세포(endothelium)의 neonatal Fc receptor (FcRn)에 의해 매개되는 재순환 작용으로 인해 체내에서 분해되는 주기가 길기 때문이다.In one embodiment, 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
일 실시예에서, 알부민은 인간 혈청 알부민일 수 있고, 이때, 인간 혈청 알부민은 하기의 아미노산 서열을 포함할 수 있다. In one embodiment, albumin may be human serum albumin, and in this case, human serum albumin may include the following amino acid sequence.
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 4)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 4)
다른 실시예에서, 알부민은 인간 혈청 알부민 또는 이의 변이체일 수 있고, 인간 혈청 알부민 또는 이의 변이체는 하기의 서열 중 선택된 어느 하나의 서열을 포함할 수 있다:In another embodiment, 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:
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQMSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 5);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQMSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 5);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSAPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 6);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSAPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 6);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNARTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 7);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNARTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 7);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 8);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 8);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 9);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 9);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGYKLVAASQAALGL (서열번호 10);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGYKLVAASQAALGL (서열번호 10);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLIEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 11);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLIEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 11);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRDLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 12);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRDLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 12);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCVEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 13);DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCVEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 13);
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKMPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 14); 및DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKMPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 14); and
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFTAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 15).DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFTAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (서열번호 15).
일 실시예에서, 인간 혈청 알부민 또는 이의 변이체에 포함된 시스테인의 티올 잔기는 링커의 일 말단의 제2 반응 작용기와 반응할 수 있다. 보다 구체적으로, 인간 혈청 알부민 또는 이의 변이체에 포함된 시스테인의 티올 잔기는 링커의 일 말단의 티올 반응성 그룹과 반응할 수 있다. 이때, 링커의 일 말단과 반응하는 시스테인은 34번 시스테인(Cys 34)일 수 있다. In one embodiment, 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).
링커linker
링커는 요산산화효소 변이체 및 알부민과 반응하여, 요산산화효소 변이체와 알부민을 연결하는 역할을 하는 구조를 의미한다. 예를 들어, 링커는 알부민과 반응하여 알부민-링커 컨쥬게이트를 형성하고, 생성된 알부민-링커 컨쥬게이트는 요산산화효소 변이체와 반응하여 요산산화효소-알부민 컨쥬게이트를 형성할 수 있다. 다른 예로, 링커는 요산산화효소 변이체와 반응하여 링커-요산산화효소 복합체를 형성하고, 생성된 링커-요산산화효소 복합체는 알부민과 반응하여 요산산화효소-알부민 컨쥬게이트를 형성할 수 있다. 또 다른 예로, 요산산화효소-알부민 컨쥬게이트는 링커, 요산산화효소 변이체, 및 알부민의 반응에 의해 형성될 수 있다. 즉, 링커의 일 말단은 비천연아미노산을 포함하는 요산산화효소 변이체와 반응할 수 있도록 구성되고, 다른 말단은 알부민과 반응할 수 있도록 구성된다. The linker means a structure that reacts with uric acid oxidase variants and albumin and serves to connect uric acid oxidase variants and albumin. For example, 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. As another example, 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. As another example, 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.
일 실시예에서, 상기 링커는 요산산화효소 변이체와 반응하는 구조, 알부민과 반응하는 구조 및/또는 두 구조 사이의 연결구조를 포함할 수 있다. In one embodiment, 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.
이때, 상기 링커는 요산산화효소 변이체의 비천연아미노산과 반응할 수 있다. 상기 반응은 링커의 제1 반응 작용기와 요산산화효소 변이체의 비천연아미노산 간의 반응이다. 일 실시예로, 상기 제1 반응 작용기는 제1 클릭화학작용기를 포함한다. 구체적인 일 실시예로, 상기 제1 반응 작용기는 제1 클릭화학작용기를 포함할 수 있고, 이때 제1 클릭화학작용기는 디벤조시클로옥틴, 아자이드, 테트라진, 트랜스시클로옥틴, 및 비시클로노닌 중에서 선택될 수 있다.In this case, 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. In one embodiment, the first reaction functional group includes a first click chemistry functional group. As a specific embodiment, 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
또한, 링커는 알부민의 시스테인 잔기와 반응할 수 있다. 상기 반응은 링커의 제2 반응 작용기와 알부민의 시스테인의 티올기 간의 반응이다. 일 실시예로, 상기 제2 반응 작용기는 시스테인 잔기의 티올에 대한 반응성을 가지는 작용기를 포함한다. 일 실시예에서, 상기 제2 반응 작용기는 티올 반응성 그룹을 포함할 수 있다. 구체적인 일 실시예로, 상기 제2 반응 작용기는 말레이미드 (Maleimide; MAL), 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles; APN)), 할로아세탈 (haloacetal), 피리딜 디설파이드 (pyridyl disulfide), 및 기타 공지된 티올과 반응할 수 있는 작용기를 포함한다.In addition, 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. In one embodiment, the second reactive functional group includes a functional group having reactivity to a thiol of a cysteine residue. In one embodiment, the second reactive functional group may include a thiol reactive group. In a specific embodiment, the second reactive functional group is maleimide (MAL), 3-arylpropiolonitriles (APN)), haloacetal, pyridyl disulfide, and functional groups capable of reacting with other known thiols.
전술한 바와 같이, 요산산화효소-알부민 컨쥬게이트의 제조에는 링커가 사용된다. As described above, a linker is used to prepare the uric acid oxidase-albumin conjugate.
일 실시예에서, 본 출원의 링커는 하기의 화학식 1의 구조를 가질 수 있다. In one embodiment, the linker of the present application may have a structure of Formula 1 below.
[화학식 1][Formula 1]
F1 - L - F2,F 1 - L - F 2 ,
이때, At this time,
F1은 제1 반응 작용기이고, F2는 제2 반응 작용기이고, L은 링커 모이어티이다. F 1 is a first reactive functional group, F 2 is a second reactive functional group, and L is a linker moiety.
일 실시예에서, F1, 즉, 상기 제1 반응 작용기는 제1 클릭화학작용기를 포함할 수 있다. 일 실시예에서, 상기 제1 클릭화학작용기는 디벤조시클로옥틴 그룹, 아자이드 그룹, 테트라진 그룹, 트랜스시클로옥틴 그룹, 및 비시클로노닌 그룹 중에서 선택되는 어느 하나일 수 있으나, 이에 제한되지 않고 당업계에서 통상적으로 클릭화학작용기로 사용되는 그룹일 수 있다. 특정한 실시예에서, 상기 제1 클릭화학작용기는 비시클로노닌일 수 있다. In one embodiment, F 1 , that is, the first reactive functional group may include a first click chemical functional group. In one embodiment, 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.
특정한 실시예에서, 제1 클릭화학작용기는 하기의 구조 중 어느 하나로 표현될 수 있다. In a specific embodiment, the first click chemistry functional group can be represented by any one of the following structures.
Figure PCTKR2022009319-appb-I000012
Figure PCTKR2022009319-appb-I000012
Figure PCTKR2022009319-appb-I000013
Figure PCTKR2022009319-appb-I000013
Figure PCTKR2022009319-appb-I000014
Figure PCTKR2022009319-appb-I000014
Figure PCTKR2022009319-appb-I000015
및
Figure PCTKR2022009319-appb-I000015
and
Figure PCTKR2022009319-appb-I000016
Figure PCTKR2022009319-appb-I000016
일 실시예에서, F2, 즉, 상기 제2 반응 작용기는 티올 그룹에 대한 반응성을 가지는 작용기를 포함할 수 있다. 일 실시예에서, 상기 제2 반응 작용기는 티올 반응성 그룹을 포함할 수 있다. 티올 반응성 그룹은 말레이미드 (Maleimide; MAL) 그룹, 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles) 그룹, 할로아세탈 (haloacetal) 그룹, 및 피리딜 디설파이드 (pyridyl disulfide) 그룹 중에서 선택되는 어느 하나일 수 있으나, 이에 제한되지 않는다. 다른 실시예에서, 상기 제2 반응 작용기는 아민 그룹에 대한 반응성을 가지는 작용기를 포함할 수 있다. 이때, 상기 제2 반응 작용기는 N-히드록시석신이미드 에스터 (N-hydroxysuccinimide ester; NHS), 및 이미도에스터(imidoester)를 포함할 수 있으며, 이에 제한되지 않고 통상적으로 아민기와 반응할 수 있는 작용기를 포함할 수 있다. In one embodiment, F 2 , that is, the second reactive functional group may include a functional group having reactivity to a thiol group. In one embodiment, 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. However, it is not limited thereto. In another embodiment, the second reactive functional group may include a functional group having reactivity to an amine group. In this case, 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. can include
특정한 실시예에서, 티올 반응성 그룹은 하기의 구조로 표현될 수 있다:In certain embodiments, thiol-reactive groups can be represented by the structure:
Figure PCTKR2022009319-appb-I000017
,
Figure PCTKR2022009319-appb-I000017
,
Figure PCTKR2022009319-appb-I000018
, 및
Figure PCTKR2022009319-appb-I000018
, and
Figure PCTKR2022009319-appb-I000019
.
Figure PCTKR2022009319-appb-I000019
.
일 실시예에서, L, 즉, 링커 모이어티는 알킬렌, 알케닐렌, 알키닐렌, 아랄킬렌, 아릴알킬렌 또는 (C2H4O)np을 포함할 수 있고, 이때 np는 1 이상 6 이하의 정수일 수 있다. 일 실시예에서, L은 치환 또는 비치환된 C1-50 알킬렌, 치환 또는 비치환된 C1-50 헤테로알킬렌, 치환 또는 비치환된 C2-50 알케닐렌, 치환 또는 비치환된 C2-50 헤테로알케닐렌, 치환 또는 비치환된 C2-50 알키닐렌, 치환 또는 비치환된 C2-50 헤테로알키닐렌일 수 있다. 이때, 상기 헤테로알킬렌, 헤테로알케닐렌, 및 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함할 수 있다. 일 실시예에서, 헤테로원자는, 각각 독립적으로, O, S, 및 N 중에서 선택될 수 있다. 특정한 실시예에서, L은 치환 또는 비치환된 C10-30 알킬렌, 치환 또는 비치환된 C10-30 헤테로알킬렌, 치환 또는 비치환된 C10-30 알케닐렌, 치환 또는 비치환된 C10-30 헤테로알케닐렌, 치환 또는 비치환된 C10-30 알키닐렌, 치환 또는 비치환된 C10-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L은 (C2H4O)np을 포함하는, 치환 또는 비치환된 C10-30 알킬렌, 치환 또는 비치환된 C10-30 헤테로알킬렌, 치환 또는 비치환된 C10-30 알케닐렌, 치환 또는 비치환된 C10-30 헤테로알케닐렌, 치환 또는 비치환된 C10-30 알키닐렌, 치환 또는 비치환된 C10-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L은 치환 또는 비치환된 C12-20 알킬렌, 치환 또는 비치환된 C12-20 헤테로알킬렌, 치환 또는 비치환된 C12-20 알케닐렌, 치환 또는 비치환된 C12-20 헤테로알케닐렌, 치환 또는 비치환된 C12-20 알키닐렌, 치환 또는 비치환된 C12-20 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L은 (C2H4O)np을 포함하는, 치환 또는 비치환된 C12-20 알킬렌, 치환 또는 비치환된 C12-20 헤테로알킬렌, 치환 또는 비치환된 C12-20 알케닐렌, 치환 또는 비치환된 C12-20 헤테로알케닐렌, 치환 또는 비치환된 C12-20 알키닐렌, 치환 또는 비치환된 C12-20 헤테로알키닐렌일 수 있다. 이때, 상기 헤테로알킬렌, 헤테로알케닐렌, 및 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함할 수 있다. 일 실시예에서, 헤테로원자는, 각각 독립적으로, O, S, 및 N 중에서 선택될 수 있다.In one embodiment, L, ie the linker moiety, 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 In one embodiment, 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. In this case, the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms. In one embodiment, the heteroatoms can each independently be selected from O, S, and N. In certain embodiments, 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. 10-30 heteroalkenylene, substituted or unsubstituted C 10-30 alkynylene, or substituted or unsubstituted C 10-30 heteroalkynylene. In certain embodiments, 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 . 10-30 alkenylene, substituted or unsubstituted C 10-30 heteroalkenylene, substituted or unsubstituted C 10-30 alkynylene, or substituted or unsubstituted C 10-30 heteroalkynylene. In certain embodiments, 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. 12-20 heteroalkenylene, substituted or unsubstituted C 12-20 alkynylene, or substituted or unsubstituted C 12-20 heteroalkynylene. In certain embodiments, 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 . 12-20 alkenylene, substituted or unsubstituted C 12-20 heteroalkenylene, substituted or unsubstituted C 12-20 alkynylene, or substituted or unsubstituted C 12-20 heteroalkynylene. In this case, the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms. In one embodiment, the heteroatoms can each independently be selected from O, S, and N.
특정한 실시예에서, 링커 모이어티(L)는 하기의 구조 중 어느 하나로 표현될 수 있다:In certain embodiments, the linker moiety (L) can be represented by any of the following structures:
Figure PCTKR2022009319-appb-I000020
, 및
Figure PCTKR2022009319-appb-I000020
, and
Figure PCTKR2022009319-appb-I000021
.
Figure PCTKR2022009319-appb-I000021
.
이때, 링커 모이어티의 1'는 F1과 연결되고, 2'는 F2와 연결된다.In this case, 1' of the linker moiety is connected to F 1 , and 2' is connected to F 2 .
일 실시예에서, L1은 결합(bond)이거나, 치환 또는 비치환된 C1-6 알킬렌, 치환 또는 비치환된 C1-6 헤테로알킬렌, 치환 또는 비치환된 C2-6 알케닐렌, 치환 또는 비치환된 C2-6 헤테로알케닐렌, 치환 또는 비치환된 C2-6 알키닐렌, 또는 치환 또는 비치환된 C2-6 헤테로알키닐렌일 수 있다. 일 실시양태에서, L1은 -O-, -NH- 또는 -S- 일 수 있다. 특정한 실시예에서, L1은 결합(bond)이거나, 비치환된 C1-3알킬렌, 또는 비치환된 C1-3 헤테로알킬렌일 수 있다.In one embodiment, 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. In one embodiment, L 1 can be -O-, -NH- or -S-. In certain embodiments, L 1 can be a bond, unsubstituted C 1-3 alkylene, or unsubstituted C 1-3 heteroalkylene.
일 실시예에서, L2는 알킬렌, 알케닐렌, 알키닐렌, 아랄킬렌, 아릴알킬렌 또는 (C2H4O)np을 포함할 수 있고, 이때 np는 1 이상 6 이하의 정수일 수 있다. 일 실시예에서, L2는 치환 또는 비치환된 C1-30 알킬렌, 치환 또는 비치환된 C1-30 헤테로알킬렌, 치환 또는 비치환된 C2-30 알케닐렌, 치환 또는 비치환된 C2-30 헤테로알케닐렌, 치환 또는 비치환된 C2-30 알키닐렌, 또는 치환 또는 비치환된 C2-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L2는 (C2H4O)np을 포함하는, 치환 또는 비치환된 C1-30 알킬렌, 치환 또는 비치환된 C1-30 헤테로알킬렌, 치환 또는 비치환된 C2-30 알케닐렌, 치환 또는 비치환된 C2-30 헤테로알케닐렌, 치환 또는 비치환된 C2-30 알키닐렌, 또는 치환 또는 비치환된 C2-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L2는 치환 또는 비치환된 C10-20 알킬렌, 치환 또는 비치환된 C10-20 헤테로알킬렌, 치환 또는 비치환된 C10-20 알케닐렌, 치환 또는 비치환된 C10-20 헤테로알케닐렌, 치환 또는 비치환된 C10-20 알키닐렌, 또는 치환 또는 비치환된 C10-20 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L2는 (C2H4O)np을 포함하는, 치환 또는 비치환된 C10-20 알킬렌, 치환 또는 비치환된 C10-20 헤테로알킬렌, 치환 또는 비치환된 C10-20 알케닐렌, 치환 또는 비치환된 C10-20 헤테로알케닐렌, 치환 또는 비치환된 C10-20 알키닐렌, 또는 치환 또는 비치환된 C10-20 헤테로알키닐렌일 수 있다.In one embodiment, 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. In one embodiment, 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. In certain embodiments, 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 . C 2-30 alkenylene, substituted or unsubstituted C 2-30 heteroalkenylene, substituted or unsubstituted C 2-30 alkynylene, or substituted or unsubstituted C 2-30 heteroalkynylene. In certain embodiments, 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. C 10-20 heteroalkenylene, substituted or unsubstituted C 10-20 alkynylene, or substituted or unsubstituted C 10-20 heteroalkynylene. In certain embodiments, 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.
일 실시예에서, L3는 결합(bond)이거나, 치환 또는 비치환된 C1-6 알킬렌, 치환 또는 비치환된 C1-6 헤테로알킬렌, 치환 또는 비치환된 C2-6 알케닐렌, 치환 또는 비치환된 C2-6 헤테로알케닐렌, 치환 또는 비치환된 C2-6 알키닐렌, 또는 치환 또는 비치환된 C2-6 헤테로알키닐렌일 수 있다. 일 실시예에서, L3는 -O-, -NH-, 또는 -S- 일 수 있다. 특정한 실시예에서, L3는 결합(bond)이거나, 비치환된 C1-3알킬렌, 또는 비치환된 C1-3 헤테로알킬렌일 수 있다.In one embodiment, 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. In one embodiment, L 3 may be -O-, -NH-, or -S-. In certain embodiments, L 3 can be a bond, unsubstituted C 1-3 alkylene, or unsubstituted C 1-3 heteroalkylene.
특정한 실시예에서, 화학식 1은 화학식 1-1로 표현될 수 있고, 링커는 하기의 화학식 1-1의 구조를 가질 수 있다:In a specific embodiment, Formula 1 may be represented by Formula 1-1, and the linker may have the structure of Formula 1-1:
[화학식 1-1][Formula 1-1]
Figure PCTKR2022009319-appb-I000022
,
Figure PCTKR2022009319-appb-I000022
,
이때, F1, L1, L2, L3, 및 F2 각각은 상술한 바와 같다. In this case, each of F 1 , L 1 , L 2 , L 3 , and F 2 is as described above.
특정한 실시예에서, 화학식 1-1은 화학식 1-2로 표현될 수 있고, 링커는 하기의 화학식 1-2의 구조를 가질 수 있다:In a specific embodiment, Formula 1-1 may be represented by Formula 1-2, and the linker may have a structure of Formula 1-2:
[화학식 1-2][Formula 1-2]
Figure PCTKR2022009319-appb-I000023
,
Figure PCTKR2022009319-appb-I000023
,
이때, F1, L1, L2, 및 F2 각각은 상술한 바와 같고, np는 1 이상 6 이하의 정수일 수 있다. In this case, 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.
특정한 실시예에서, 화학식 1-2는 화학식 1-3으로 표현될 수 있고, 링커는 하기의 화학식 1-3의 구조를 가질 수 있다:In a specific embodiment, Formula 1-2 may be represented by Formula 1-3, and the linker may have a structure of Formula 1-3:
[화학식 1-3][Formula 1-3]
Figure PCTKR2022009319-appb-I000024
,
Figure PCTKR2022009319-appb-I000024
,
이때, np는 1 이상 6 이하의 정수이다. At this time, np is an integer of 1 or more and 6 or less.
특정한 실시예에서, 링커는 하기의 화학식 1-4의 구조를 가질 수 있다:In certain embodiments, a linker can have the structure of Formulas 1-4:
[화학식 1-4][Formula 1-4]
Figure PCTKR2022009319-appb-I000025
,
Figure PCTKR2022009319-appb-I000025
,
이때, np는 1 이상 6 이하의 정수이다. At this time, np is an integer of 1 or more and 6 or less.
특정한 실시예에서, 링커는 하기의 화학식 중 선택되는 어느 하나의 화학식의 구조를 가질 수 있다:In certain embodiments, the linker may have a structure of any one of the following formulas:
[화학식 1-5][Formula 1-5]
Figure PCTKR2022009319-appb-I000026
;
Figure PCTKR2022009319-appb-I000026
;
[화학식 1-6][Formula 1-6]
Figure PCTKR2022009319-appb-I000027
; 및
Figure PCTKR2022009319-appb-I000027
; and
[화학식 1-7][Formula 1-7]
Figure PCTKR2022009319-appb-I000028
.
Figure PCTKR2022009319-appb-I000028
.
이하에서, 요산산화효소-알부민 컨쥬게이트의 제조 방법을 개시한다. Hereinafter, a method for preparing a uric acid oxidase-albumin conjugate is disclosed.
1. 비천연아미노산을 포함하는 요산산화효소 변이체 생산1. Production of urate oxidase variants containing non-natural amino acids
위치특이적으로 알부민이 결합된 요산산화효소-알부민 컨쥬게이트를 생산하기 위해, 비천연아미노산을 포함하는 요산산화효소 변이체를 생산한다. 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.
1) 위치특이적으로 비천연아미노산을 포함하는 요산산화효소 변이체 생산을 위한 발현 벡터의 제작 및 형질전환1) Construction and transformation of expression vector for site-specific production of uric acid oxidase variants containing non-natural amino acids
비천연 아미노산을 포함하는 요산산화효소 변이체를 생산하기 위해, 야생형 요산산화효소의 아미노산 서열에서 하나 이상의 잔기를 비천연아미노산으로 변경할 수 있다. 이를 위해, 본래 요산산화효소의 구조와 기능에 가능한 영향을 미치지 않는 최적의 위치를 결정하는 것이 필요하다. 상기 위치는 페닐알라닌, 트립토판, 타이로신 중에서 선택될 수 있다. 또한, 링커와의 효율적인 결합이 이루어지게 하기 위해 높은 용매 접근성을 가지는 위치 중에서 선택될 수 있다. To produce a uric acid oxidase variant comprising an unnatural amino acid, one or more residues in the amino acid sequence of wild type uric acid oxidase can be changed to an unnatural amino acid. To this end, it is necessary to determine an optimal location that does not possibly affect the structure and function of native uric acid oxidase. The position may be selected from phenylalanine, tryptophan, and tyrosine. In addition, it may be selected from positions having high solvent accessibility in order to achieve efficient binding with the linker.
일 실시예로, 상기 비천연 아미노산으로의 변경은 서열번호 1의 아미노산 서열의 하나 이상의 잔기가 치환되는 것일 수 있다. 이때, 상기 서열번호 1은 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 Ser Glu Glu Lys Arg Asn Val Gln Val Asp Val Val Glu Gly Lys Gly Ile Asp Ile Lys Ser Ser Leu Ser Gly Leu Thr Val Leu Lys Ser Thr Asn Ser Gln Phe Trp Gly Phe Leu Arg Asp Glu Tyr Thr Thr Leu Lys Glu Thr Trp Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Gln Trp Lys Asn Phe Ser Gly Leu Gln Glu Val Arg Ser His Val Pro Lys Phe Asp Ala Thr Trp Ala Thr Ala Arg Glu Val Thr Leu Lys Thr Phe Ala Glu Asp Asn Ser Ala Ser Val Gln Ala Thr Met Tyr Lys Met Ala Glu Gln Ile Leu Ala Arg Gln Gln Leu Ile Glu Thr Val Glu Tyr Ser Leu Pro Asn Lys His Tyr Phe Glu Ile Asp Leu Ser Trp His Lys Gly Leu Gln Asn Thr Gly Lys Asn Ala Glu Val Phe Ala Pro Gln Ser Asp Pro Asn Gly Leu Ile Lys Cys Thr Val Gly Arg Ser Ser Leu Lys Ser Lys Leu로 표시된다. 구체적인 일 실시예로, 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환되는 것일 수 있다. 보다 더 구체적인 일 실시예로, 서열번호 1의 아미노산 서열의 160번째 트립토판, 또는 174번째 트립토판 중 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환되는 것일 수 있다. In one embodiment, 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. In this case, 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 Ser Glu Glu Lys Arg Asn Val Gln Val Asp Val Val Val Glu Gly Lys Gly Ile Asp Ile Lys Ser Ser Leu Ser Gly Leu Thr Val Leu Lys Ser Thr Asn Ser Gln Phe Trp Gly Phe Leu Arg Asp Glu Tyr Thr Thr Leu Lys Glu Thr Trp Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Gln Trp Lys Asn Phe Ser Gly Leu Gln Glu Val Arg Ser His Val Pro Lys Phe Asp Ala Thr Trp Ala Thr Ala Arg Glu Val Thr Leu Lys Thr Phe Ala Glu Asp Asn Ser Ala Ser Val Gln Ala Thr Met Tyr Lys Met Ala Glu Gln Ile Leu Ala Arg Gln Gln Leu Ile Glu T hr Val Glu Tyr Ser Leu Pro Asn Lys His Tyr Phe Glu Ile Asp Leu Ser Trp His Lys Gly Leu Gln Asn Thr Gly Lys Asn Ala Glu Val Phe Ala Pro Gln Ser Asp Pro Asn Gly Leu Ile Lys Cys Thr Val Gly Arg Ser Ser It is represented by Leu Lys Ser Lys Leu. In a specific embodiment, 8th tyrosine, 16th tyrosine, 30th tyrosine, 46th tyrosine, 65th tyrosine, 79th phenylalanine, 87th phenylalanine, 91st tyrosine of the amino acid sequence of SEQ ID NO: 1 , 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, position 208 tryptophan, 219 At least one residue selected from the group consisting of phenylalanine at position 233, tyrosine at position 233, tyrosine at position 258, tyrosine at position 258, phenylalanine at position 259, tryptophan at position 265, and phenylalanine at position 279 may be substituted with a non-natural amino acid. In a more specific embodiment, one or more residues selected from tryptophan at position 160 and tryptophan at position 174 of the amino acid sequence of SEQ ID NO: 1 may be substituted with a non-natural amino acid.
또 다른 일 실시예로, 서열번호 2의 아미노산 서열의 하나 이상의 잔기가 비천연 아미노산으로 치환되는 것일 수 있다. 이때, 상기 서열번호 2는 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 His Glu Gly Gly Glu Lys Arg Ile Thr Asp Leu Tyr Tyr Lys Arg Ser Gly Asp Tyr Lys Leu Ser Ser Ala Ile Lys Asp Leu Thr Val Leu Lys Ser Thr Gly Ser Met Phe Tyr Gly Tyr Asn Lys Cys Asp Phe Thr Thr Leu Gln Pro Thr Thr Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Val Trp Asp Asn Lys Lys Ile Gly Ser Val Tyr Asp Ile Ala Lys Ala Ala Asp Lys Gly Ile Phe Asp Asn Val Tyr Asn Gln Ala Arg Glu Ile Thr Leu Thr Thr Phe Ala Leu Glu Asn Ser Pro Ser Val Gln Ala Thr Met Phe Asn Met Ala Thr Gln Ile Leu Glu Lys Ala Cys Ser Val Tyr Ser Val Ser Tyr Ala Leu Pro Asn Lys His Tyr Phe Leu Ile Asp Leu Lys Trp Lys Gly Leu Glu Asn Asp Asn Glu Leu Phe Tyr Pro Ser Pro His Pro Asn Gly Leu Ile Lys Cys Thr Val Val Arg Lys Glu Lys Thr Lys Leu로 표시된다.In another embodiment, one or more residues of the amino acid sequence of SEQ ID NO: 2 may be substituted with a non-natural amino acid. At this time, 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 His Glu Gly Gly Glu Lys Arg Ile Thr Asp Leu Tyr Tyr Lys Arg Ser Gly Asp Tyr Lys Leu Ser Ser Ala Ile Lys Asp Leu Thr Val Leu Lys Ser Thr Gly Ser Met Phe Tyr Gly Tyr Asn Lys Cys Asp Phe Thr Thr Leu Gln Pro Thr Thr Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Val Trp Asp Asn Lys Lys Ile Gly Ser Val Tyr Asp Ile Ala Lys Ala Ala Asp Lys Gly Ile Phe Asp Asn Val Tyr Asn Gln Ala Arg Glu Ile Thr Leu Thr Thr Phe Ala Leu Glu Asn Ser Pro Ser Val Gln Ala Thr Met Phe Asn Met Ala Thr Gln Ile Leu G lu Lys Ala Cys Ser Val Tyr Ser Val Ser Tyr Ala Leu Pro Asn Lys His Tyr Phe Leu Ile Asp Leu Lys Trp Lys Gly Leu Glu Asn Asp Asn Glu Leu Phe Tyr Pro Ser Pro His Pro Asn Gly Leu Ile Lys Cys Thr Val Val Arg Lys Glu Lys Thr Lys Leu.
구체적인 일 실시예로, 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신 중 선택되는 하나 이상의 잔기가 비천연아미노산으로 치환되는 것일 수 있다. 보다 더 구체적인 일 실시예로, 서열번호 2의 아미노산 서열의 163번째 타이로신(tyrosine), 170번째 페닐알라닌 (phenylalanine), 200번째 타이로신, 및 271번째 트립토판(tryptophan) 중 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환되는 것일 수 있다.As a specific embodiment, 10th tyrosine, 163rd tyrosine, 17th phenylalanine, 45th phenylalanine, 59th tyrosine, 77th tryptophan, 82 phenylalanine at position 90, phenylalanine at position 94, tyrosine position 94, tryptophan position 109, tyrosine position 112, phenylalanine position 123, tyrosine position 136, tyrosine position 137, tyrosine position 143, phenylalanine position 162, phenylalanine position 163 Tyrosine, position 165, phenylalanine position 170, tryptophan position 189, tryptophan position 191, tyrosine position 200, phenylalanine position 211, tyrosine position 215, phenylalanine position 226, phenylalanine position 239, tyrosine position 253, tyrosine position 257, tyrosine position 264, At least one residue selected from phenylalanine at position 265, tryptophan at position 271, phenylalanine at position 281, and tyrosine at position 282 may be substituted with a non-natural amino acid. In a more specific embodiment, 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
이때, 상기 비천연 아미노산은 통상적인 20가지의 아미노산, 파이로리신, 셀레노시스테인 중 하나가 아닌 아미노산을 지칭한다. 상기 비천연 아미노산은 링커의 제1 반응 작용기와 반응할 수 있는 구조를 포함한다. 상기 링커의 제1 반응 작용기는 제1 클릭화학작용기를 포함한다. 일 예로, 상기 비천연 아미노산은 p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), LHomopropargylglycine(HPG), O-propargyl-L-tyrosine (oPa), ppropargyloxyphenylalanine(pPa), 및 4-(1,2,4,5-tetrazin-3-yl) phenylalanine (frTet) 중 선택되는 하나 이상일 수 있다. In this case, 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. For example, 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).
비천연 아미노산을 포함하는 요산산화효소 변이체의 생산을 위한 발현 벡터 제작 및 생산 균주에 관한 내용은 대한민국등록특허공보 10-1637010에 기재되어 있으며, 해당 내용은 본 명세서에 참조로 통합된다. Information on the construction of expression vectors and production strains for the production of variants of uric acid oxidase containing non-natural amino acids is described in Korean Patent Registration Publication No. 10-1637010, the contents of which are incorporated herein by reference.
일 실시예로, Uox의 코딩서열을 PCR로 얻어낸 다음, pQE80에 클로닝하여 pQE80-Uox를 제작할 수 있다. 그 다음, 요산산화효소의 특정 위치의 아미노산 잔기를 amber 코돈으로 대체하기 위하여 상기 pQE80-Uox를 템플레이트로 site-directed mutagenic PCR을 수행할 수 있다. 또한, Methanococcus jannaschii로부터 기원한 tyrosyl-tRNA synthetase와 amber suppressor tRNA로 이루어진 AzF 특이적인 engineered pair를 포함하고 있는 pEVOL-pAzF 플라스미드(Plasmid ID: 31186)를 이용할 수 있다. 구체적인 일 실시예로, 비천연아미노산을 포함하는 요산산화효소 변이체를 발현시키기 위해 특정 위치, 예를 들어 X위치에 amber 코돈이 도입된 pQE80-Uox.Xamb와 pEVOL-pAzF로 동시에 생산 균주를 형질전환 시킬 수 있다.In one embodiment, 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. In addition, 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. In a specific embodiment, in order to express a variant of uric acid oxidase containing an unnatural amino acid, the production strain 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
2) 비천연아미노산을 포함하는 요산산화효소 변이체 생산 균주의 대량 생산2) Mass production of strains producing uric acid oxidase mutants containing non-natural amino acids
본 출원에서 개시하는 방법에서는, 비천연아미노산을 포함하는 요산산화효소 변이체의 대량 생산을 위해, 요산산화효소의 생산 균주를 효과적으로 배양함을 포함한다. 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.
상기 생산 균주는 박테리아를 포함한다. 상기 박테리아는 일 실시예로, 에스케리키아 (Escherichia) 속, 어위니아 (Erwinia) 속, 세라티아 (Serratia) 속, 프로비덴시아 (Providencia) 속, 코리네박테리움 (Corynebacterium) 속, 슈도모나스 (Pseudomonas) 속, 렙토스피라(Leptospira) 속, 살모넬라 (Salmonellar) 속 및 브레비박테리움 (Brevibacterium) 속, 하이포모나스 (hyphomonas) 속, 크로모박테리움 (Chromobactorium) 속, 노카디아(Norcardia) 속 또는 펀자이류 (fungi), 또는 효모류 (yeast)에서 선택될 수 있다. The production strain includes bacteria. In one embodiment, 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).
상기 박테리아를 배양하기 위해, 탄소원과 질소원을 일정한 속도로 공급해주며 유가식 배양(Fed-batch)공정을 실시할 수 있다. 상기 배양은 종배양 및 본배양을 포함할 수 있다. 상기 배양을 위한 배지는 soy peptone, yeast extract, KH2PO4, K2HPO4를 포함할 수 있고, 추가적으로, 카나마이신, 클로람페니콜과 같은 항생제를 포함할 수 있다.In order to culture the bacteria, 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.
상기 배양 과정에서 탄소원 및 질소원이 공급될 수 있는데, 일 실시예로, 탄소원으로 glucose, MgSO4, 질소원으로 yeast extract, (NH4)2SO4가 공급될 수 있다. Carbon and nitrogen sources may be supplied during the culturing process. In one embodiment, glucose and MgSO 4 may be supplied as carbon sources, and yeast extract and (NH 4 ) 2 SO 4 may be supplied as nitrogen sources.
상기 배양은 발효기 내에서 이루어지는데, 비천연아미노산을 포함하는 요산산화효소 변이체의 생산 수율을 높이기 위해 발효기의 교반 속도가 변경될 수 있다. 일 실시예로, 교반 속도를 약 300rpm, 400rpm, 500rpm, 600rpm, 700rpm으로 설정할 수 있다. 구체적인 일 실시예로, 50L 대량 배양을 위해 교반 속도를 약 600rpm으로 설정할 수 있다.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. In one embodiment, 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.
비천연아미노산을 포함하는 요산산화효소 변이체의 생산 수율을 높이기 위해 발효기의 내압조건이 변경될 수 있다. 일 실시예로, 내압을 약 100mbar, 200mbar, 300mbar, 400mbar, 500mbar로 설정할 수 있다. 구체적인 일 실시예로, 50L 대량 배양을 위해 내압 조건을 약 400mbar로 설정할 수 있다.In order to increase the production yield of uric acid oxidase variants containing non-natural amino acids, the pressure conditions of the fermentor may be changed. In one embodiment, the internal pressure may be set to about 100 mbar, 200 mbar, 300 mbar, 400 mbar, or 500 mbar. In a specific embodiment, the internal pressure condition may be set to about 400 mbar for 50L mass culture.
비천연아미노산을 포함하는 요산산화효소 변이체의 생산 수율을 높이기 위해 공기 공급조건을 변경할 수 있다. 일 실시예로, 공기 공급 조건은 약 1vvm, 1.5vvm, 2vvm 일 수 있다. 구체적인 일 실시예로, 50L 대량 배양을 위해 공기 공급 조건을 약 1.5vvm으로 설정할 수 있다.Air supply conditions may be changed to increase the production yield of the uric acid oxidase variants containing non-natural amino acids. In one embodiment, 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.
이때, 상기 공기는 일 실시예로, 질소 78%, 산소 21% 기타 1%의 조성을 가지는 것을 의미할 수 있다.In this case, the air may mean having a composition of 78% nitrogen, 21% oxygen, and the like, in one embodiment, 1%.
3) 비천연아미노산을 포함하는 요산산화효소 변이체 생산 균주 파쇄3) Disruption of strains producing uric acid oxidase mutants containing non-natural amino acids
다음으로, 상기 방법은 비천연아미노산을 포함하는 요산산화효소 변이체를 수득하기 위해 균주를 파쇄함을 포함할 수 있다. Next, the method may include disrupting the strain to obtain a urate oxidase variant containing an unnatural amino acid.
요산산화효소 변이체의 수득율 및 효소 활성을 높이기 위해 적절한 파쇄 조건을 설정할 수 있다. 일 실시예로, 적절한 파쇄 조건을 위해 파쇄 압력을 변경할 수 있다. 구체적인 일 실시예로, 약 15,000, 16,000, 17,000, 18,000, 19,000, 20,000psi로 세포 파쇄 압력을 설정할 수 있다. 또 다른 일 실시예로, 적절한 파쇄 조건을 위해 파쇄 버퍼의 부피를 변경할 수 있다. 구체적인 일 실시예로, wet-cell 1g 기준 약 5ml, 10ml, 15ml의 버퍼를 첨가할 수 있다. 또 다른 일 실시예로, 적절한 파쇄 조건을 위해 파쇄 사이클의 수를 변경할 수 있다. 구체적인 일 실시예로, 파쇄 사이클을 1회, 2회, 3회, 4회, 5회로 설정할 수 있다. 요산산화효소 변이체의 수득율 및 효소 활성을 높이기 위한 적절한 파쇄 조건의 구체적인 일 실시예로, wet-cell 1g 당 10ml의 버퍼를 첨가, 20,000psi의 압력, 2 파쇄 사이클 조건이 설정될 수 있다.Appropriate disruption conditions may be set to increase the yield and enzymatic activity of uric acid oxidase variants. In one embodiment, the crushing pressure may be varied for appropriate crushing conditions. In a specific embodiment, the cell disruption pressure may be set to about 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 psi. In another embodiment, the volume of the disruption buffer may be changed for appropriate disruption conditions. In a specific embodiment, about 5 ml, 10 ml, and 15 ml of buffer based on 1 g of wet-cell may be added. In another embodiment, the number of crushing cycles can be varied for suitable crushing conditions. In a specific embodiment, the crushing cycle may be set to 1 time, 2 times, 3 times, 4 times, and 5 times. As a specific example of appropriate disruption conditions for increasing the yield and enzyme activity of the uric acid oxidase variant, 10 ml of buffer per 1 g of wet-cell is added, a pressure of 20,000 psi, and two disruption cycle conditions may be set.
4) 비천연아미노산을 포함하는 요산산화효소 변이체의 분리 정제4) Separation and purification of uric acid oxidase variants containing non-natural amino acids
이 때, 상기 방법은, 비천연아미노산을 포함하는 요산산화효소 변이체의 분리 및 정제를 위해 크로마토그래피를 수행함을 추가로 더 포함할 수 있다. 일 실시예로, 상기 분리 및 정제를 위해 2단계 이상의 크로마토그래피가 수행될 수 있다. 상기 크로마토그래피는 일 실시예로, 양이온 크로마토그래피, 음이온크로마토그래피, 크기배제 크로마토그래피를 포함할 수 있다. 일 실시예로, 상기 분리 및 정제를 위해 1차 음이온 크로마토그래피 및 2차 양이온 크로마토그래피가 수행될 수 있다. 이때, 요산산화효소 변이체의 분리 및 정제의 최적화를 위해 elution buffer의 조건이 변경될 수 있다. 일 실시예로, 1차 음이온 크로마토그래피에서 elution buffer의 조건은 20mM sodium phosphate pH 6.0 + 0.1M NaCl일 수 있다. 일 실시예로, 2차 양이온 크로마토그래피에서 elution buffer의 조건은 20mM sodium phosphate pH 8.5일 수 있다.At this time, the method may further include performing chromatography to separate and purify the variant uric acid oxidase containing the non-natural amino acid. In one embodiment, two or more steps of chromatography may be performed for the separation and purification. In one embodiment, the chromatography may include cation chromatography, anion chromatography, and size exclusion chromatography. In one embodiment, primary anion chromatography and secondary cation chromatography may be performed for the separation and purification. At this time, the conditions of the elution buffer may be changed to optimize the separation and purification of uric acid oxidase variants. In one embodiment, the 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.
2. 알부민-링커 컨쥬게이트 생산2. Production of albumin-linker conjugates
본 출원의 컨쥬게이트 제조 방법은, 요산산화효소 변이체와 알부민을 결합시키기 위해, 알부민과 링커를 연결하는 과정을 수행함을 포함한다. 상기 알부민-링커 컨쥬게이트 생산은 3. 비천연 아미노산을 포함하는 요산산화효소 변이체와 알부민-링커 컨쥬게이트 결합 전에 수행된다. 상기 알부민-링커 컨쥬게이트 생산은 1. 비천연아미노산을 포함하는 요산산화효소 변이체 생산 전 또는 생산 과정 중간 또는 생산 후에 수행될 수 있다.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.
1) 제2 반응 작용기 포함하는 링커와 알부민 연결1) linking a linker containing a second reactive functional group with albumin
상기 링커는 알부민의 특정 아미노산 잔기를 통해 연결될 수 있다. The linker may be connected through a specific amino acid residue of albumin.
일 실시예로, 시스테인 잔기를 통해 알부민과 연결될 수 있으며, 상기 연결은 링커의 제2 반응 작용기와 알부민의 시스테인 잔기와의 반응에 의해 형성될 수 있다. 구체적인 일 실시예로, 알부민은 알부민의 시스테인 잔기의 티올에 대한 반응성을 가지는 제2 반응 작용기를 포함하는 링커와 연결된다. 보다 더 구체적인 일 실시예로, 알부민은 말레이미드 (Maleimide; MAL), 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles), 할로아세탈 (haloacetal), 피리딜 디설파이드 (pyridyl disulfide), 또는 기타 공지된 티올기와 반응할 수 있는 반응기를 포함하는 링커와 연결된다. 보다 더 구체적인 일 실시예로, 알부민은 말레이미드 (Maleimide; MAL) 또는 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles)를 포함하는 링커와 연결된다. 특정한 실시예에서, 인간 혈청 알부민(human serum albumin, HSA)에 포함된 시스테인의 티올기와 링커의 제2 반응 작용기가 반응하여 알부민-링커 컨쥬게이트가 제조될 수 있다. 특정한 실시예에서, 인간 혈청 알부민(human serum albumin, HSA)의 34번 시스테인 (Cys 34)의 티올기와 링커의 제2 반응 작용기에 포함된 티올 반응성 그룹이 반응하여 알부민-링커 컨쥬게이트가 제조될 수 있다. 특정한 실시예에서, 인간 혈청 알부민 또는 이의 변이체의 Cys 34의 티올기와 링커의 제2 반응작용기에 포함된 말레이미드 그룹이 반응하여 알부민-링커 컨쥬게이트가 제조될 수 있다. 이때, 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles)를 포함하는 링커와 연결되는 경우, 체내 안정성이 증가하는 효과를 가질 수 있다. 다만, 이에 제한되지 않고, 통상적으로 사용되는 결합 반응에 의해 연결되는 것을 모두 포함한다.In one embodiment, it 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. In a specific embodiment, albumin is linked with a linker including a second reactive functional group having reactivity to a thiol of a cysteine residue of albumin. In a more specific embodiment, 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. In a more specific embodiment, albumin is linked with a linker including Maleimide (MAL) or 3-Arylpropiolonitrile. In a specific example, 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. In a specific example, 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. there is. In a specific embodiment, 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. At this time, when connected with a linker containing 3-arylpropiolonitriles, it may have an effect of increasing stability in the body. However, it is not limited thereto, and includes all those connected by commonly used coupling reactions.
일 실시예에서, 링커와 알부민의 반응으로부터 생성된 알부민-링커 컨쥬게이트는 하기의 화학식 2의 구조를 가질 수 있다:In one embodiment, an albumin-linker conjugate resulting from the reaction of a linker with albumin may have the structure of Formula 2 below:
[화학식 2][Formula 2]
F1 - L - X2 - A. F 1 - L - X 2 - A.
이때, At this time,
F1은 제1 반응 작용기이고, F 1 is a first reactive functional group;
L은 링커 모이어티이고, L is a linker moiety,
X2는 제2 반응 작용기와 알부민의 반응에 의해 형성된 구조이고, X 2 is a structure formed by the reaction of the second reactive functional group with albumin,
A는 알부민이다. A is albumin.
이때, F1 및 L은 링커로부터 유래되며, 관련 단락에서 설명된 바와 같다. wherein F 1 and L are derived from a linker and are as described in the relevant paragraphs.
특정한 실시예에서, 화학식 2는 화학식 2-1로 표현될 수 있고, 알부민-링커 컨쥬게이트는 하기의 화학식 2-1의 구조를 가질 수 있다:In certain embodiments, Formula 2 may be represented by Formula 2-1, and the albumin-linker conjugate may have the structure of Formula 2-1:
[화학식 2-1][Formula 2-1]
Figure PCTKR2022009319-appb-I000029
,
Figure PCTKR2022009319-appb-I000029
,
이때, F1, L1, 및 L3는 관련 단락에서 설명된 바와 같다. 이때, np는 1 이상 6 이하의 정수이다. At this time, F 1 , L 1 , and L 3 are as described in the related paragraph. At this time, np is an integer of 1 or more and 6 or less.
일 실시예에서, 상기 X2는 알부민에 대한 반응 작용기와 알부민이 반응하여 형성된 구조이다. 즉, 상기 X2는 링커의 제2 반응 작용기와 알부민이 반응하여 형성된 구조일 수 있다. 상기 제2 반응 작용기는 일 예시로, 알부민의 시스테인 잔기의 티올에 대한 반응성을 갖는 반응 작용기 일 수 있다. 이 때, 상기 제2 반응 작용기는 말레이미드 (Maleimide; MAL), 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles), 할로아세탈 (haloacetal), 또는 피리딜 디설파이드 (pyridyl disulfide)를 포함하며, 이에 제한되지 않고 통상적으로 티올기와 반응할 수 있는 작용기를 포함할 수 있다. 또 다른 일 예시로, 상기 제2 반응 작용기는 알부민의 라이신 잔기에 대한 반응성을 갖는 것일 수 있다. 이때, 상기 제2 반응 작용기는 N-히드록시석신이미드 에스터 (N-hydroxysuccinimide ester; NHS), 이미도에스터(imidoester)를 포함하며, 이에 제한되지 않고 통상적으로 아민기와 반응할 수 있는 작용기를 포함할 수 있다. In one embodiment, 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. At this time, 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. As another example, the second reactive functional group may have reactivity to the lysine residue of albumin. At this time, 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
일 실시예에서, 상기 X2는 제2 반응 작용기에 포함된 티올 반응성 그룹과 인간 혈청 알부민(human serum albumin; HSA)의 34번 시스테인 (Cys 34)의 티올 잔기가 반응하여 형성된 구조일 수 있다. In one embodiment, 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).
특정한 실시예에서, X2는 하기의 구조로 표현될 수 있다:In certain embodiments, X 2 can be represented by the structure:
Figure PCTKR2022009319-appb-I000030
,
Figure PCTKR2022009319-appb-I000030
,
Figure PCTKR2022009319-appb-I000031
, 및
Figure PCTKR2022009319-appb-I000031
, and
Figure PCTKR2022009319-appb-I000032
.
Figure PCTKR2022009319-appb-I000032
.
이때, S는 알부민의 시스테인의 티올그룹으로부터 유래된다. 특정한 실시예에서, S는 인간 혈청 알부민의 Cys 34로부터 유래된 것일 수 있다. At this time, S is derived from the thiol group of cysteine of albumin. In certain embodiments, S may be derived from Cys 34 of human serum albumin.
상기 링커와 알부민을 결합시키기 위해 알부민과 링커를 일정한 비율로 반응시킬 수 있다. 예를 들어, 1:1, 1:2, 1;3, 1:4, 1:5, 1:6, 1:7 또는 1:8의 비율로 반응시킬 수 있다. 일 실시예로, HSA와 MAL을 포함하는 링커와 1:4의 몰비율로 반응시킬 수 있다. In order to bind the linker and 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.
2) 미반응 링커 제거2) Removal of unreacted linkers
알부민과 링커의 결합 반응 이후에, 미반응 링커를 제거하는 과정이 추가로 수행될 수 있다. 일 실시예로, 미반응 링커를 제거하기 위해 PD-10으로 desalting하는 과정이 수행될 수 있다. 구체적인 실시예로, 10 내지 30 mM의 sodium phosphate를 이용하여 pH 5 내지 8에서 desalting이 일어날 수 있다. 더 구체적으로, 20 mM의 sodium phosphate를 이용하여 pH 6에서 desalting이 일어날 수 있다.After the binding reaction between albumin and the linker, a process of removing the unreacted linker may be additionally performed. In one embodiment, a process of desalting with PD-10 may be performed to remove unreacted linkers. As a specific example, 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.
3. 비천연 아미노산을 포함하는 요산산화효소 변이체와 알부민-링커 컨쥬게이트 결합3. Combination of uric acid oxidase variants containing non-natural amino acids with albumin-linker conjugates
본 출원의 컨쥬게이트 제조 방법은, 요산산화효소 변이체와 알부민을 결합시키기 위해, 알부민과 결합된 링커를 요산산화효소 변이체와 연결하는 과정을 포함한다. 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.
일 실시예에서, 요산산화효소-알부민 컨쥬게이트는 요산산화효소 변이체에 1개 내지 4개의 알부민-링커와 컨쥬게이트되어 제조될 수 있다. In one embodiment, uric acid oxidase-albumin conjugates can be prepared by conjugated uric acid oxidase variants with 1 to 4 albumin-linkers.
다른 실시예에서, 요산산화효소-알부민 컨쥬게이트는 다음을 포함하는 방법으로 제조될 수 있다: 요산산화효소 변이체 서브유닛에 하나의 알부민이 접합된, 서브유닛-알부민 컨쥬게이트가 제조됨; 및 4개의 상기 서브유닛-알부민 컨쥬게이트가 사량체를 구성하거나 또는 상기 서브유닛-알부민 컨쥬게이트 및 요산산화효소 변이체 서브유닛이 사량체를 구성하여, 요산산화효소-알부민 컨쥬게이트가 제조됨, 이때 상기 요산산화효소-알부민 컨쥬게이트는 하나 내지 4개의 서브유닛-알부민 컨쥬게이트를 포함함. In another embodiment, 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.
1) 클릭화학반응1) Click chemical reaction
상기 방법에서, 한쪽 말단에 알부민에 결합되어 있는 전술한 링커는, 다른 말단의 반응 작용기를 이용하여 요산산화효소 변이체와 컨쥬게이팅한다. 구체적으로, 상기 링커는 요산산화효소 변이체의 비천연아미노산을 통해 요산산화효소 변이체와 연결될 수 있으며, 이 때, 상기 연결은 링커의 제1 반응 작용기와 요산산화효소 변이체의 비천연아미노산과의 반응에 의해 형성될 수 있다.In the above method, the linker linked to albumin at one end is conjugated with a urate oxidase variant using a reactive functional group at the other end. Specifically, 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. can be formed by
이 때, 상기 요산산화효소 변이체의 비천연아미노산과 알부민-링커 컨쥬게이트는 클릭화학 반응을 통해 연결될 수 있다. 예를 들어, 요산산화효소-알부민 컨쥬게이트는 요산산화효소 변이체에 포함된 비천연아미노산의 제2 클릭화학작용기와 알부민-링커 컨쥬게이트의 제1 반응 작용기에 포함된 제1 클릭화학작용기의 클릭화학반응에 의해 형성될 수 있다. 이때, 상기 제2 클릭화학작용기는 상기 제1 클릭화학작용기와 클릭화학반응을 할 수 있는 그룹일 수 있다. 예를 들어, 상기 제1 클릭화학작용기는 비시클로노닌이고, 상기 제2 클릭화학작용기는 아자이드일 수 있으나, 달리 제한되지 않는다. In this case, the non-natural amino acid of the uric acid oxidase variant and the albumin-linker conjugate may be linked through a click chemistry reaction. For example, in the uric acid oxidase-albumin conjugate, 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. can be formed by reaction. In this case, the second click chemical functional group may be a group capable of performing a click chemical reaction with the first click chemical functional group. For example, the first click chemical functional group may be a bicyclononine, and the second click chemical functional group may be an azide, but is not limited thereto.
일 실시예에서, 알부민-링커 컨쥬게이트는 제1 클릭화학작용기를 포함한다. 이때, 상기 제1 클릭화학작용기는 말단 알킨 (terminal alkyne), 아자이드 (azide), 스트레인된 알킨(strained alkyne), 다이엔 (diene), 친다이엔체 (dienophile), 트랜스 시클로옥틴(trans-cyclooctene), 알켄 (alkene), 티올 (thiol), 테트라진 (tetrazine), DBCO(dibenzocyclooctyne) 및 비시클로노닌(bicyclononyne) 중에서 선택되는 어느 하나일 수 있으나, 이에 제한되지 않는다. In one embodiment, the albumin-linker conjugate includes a first click chemofunctional group. At this time, 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.
상기 요산산화효소 변이체의 비천연아미노산은 제2 클릭화학작용기를 포함한다. 이때, 상기 제2 클릭화학작용기는 말단 알킨 (terminal alkyne), 아자이드 (azide), 스트레인된 알킨(strained alkyne), 다이엔 (diene), 친다이엔체 (dienophile), 트랜스 시클로옥틴(trans-cyclooctene), 알켄 (alkene), 티올 (thiol), 테트라진 (tetrazine), DBCO(dibenzocyclooctyne) 및 비시클로노닌(bicyclononyne) 중에서 선택되는 어느 하나일 수 있으나, 이에 제한되지 않는다. The non-natural amino acid of the uric acid oxidase variant includes a second click chemical functional group. At this time, 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.
일 실시예에서, 상기 제1 클릭화학작용기는 상기 제2 클릭화학작용기와 클릭화학반응을 할 수 있는 작용기일 수 있다. 일 실시예에서, 상기 제2 클릭화학작용기는 상기 제1 클릭화학작용기와 클릭화학반응을 할 수 있는 작용기일 수 있다. 예를 들어, 상기 제2 클릭화학작용기는 아자이드이고, 상기 제1 클릭화학작용기는 비시클로노닌일 수 있다. In one embodiment, 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. In one embodiment, 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. For example, the second click chemical functional group may be an azide, and the first click chemical functional group may be a bicyclononine.
특정한 실시예에서, 상기 요산산화효소 변이체의 비천연아미노산이 포함하는 아자이드와 알부민-링커 컨쥬게이트가 포함하는 비시클로노닌이 클릭화학 반응을 통해 연결될 수 있다. 상기 비시클로노닌을 이용한 클릭화학반응으로 비천연아미노산을 포함하는 요산산화효소 변이체와 알부민-링커 컨쥬게이트를 결합하는 것은 결합하는 알부민의 개수가 증가하는 장점을 가질 수 있다(표1 참고). In a specific embodiment, 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).
2) 결합 조건2) Combination conditions
상기 클릭화학 반응을 수행함에 있어서, In performing the click chemical reaction,
요산산화효소 변이체와 알부민-링커 컨쥬게이트는 효율적인 연결을 위해 일정한 비율로 반응시킬 수 있다. 일 실시예로, 요산산화효소 변이체와 알부민-링커 컨쥬게이트는 1:1, 1:2, 1;3, 1:4, 1:5, 1:6, 1:7 또는 1:8의 몰비율로 결합 반응시킬 수 있다. 구체적인 일 실시예로, 요산산화효소 변이체와 알부민-링커 컨쥬게이트는 1:4 또는 1:8의 몰비율로 결합 반응시킬 수 있다.The uric acid oxidase variant and the albumin-linker conjugate can be reacted at a constant ratio for efficient linkage. In one embodiment, 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. In a specific embodiment, the uric acid oxidase variant and the albumin-linker conjugate may be combined at a molar ratio of 1:4 or 1:8.
4. 요산산화효소-알부민 컨쥬게이트 분리 및 정제4. Isolation and purification of uric acid oxidase-albumin conjugate
본 출원의 컨쥬게이트 제조 방법에서, 요산산화효소 변이체와 알부민이 결합된 컨쥬게이트를 분리 및 정제하기 위해, 추가로 크로마토그래피가 수행될 수 있다. In the conjugate preparation method of the present application, chromatography may be additionally performed to isolate and purify the conjugate in which the uric acid oxidase variant and albumin are bound.
일 실시예로, 상기 분리 및 정제를 위해 2단계 이상의 크로마토그래피가 수행될 수 있다. 상기 크로마토그래피는 일 실시예로, 양이온 크로마토그래피, 음이온크로마토그래피, 크기배제 크로마토그래피를 포함할 수 있다. 일 실시예로, 요산산화효소-알부민 컨쥬게이트를 분리 및 정제하기 위해 1차 양이온 크로마토그래피 및 2차 음이온 크로마토그래피가 수행될 수 있다. In one embodiment, two or more steps of chromatography may be performed for the separation and purification. In one embodiment, the chromatography may include cation chromatography, anion chromatography, and size exclusion chromatography. In one embodiment, primary cation chromatography and secondary anion chromatography may be performed to isolate and purify the uric acid oxidase-albumin conjugate.
구체적인 일 실시예로, 1차 양이온 크로마토그래피에서, flow rate 100cm/h, binding buffer 20mM sodium phosphate pH6.0, elution buffer 20mM sodium phosphate pH6.0 +0.5M NaCl이 사용될 수 있다. 구체적인 일 실시예로, 2차 음이온 크로마토그래피에서, flow rate 100cm/h, binding buffer 20mM bis-tris pH 6.5, elution buffer 20mM bis-tris pH 6.5 + 0.3M NaCl이 사용될 수 있다. 이때, 일 실시예로, 크기배제크로마토그래피가 추가로 수행될 수 있다. As a specific example, in primary cation chromatography, 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. As a specific example, in secondary anion chromatography, 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. At this time, in one embodiment, size exclusion chromatography may be additionally performed.
상기 크기배제크로마토그래피 (size exclusion chromatography, SEC)라 함은 다양한 크기의 용질이 다공성 매트릭스를 통과하는 속도 (투과도)를 기반으로 혼합물을 분리하는 기술을 의미한다. 즉 분석대상 시료를 젤, 매트릭스, 구슬 (bead)과 같은 다공성 정지상 (stationary phase)이 채워진 컬럼을 통과시키면, 컬럼의 구멍을 통과할 수 없는 큰 분자들은 구멍에 들어가지 못하고 주변의 빈 공간을 통해 빠르게 컬럼을 빠져나오는 반면, 작은 분자들은 컬럼의 구멍을 통해 나오면서 상대적으로 천천히 이동하여 컬럼을 빠져나오는 원리를 이용한 것이다. 이 방법은 일반적으로 완충액 교환 (buffer exchange)을 위한 탈염화 (desalting), 정제를 위한 분리 또는 용질 크기에 따른 분자량 측정에 사용된다. 크기배제 크로마토그래피에서 가장 널리 사용되는 젤은 세파로즈(Sepharose; GE Healthcare), 수퍼로즈 (Superose; GE Healthcare), 세파덱스(Sephadex; Pharmacia), Bio-Gel P(Bio-Rad), 수퍼덱스 ®(superdex' GE Healthcare)와 TSKgel® (silicabased; Sigma) 등의 계열이다. 구체적인 일 실시예로, Superdex 200 increase 10/300 GL를 사용할 수 있다.The size exclusion chromatography (SEC) refers to a technique for separating mixtures based on the rate (permeability) of solutes of various sizes passing through a porous matrix. In other words, when 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). As a specific example, Superdex 200 increase 10/300 GL may be used.
5. 요산산화효소-알부민 컨쥬게이트 생산 방법의 장점5. Advantages of the production method of uric acid oxidase-albumin conjugate
1) 요산산화효소에 결합되는 알부민의 개수 증가1) Increase in the number of albumin bound to uric acid oxidase
본 명세서에서 개시하는 비시클로노닌(BCN)을 이용한 요산산화효소-알부민 컨쥬게이트 생산 방법은 디벤조시클로옥틴(DBCO)를 이용한 요산산화효소-알부민 컨쥬게이트 생산 방법과 비교하여 multi-HSA의 생산 수율이 높다. 즉, BCN을 이용하여 요산산화효소-알부민 컨쥬게이트를 생산하는 경우, 2개 이상의 알부민이 결합된 요산산화효소-알부민 컨쥬게이트의 생산 수율이 증가한다.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.
[표 1] DBCO와 BCN의 multi-HSA 요산산화효소 수율 차이[Table 1] Difference in multi-HSA uric acid oxidase yield between DBCO and BCN
Figure PCTKR2022009319-appb-I000033
Figure PCTKR2022009319-appb-I000033
표1은 DBCO를 이용하여 요산산화효소-알부민 컨쥬게이트를 생산하는 방법과 BCN을 이용하여 요산산화효소-알부민 컨쥬게이트를 생산하는 방법에 따라, 생산된 컨쥬게이트를 비교한 결과이다. 표1을 보면, BCN을 이용하는 경우, 2개 이상의 알부민이 결합된 요산산화효소-알부민 컨쥬게이트가 많이 생산되는 것을 확인할 수 있다. Multi-의 경우 BCN이 DBCO보다 1.5배 높은 수율을 가지며, di-의 경우 2.2배 높은 수율을 가지는 것을 확인할 수 있다.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. Referring to Table 1, it can be seen that when BCN is used, a large number of uric acid oxidase-albumin conjugates in which two or more albumins are bound are produced. In the case of Multi-, it can be seen that 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.
요산산화효소-알부민 컨쥬게이트 생산 방법의 다른 실시예Another embodiment of the method for producing uric acid oxidase-albumin conjugate
링커와 알부민을 접촉하여 알부민-링커 컨쥬게이트를 제조하고, 상기 제조된 알부민-링커 컨쥬게이트와 요산산화효소 변이체를 접촉하여 요산산화효소-알부민 컨쥬게이트를 제조하는 방법에 대하여는 전술하였다. 그러나 이에 제한되지 않고, 요산산화효소-알부민 컨쥬게이트는 하기의 단계를 포함하는 방법에 의해 제조될 수 있다: 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. However, without being limited thereto, the uric acid oxidase-albumin conjugate can be prepared by a method comprising the following steps:
요산산화효소 변이체와 링커를 접촉함, 이때 요산산화효소-링커 컨쥬게이트가 제조됨; 및contacting the uric acid oxidase variant with a linker, wherein a uric acid oxidase-linker conjugate is prepared; and
상기 요산산화효소-링커 컨쥬게이트와 알부민을 접촉함, 이때 요산산화효소-알부민 컨쥬게이트가 제조됨. Contacting the uric acid oxidase-linker conjugate with albumin, whereby the uric acid oxidase-albumin conjugate is prepared.
각 단계에 대한 상세한 설명을 위해, 전술한 요산산화효소-알부민 컨쥬게이트 생산 방법이 참조될 수 있다.For a detailed description of each step, reference may be made to the method for producing the uric acid oxidase-albumin conjugate described above.
일 실시예에서, 요산산화효소-링커 컨쥬게이트는 하기의 화학식 5의 구조를 가질 수 있다:In one embodiment, the uric acid oxidase-linker conjugate may have the structure of Formula 5:
[화학식 5][Formula 5]
Uoxv - X1 - L - F2, Uoxv - X 1 - L - F 2 ,
이때, At this time,
Uoxv는 요산산화효소 변이체이고, Uoxv is a uric acid oxidase variant,
X1은 제1 반응 작용기와 요산산화효소 변이체의 비천연아미노산의 반응에 의해 형성된 구조이고, 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,
L은 링커 모이어티이고, L is a linker moiety,
F2는 제2 반응 작용기이다. F 2 is a second reactive functional group.
이때, F2 및 L은 링커로부터 유래되며, 관련 단락에서 설명된 바와 같다. where F 2 and L are derived from the linker and are as described in the relevant paragraphs.
Ⅱ. 요산산화효소-알부민 컨쥬게이트II. Uric acid oxidase-albumin conjugate
전술한 바와 같이, 요산산화효소-알부민 컨쥬게이트는 요산산화효소 변이체와 알부민이 결합된 복합체를 의미할 수 있다. 일 실시예에서, 요산산화효소-알부민 컨쥬게이트는 1개 내지 8개의 알부민을 포함할 수 있다. 특정한 실시예에서, 요산산화효소-알부민 컨쥬게이트는 1개 내지 4개의 알부민을 포함할 수 있다. 특정한 실시양태에서, 요산산화효소-알부민 컨쥬게이트는 사량체의 요산산화효소와 하나 이상의 알부민이 결합된 형태일 수 있다. 이때, 하나의 서브유닛에 0 또는 1개의 알부민이 결합되어 있을 수 있다. 예를 들어, 요산산화효소-알부민 컨쥬게이트가 4개의 알부민을 포함하는 경우, 4개의 서브유닛 각각에 하나의 알부민이 결합되어 있을 수 있다. 다른 예로, 요산산화효소-알부민 컨쥬게이트가 3개의 알부민을 포함하는 경우, 4개의 서브유닛 중 3개의 서브유닛 각각에 하나의 알부민이 결합되어 있고, 나머지 하나의 서브유닛에는 알부민이 결합되어 있지 않을 수 있다. 이때, 요산산화효소와 각각의 알부민은 링커를 통해 결합된다. 이하에서, 본 출원에 의해 제공되는 요산산화효소-알부민 컨쥬게이트를 개시한다. As described above, the uric acid oxidase-albumin conjugate may refer to a complex in which a variant of uric acid oxidase and albumin are bound. In one embodiment, the uric acid oxidase-albumin conjugate may contain 1 to 8 albumins. In certain embodiments, a uric acid oxidase-albumin conjugate may contain 1 to 4 albumins. In certain embodiments, 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. For example, when the uric acid oxidase-albumin conjugate includes four albumins, one albumin may be bound to each of the four subunits. As another example, when 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. can At this time, uric acid oxidase and each albumin are bonded through a linker. Hereinafter, the uric acid oxidase-albumin conjugate provided by the present application is disclosed.
1. 요산산화효소-알부민 컨쥬게이트1. Uric acid oxidase-albumin conjugate
1) 구조1) Rescue
본 출원은 하기의 화학식 3의 구조를 갖는 요산산화효소-알부민 컨쥬게이트를 제공한다:The present application provides a uric acid oxidase-albumin conjugate having the structure of Formula 3 below:
[화학식 3][Formula 3]
Uoxv - [X1 - L - X2 - A]n, Uoxv - [X 1 - L - X 2 - A] n ,
이때, n은 1 이상의 정수이다. 일 실시예에서, n은 1 이상 8 이하의 정수일 수 있다. 특정한 실시예에서, n은 1 이상 4 이하의 정수일 수 있다. At this time, 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.
i) Uoxvi) Uoxv
화학식 3에서, Uoxv는 요산산화효소 변이체를 의미한다. In Formula 3, Uoxv means a uric acid oxidase variant.
전술한 바와 같이, 요산산화효소 변이체는 4개의 서브유닛을 포함하는 사량체 단백질일 수 있다. 상기 요산산화효소 변이체는 1개 내지 4개의 요산산화효소 변이체 서브유닛을 포함하며, 상기 요산산화효소 변이체 서브유닛은 그 원형이 되는 야행성 요산산화효소 서브유닛에서 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것을 특징으로 한다. 일 실시예로, 상기 요산산화효소 변이체는 1개의 요산산화효소 변이체 서브유닛, 및 3개의 야생형 요산산화효소 서브유닛을 포함할 수 있다. 일 실시예로, 상기 요산산화효소 변이체는 2개의 요산산화효소 변이체 서브유닛, 및 1개의 야생형 요산산화효소 서브유닛을 포함할 수 있다. 다른 실시예로, 상기 요산산화효소 변이체는 3개의 요산산화효소 변이체 서브유닛, 및 1개의 야생형 요산산화효소 서브유닛을 포함할 수 있다. 또 다른 실시예로, 상기 요산산화효소 변이체는 4개의 요산산화효소 변이체 서브유닛을 포함할 수 있다. 요산산화효소 변이체 서브유닛은 하나 이상의 비천연아미노산을 포함하며, 관련 단락에서 전술한 바와 같다. 특정한 실시예에서, 요산산화효소 변이체 서브유닛은 AzF를 포함할 수 있다. As described above, 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. characterized by In one embodiment, the uric acid oxidase variant may include one uric acid oxidase variant subunit and three wild-type uric acid oxidase subunits. In one embodiment, 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.
일 실시예에서, 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. 구체적으로, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환된 것일 수 있다. 보다 더 구체적으로, 서열번호 1의 아미노산 서열의 160번째 트립토판, 또는 174번째 트립토판 중 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환된 것일 수 있다.In one embodiment, 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. Specifically, 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 group consisting of tryptophan at position 208, phenylalanine at position 219, tyrosine at position 233, tyrosine at position 251, tyrosine at position 258, phenylalanine at position 259, tryptophan at position 265 and phenylalanine at position 279 may be substituted with an unnatural amino acid. . More specifically, one or more residues selected from tryptophan at position 160 or tryptophan at position 174 of the amino acid sequence of SEQ ID NO: 1 may be substituted with a non-natural amino acid.
다른 실시예에서, 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. 구체적으로, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신 중 선택되는 하나 이상의 잔기가 비천연아미노산으로 치환된 것일 수 있다. 보다 더 구체적으로, 서열번호 2의 아미노산 서열의 163번째 타이로신(tyrosine), 170번째 페닐알라닌 (phenylalanine), 200번째 타이로신, 및 271번째 트립토판(tryptophan) 중 선택되는 하나 이상의 잔기가 비천연 아미노산으로 치환된 것일 수 있다.In another embodiment, 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. Specifically, 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 189 tryptophan, position 191 tryptophan, position 200 tyrosine, position 211 phenylalanine, position 215 tyrosine, position 226 phenylalanine, position 239 phenylalanine, position 253 tyrosine, position 257 tyrosine , At least one residue selected from tyrosine at position 264, phenylalanine at position 265, tryptophan at position 271, phenylalanine at position 281, and tyrosine at position 282 may be substituted with a non-natural amino acid. More specifically, 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
다른 실시예에서, 요산산화효소 변이체 서브유닛은 서열번호 3의 아미노산 서열의 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. 구체적으로, 상기 요산산화효소 변이체 서브유닛은 서열번호 3의 아미노산 서열의 20번째 타이로신, 52번째 페닐알라닌, 75번째 타이로신, 77번째 페닐알라닌, 82번째 페닐알라닌, 88번째 페닐알라닌, 96번째 페닐알라닌, 100번째 페닐알라닌, 102번째 트립토판, 108번째 트립토판, 113번째 페닐알라닌, 114번째 페닐알라닌, 115번째 트립토판, 125번째 페닐알라닌, 163번째 페닐알라닌, 166번째 페닐알라닌, 171번째 타이로신, 190번째 트립토판, 192번째 타이로신, 199번째 페닐알라닌, 203번째 타이로신, 214번째 페닐알라닌, 227번째 타이로신, 253번째 페닐알라닌, 260번째 페닐알라닌, 269번째 페닐알라닌, 270번째 타이로신, 276번째 타이로신, 295번째 트립토판, 및 301번째 페닐알라닌 중 선택된 하나 이상의 아미노산이 비천연 아미노산으로 치환된 것일 수 있다. In another embodiment, 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. Specifically, 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 selected from tyrosine, 214th phenylalanine, 227th tyrosine, 253rd phenylalanine, 260th phenylalanine, 269th phenylalanine, 270th tyrosine, 276th tyrosine, 295th tryptophan, and 301st phenylalanine are substituted with a non-natural amino acid. it could be
ii) X1 ii) X 1
상기 X1은 요산산화효소 변이체의 비천연아미노산에 포함된 제2 클릭화학작용기와 링커 또는 알부민-링커 컨쥬게이트의 제1 반응 작용기에 포함된 제1 클릭화학작용기 간의 클릭화학반응에 의해 형성된 구조를 포함한다. 제1 클릭화학작용기 및 제2 클릭화학작용기 각각은 관련 단락에서 전술한 바와 같다. 특정한 실시예에서, X1은 비천연아미노산에 포함된 아자이드 그룹과 링커 또는 알부민-링커 컨쥬게이트의 제1 반응 작용기에 포함된 비시클로노닌 그룹의 반응에 의해 형성된 구조를 포함할 수 있다. 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. include Each of the first click chemical functional group and the second click chemical functional group is as described above in the relevant paragraph. In a specific embodiment, 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.
일 실시예에서, X1은 하기의 구조 중 선택되는 어느 하나의 구조로 표현될 수 있다:In one embodiment, X 1 may be represented by any one structure selected from the following structures:
Figure PCTKR2022009319-appb-I000034
Figure PCTKR2022009319-appb-I000034
Figure PCTKR2022009319-appb-I000035
,
Figure PCTKR2022009319-appb-I000035
,
Figure PCTKR2022009319-appb-I000036
Figure PCTKR2022009319-appb-I000036
Figure PCTKR2022009319-appb-I000037
및
Figure PCTKR2022009319-appb-I000037
and
Figure PCTKR2022009319-appb-I000038
.
Figure PCTKR2022009319-appb-I000038
.
일 실시예로, X1은 아자이드와 BCN의 클릭화학반응으로 인해 형성된 구조를 포함할 수 있다. In one embodiment, X 1 may include a structure formed by a click chemical reaction between azide and BCN.
iii) Liii) L
상기 L은 링커 모이어티이고, 링커로부터 유래된다. 일 실시예에서, L은 X1과 X2를 연결하는 구조이다. 일 실시예에서, 상기 L은 알킬렌, 알케닐렌, 알키닐렌, 아랄킬렌, 아릴알킬렌 또는 (C2H4O)np을 포함할 수 있다. 이 때, np은 1 내지 6 중에 선택될 수 있다. 일 실시예에서, L은 치환 또는 비치환된 C1-50 알킬렌, 치환 또는 비치환된 C1-50 헤테로알킬렌, 치환 또는 비치환된 C2-50 알케닐렌, 치환 또는 비치환된 C2-50 헤테로알케닐렌, 치환 또는 비치환된 C2-50 알키닐렌, 치환 또는 비치환된 C2-50 헤테로알키닐렌일 수 있다. 이때, 상기 헤테로알킬렌, 헤테로알케닐렌, 및 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함할 수 있다. 일 실시예에서, 헤테로원자는, 각각 독립적으로, O, S, 및 N 중에서 선택될 수 있다. 특정한 실시예에서, L은 치환 또는 비치환된 C10-30 알킬렌, 치환 또는 비치환된 C10-30 헤테로알킬렌, 치환 또는 비치환된 C10-30 알케닐렌, 치환 또는 비치환된 C10-30 헤테로알케닐렌, 치환 또는 비치환된 C10-30 알키닐렌, 치환 또는 비치환된 C10-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L은 치환 또는 비치환된 C12-20 알킬렌, 치환 또는 비치환된 C12-20 헤테로알킬렌, 치환 또는 비치환된 C12-20 알케닐렌, 치환 또는 비치환된 C12-20 헤테로알케닐렌, 치환 또는 비치환된 C12-20 알키닐렌, 치환 또는 비치환된 C12-20 헤테로알키닐렌일 수 있다. 이때, 상기 헤테로알킬렌, 헤테로알케닐렌, 및 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함할 수 있다. 일 실시예에서, 헤테로원자는, 각각 독립적으로, O, S, 및 N 중에서 선택될 수 있다. The L is a linker moiety and is derived from a linker. In one embodiment, L is a structure connecting X 1 and X 2 . In one embodiment, 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. In one embodiment, 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. In this case, the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms. In one embodiment, the heteroatoms can each independently be selected from O, S, and N. In certain embodiments, 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. 10-30 heteroalkenylene, substituted or unsubstituted C 10-30 alkynylene, or substituted or unsubstituted C 10-30 heteroalkynylene. In certain embodiments, 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. 12-20 heteroalkenylene, substituted or unsubstituted C 12-20 alkynylene, or substituted or unsubstituted C 12-20 heteroalkynylene. In this case, the heteroalkylene, heteroalkenylene, and heteroalkynylene may each independently contain one or more heteroatoms. In one embodiment, the heteroatoms can each independently be selected from O, S, and N.
특정한 실시예에서, 링커 모이어티(L)는 하기의 구조 중 어느 하나로 표현될 수 있다:In certain embodiments, the linker moiety (L) can be represented by any of the following structures:
Figure PCTKR2022009319-appb-I000039
, 및
Figure PCTKR2022009319-appb-I000039
, and
Figure PCTKR2022009319-appb-I000040
,
Figure PCTKR2022009319-appb-I000040
,
이때, 1''은 X1과 연결되고, 2''는 X2와 연결된다. At this time, 1'' is connected to X 1 and 2'' is connected to X 2 .
일 실시예에서, L1은 결합(bond)이거나, 치환 또는 비치환된 C1-6 알킬렌, 치환 또는 비치환된 C1-6 헤테로알킬렌, 치환 또는 비치환된 C2-6 알케닐렌, 치환 또는 비치환된 C2-6 헤테로알케닐렌, 치환 또는 비치환된 C2-6 알키닐렌, 또는 치환 또는 비치환된 C2-6 헤테로알키닐렌일 수 있다. 일 실시양태에서, L1은 -O-, -NH- 또는 -S- 일 수 있다. 특정한 실시예에서, L1은 비치환된 C1-3알킬렌, 또는 비치환된 C1-3 헤테로알킬렌일 수 있다.In one embodiment, 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. In one embodiment, L 1 can be -O-, -NH- or -S-. In certain embodiments, L 1 can be unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
일 실시예에서, L2는 알킬렌, 알케닐렌, 알키닐렌, 아랄킬렌, 아릴알킬렌 또는 (C2H4O)np을 포함할 수 있고, 이때 np는 1 이상 6 이하의 정수일 수 있다. 일 실시예에서, L2는 치환 또는 비치환된 C1-30 알킬렌, 치환 또는 비치환된 C1-30 헤테로알킬렌, 치환 또는 비치환된 C2-30 알케닐렌, 치환 또는 비치환된 C2-30 헤테로알케닐렌, 치환 또는 비치환된 C2-30 알키닐렌, 또는 치환 또는 비치환된 C2-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L2는 (C2H4O)np을 포함하는, 치환 또는 비치환된 C1-30 알킬렌, 치환 또는 비치환된 C1-30 헤테로알킬렌, 치환 또는 비치환된 C2-30 알케닐렌, 치환 또는 비치환된 C2-30 헤테로알케닐렌, 치환 또는 비치환된 C2-30 알키닐렌, 또는 치환 또는 비치환된 C2-30 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L2는 치환 또는 비치환된 C10-20 알킬렌, 치환 또는 비치환된 C10-20 헤테로알킬렌, 치환 또는 비치환된 C10-20 알케닐렌, 치환 또는 비치환된 C10-20 헤테로알케닐렌, 치환 또는 비치환된 C10-20 알키닐렌, 또는 치환 또는 비치환된 C10-20 헤테로알키닐렌일 수 있다. 특정한 실시예에서, L2는 (C2H4O)np을 포함하는, 치환 또는 비치환된 C10-20 알킬렌, 치환 또는 비치환된 C10-20 헤테로알킬렌, 치환 또는 비치환된 C10-20 알케닐렌, 치환 또는 비치환된 C10-20 헤테로알케닐렌, 치환 또는 비치환된 C10-20 알키닐렌, 또는 치환 또는 비치환된 C10-20 헤테로알키닐렌일 수 있다.In one embodiment, 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. In one embodiment, 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. In certain embodiments, 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 . C 2-30 alkenylene, substituted or unsubstituted C 2-30 heteroalkenylene, substituted or unsubstituted C 2-30 alkynylene, or substituted or unsubstituted C 2-30 heteroalkynylene. In certain embodiments, 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. C 10-20 heteroalkenylene, substituted or unsubstituted C 10-20 alkynylene, or substituted or unsubstituted C 10-20 heteroalkynylene. In certain embodiments, 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.
일 실시예에서, L3는 결합(bond)이거나, 치환 또는 비치환된 C1-6 알킬렌, 치환 또는 비치환된 C1-6 헤테로알킬렌, 치환 또는 비치환된 C2-6 알케닐렌, 치환 또는 비치환된 C2-6 헤테로알케닐렌, 치환 또는 비치환된 C2-6 알키닐렌, 또는 치환 또는 비치환된 C2-6 헤테로알키닐렌일 수 있다. 일 실시예에서, L3는 -O-, -NH- 또는 -S- 일 수 있다. 특정한 실시예에서, L3는 비치환된 C1-3알킬렌, 또는 비치환된 C1-3 헤테로알킬렌일 수 있다. In one embodiment, 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. In one embodiment, L 3 may be -O-, -NH- or -S-. In certain embodiments, L 3 can be unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
특정한 실시예에서, 링커 모이어티(L)은 하기의 구조로 표현될 수 있다:In certain embodiments, the linker moiety (L) can be represented by the following structure:
Figure PCTKR2022009319-appb-I000041
,
Figure PCTKR2022009319-appb-I000041
,
이때, 1''은 X1과 연결되고, 2''는 X2와 연결되고, L1, L3, 및 np 각각은 관련 단락에서 전술한 바와 같다. In this case, 1'' is connected to X 1 , 2'' is connected to X 2 , and each of L 1 , L 3 , and np is as described above in the related paragraph.
iv) X2 iv) X 2
일 실시예에서, 상기 X2는 알부민에 대한 반응 작용기와 알부민이 반응하여 형성된 구조이다. 즉, 상기 X2는 링커의 제2 반응 작용기와 알부민이 반응하여 형성된 구조일 수 있다. 상기 제2 반응 작용기는 일 예시로, 알부민의 시스테인 잔기의 티올에 대한 반응성을 갖는 반응 작용기 일 수 있다. 이 때, 상기 제2 반응 작용기는 말레이미드 (Maleimide; MAL), 3-아릴프로피올로니트릴 (3-Arylpropiolonitriles), 할로아세탈 (haloacetal), 또는 피리딜 디설파이드 (pyridyl disulfide)를 포함하며, 이에 제한되지 않고 통상적으로 티올기와 반응할 수 있는 작용기를 포함할 수 있다. 또 다른 일 예시로, 상기 제2 반응 작용기는 알부민의 라이신 잔기에 대한 반응성을 갖는 것일 수 있다. 이때, 상기 제2 반응 작용기는 N-히드록시석신이미드 에스터 (N-hydroxysuccinimide ester; NHS), 이미도에스터(imidoester)를 포함하며, 이에 제한되지 않고 통상적으로 아민기와 반응할 수 있는 작용기를 포함할 수 있다. In one embodiment, 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. At this time, 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. As another example, the second reactive functional group may have reactivity to the lysine residue of albumin. At this time, 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
일 실시예에서, 상기 X2는 제2 반응 작용기에 포함된 티올 반응성 그룹과 인간 혈청 알부민(human serum albumin; HSA)의 34번 시스테인 (Cys 34)의 티올 잔기가 반응하여 형성된 구조일 수 있다. In one embodiment, 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).
특정한 실시예에서, X2는 하기의 구조 중 어느 하나로 표현될 수 있다:In certain embodiments, X 2 can be represented by any of the following structures:
Figure PCTKR2022009319-appb-I000042
,
Figure PCTKR2022009319-appb-I000042
,
Figure PCTKR2022009319-appb-I000043
, 및
Figure PCTKR2022009319-appb-I000043
, and
Figure PCTKR2022009319-appb-I000044
.
Figure PCTKR2022009319-appb-I000044
.
이때, S는 알부민의 시스테인의 티올기로부터 유래된다. 특정한 실시예에서, S는 인간 혈청 알부민 또는 이의 변이체의 Cys 34로부터 유래된 것일 수 있다. At this time, S is derived from the thiol group of cysteine of albumin. In a specific embodiment, S may be derived from Cys 34 of human serum albumin or variants thereof.
v) Av) A
화학식 A는 알부민을 의미하며, 관련 단락에서 전술한 바와 같다. 일 실시예에서, 알부민은 인간 혈청 알부민일 수 있다. 일 실시예에서 알부민은 인간 혈청 알부민의 변이체일 수 있다. 일 실시예에서 알부민은 서열번호 4 내지 15 중에 선택되는 어느 하나의 아미노산 서열을 포함할 수 있다. Formula A refers to albumin, as described above in the relevant paragraph. In one embodiment, the albumin can be human serum albumin. In one embodiment, the albumin may be a variant of human serum albumin. In one embodiment, albumin may include any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
전술한 바와 같이, 알부민에 포함된 시스테인의 티올 잔기와 링커의 일 말단의 티올 반응성 그룹의 반응을 통해, X2- 가 형성된다. 이때, 티올 반응성 그룹과 반응하는 알부민에 포함된 시스테인은 Cys 34일 수 있다. As described above, 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. At this time, the cysteine included in albumin that reacts with the thiol-reactive group may be Cys 34.
요산산화효소-알부민 컨쥬게이트의 구체예Specific examples of uric acid oxidase-albumin conjugates
일 실시예에서, 화학식 3은 하기의 화학식 3-1로 표현될 수 있고, 요산산화효소-알부민 컨쥬게이트는 화학식 3-1의 구조를 가질 수 있다:In one embodiment, 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:
[화학식 3-1][Formula 3-1]
Figure PCTKR2022009319-appb-I000045
,
Figure PCTKR2022009319-appb-I000045
,
이때 Uoxv, A, X1, X2, L1, L2, L3, 및 n 각각은 관련 단락에서 전술한 바와 같다. where Uoxv, A, X 1 , X 2 , L 1 , L 2 , L 3 , and n Each is as described above in the relevant paragraphs.
일 실시예에서, 화학식 3-1은 하기의 화학식 3-2로 표현될 수 있고, 요산산화효소-알부민 컨쥬게이트는 화학식 3-2의 구조를 가질 수 있다:In one embodiment, 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:
[화학식 3-2][Formula 3-2]
Figure PCTKR2022009319-appb-I000046
,
Figure PCTKR2022009319-appb-I000046
,
이때, Uoxv, A, X1, X2, L1, L3, n, 및 np 각각은 관련 단락에서 전술한 바와 같다. At this time, each of Uoxv, A, X 1 , X 2 , L 1 , L 3 , n, and np is as described above in the related paragraph.
특정한 실시예에서, 화학식 3-2는 하기의 화학식 3-3으로 표현될 수 있고, 요산산화효소-알부민 컨쥬게이트는 하기의 화학식 3-3의 구조를 가질 수 있다:In a specific embodiment, 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:
[화학식 3-3][Formula 3-3]
Figure PCTKR2022009319-appb-I000047
Figure PCTKR2022009319-appb-I000047
Uoxv, A, X1, X2, 및 n 각각은 관련 단락에서 전술한 바와 같다. Uoxv, A, X 1 , X 2 , and n Each is as described above in the relevant paragraphs.
일 실시예에서, 화학식 3은 하기의 화학식 3-4로 표현될 수 있고, 요산산화효소-알부민 컨쥬게이트는 화학식 3-4의 구조를 가질 수 있다:In one embodiment, 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:
[화학식 3-4][Formula 3-4]
Figure PCTKR2022009319-appb-I000048
,
Figure PCTKR2022009319-appb-I000048
,
이때 Uoxv, A, X1, X2, L1, L2, L3, 및 n 각각은 관련 단락에서 전술한 바와 같다. where Uoxv, A, X 1 , X 2 , L 1 , L 2 , L 3 , and n Each is as described above in the relevant paragraphs.
일 실시예에서, 화학식 3-4는 하기의 화학식 3-5로 표현될 수 있고, 요산산화효소-알부민 컨쥬게이트는 화학식 3-5의 구조를 가질 수 있다:In one embodiment, 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:
[화학식 3-5][Formula 3-5]
Figure PCTKR2022009319-appb-I000049
,
Figure PCTKR2022009319-appb-I000049
,
이때, Uoxv, A, X1, X2, L1, L3, np, 및 n 각각은 관련 단락에서 전술한 바와 같다. In this case, Uoxv, A, X 1 , X 2 , L 1 , L 3 , np, and n Each is as described above in the relevant paragraphs.
일 실시예에서, 상기 요산산화효소-알부민 컨쥬게이트는 하나 이상의 서브유닛-알부민 컨쥬게이트를 포함할 수 있다. 이때, 서브유닛-알부민 컨쥬게이트는 요산산화효소 변이체 서브유닛에 알부민이 접합된 것이다. 일 실시예에서, 상기 요산산화효소-알부민 컨쥬게이트는 1, 2, 3, 4, 5, 6, 7, 또는 8개의 서브유닛-알부민 컨쥬게이트를 포함할 수 있다. 이때, 상기 서브유닛-알부민 컨쥬게이트는 하기의 화학식 4의 구조를 가질 수 있다. In one embodiment, the uric acid oxidase-albumin conjugate may include one or more subunit-albumin conjugates. In this case, the subunit-albumin conjugate is one in which albumin is conjugated to a urate oxidase mutant subunit. In one embodiment, the urate oxidase-albumin conjugate may include 1, 2, 3, 4, 5, 6, 7, or 8 subunit-albumin conjugates. In this case, the subunit-albumin conjugate may have a structure represented by Chemical Formula 4 below.
[화학식 4][Formula 4]
Uoxv' - X1 - L - X2 - A,Uoxv' - X 1 - L - X 2 - A;
이때, Uoxv' 은 요산산화효소 변이체 서브유닛이고, 상기 요산산화효소 변이체 서브유닛은 관련 단락에서 전술한 바와 같다. At this time, Uoxv' is a uric acid oxidase variant subunit, and the uric acid oxidase variant subunit is as described above in the relevant paragraph.
이때, X1, L, X2, 및 A 각각은 관련 단락에서 전술한 바와 같다. At this time, each of X1, L, X2, and A is as described above in the relevant paragraph.
2. 본 명세서에서 개시하는 방법에 의해 생산된 컨쥬게이트의 특징2. Characteristics of conjugates produced by the methods disclosed herein
개선된 반감기 및 면역원성Improved half-life and immunogenicity
본 명세서에서 개시하는 요산산화효소-알부민 컨쥬게이트 생산 방법에 의해 생산된 요산산화효소-알부민 컨쥬게이트는 2개 이상의 알부민이 결합된 multi-알부민 컨쥬게이트가 높은 비율로 존재한다. In the urate oxidase-albumin conjugate produced by the method for producing a uric acid oxidase-albumin conjugate disclosed herein, a multi-albumin conjugate in which two or more albumins are bonded is present in a high proportion.
약물의 반감기를 증가시키기 위해 알부민을 결합하는 컨쥬게이트의 경우, 결합된 알부민의 개수가 많아지면 반감기가 증가하고 면역원성이 개선된다고 알려져있다. In the case of a conjugate that binds albumin to increase the half-life of a drug, it is known that the half-life increases and immunogenicity improves when the number of albumin bound increases.
따라서, 본 명세서의 방법에 의해 생산된 컨쥬게이트는 알부민이 2개 이상 결합된 요산산화효소-알부민 컨쥬게이트의 비율이 높기 때문에, 종래의 DBCO를 이용한 방법에 의해 생산된 컨쥬게이트에 비하여 개선된 효능을 갖는다.Therefore, since 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. have
3. 용도3. Usage
1) 약학적 용도1) Pharmaceutical use
본 출원은 상기 방법에 의하여 생산된 요산산화효소-알부민 컨쥬게이트를 유효성분으로 포함하는 종양용해증후군(tumor lysis syndrome, TLS), 고요산혈증, 통풍, 관절내 요산염 결정의 침착, 요산염 결정의 침착으로 인한 급성 통풍성 관절염, 요로 결석증, 신결석증 및 통풍성 신병증으로 이루어진 군으로부터 선택된 1종 이상의 질환의 예방 또는 치료용 약학적 조성물을 제공한다.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. 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.
본 출원에서 "종양용해증후군(tumor lysis syndrome, TLS)"은 항암제 투여 후 종양세포의 급격한 파괴로 인해, 세포 내 요산, 칼륨, 인이 혈류로 유리되어 발생한다. 요산 배설이 현저히 증가하면 요세관에 요산 결정이 침전되고, 이로 인해 요세관이 폐쇄되어 신부전이 유발될 수 있다. 이와 같이 종양용해증후군 환자는 대사이상으로 상태가 빠르게 악화될 수 있다. 이때, 종양용해증후군에서 가장 흔한 대상이상으로 알려져 있는 것이 고요산혈증이다. In the present application, “tumor lysis syndrome (TLS)” 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. As described above, patients with tumor lysis syndrome may rapidly deteriorate due to metabolic abnormalities. At this time, hyperuricemia is known as the most common target abnormality in tumor lysis syndrome.
본 출원에서 "고요산혈증 (Hyperuricemia)"이란 혈액에 요산의 농도가 증가되어 있는 질환이다. 이는 혈청에서 모노소디움 요산염의 농도가 제한된 용해도의 한계를 넘을 때 발생한다. 37℃에서 혈장의 요산 포화도는 약 7 mg/dl이다. 그러므로 이 농도를 넘으면 물리화학적으로 과포화 상태가 된다. 혈청 요산 농도는 정상인의 평균 혈청 요산 농도보다 +2 표준편차를 초과한 경우에 상대적으로 높은 것으로 알려져 있다. 대부분의 역학 조사에서 남자의 상위 한계는 7 mg/dl, 여자는 6 mg/dl이다. 이러한 이유 때문에 고요산혈증의 실질적인 상위 한계 수준은 7.0 mg/dl 이상인 경우로 정의되고 있다.In this application, "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. It is known that the serum uric acid concentration is relatively higher when it exceeds +2 standard deviation than the average serum uric acid concentration of normal subjects. In most epidemiologic studies, 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.
본 출원에 있어서, 상기 고요산혈증 및 고요산혈증 관련 대사 장애는 요산이 정상 수치보다 많아져서 신장이 요산을 배설시키는 능력이 저하될 경우 혈액 중에 여분의 요산이 남아있게 되고 혈중 요산치가 높아져 발생되는 질환이나 질병을 말한다.In the present application, 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
구체적으로, 상기 고요산혈증 관련 대사 장애에는 통풍, 요산 결정, 관절내 요산염 결정의 침착, 요산염 결정의 침착으로 인한 급성 통풍성 관절염, 단일 관절성 관절염, 염증성 관절염의 통증 발작, 요로 결석증, 신결석증 및 통풍성 신병증이 포함된다. 장기적인 신결석증 및 통풍성 신병증은 신장 손상 및 신부전의 위험을 증가시키는 것으로 알려져 있다.Specifically, 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.
상기 통풍은 통상적으로 급성 염증성 관절염의 재발성 발작을 특징으로 하는 의학적 상태이며, 엄지 발가락 기저부의 중족-지골 관절에서 흔하게 발생한다. 또한, 통풍은 관절, 힘줄 및 주변 조직들 내에서 결정화되고 침착 되는 혈중 요산에 의해 유발되며, 통풍 결절, 신장 결석 또는 요산염 신병증으로서 존재할 수도 있다.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.
본 출원에서 사용되는 용어, "예방"이란 본 출원에 따른 약학적 조성물의 투여에 의해 통풍, 고요산혈증, 또는 고요산혈증 관련 대사 장애를 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used in this application, the term "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.
본 출원에서 사용되는 용어, "치료"란 본 출원에 따른 약학적 조성물의 투여에 의해 통풍, 고요산혈증, 또는 고요산혈증 관련 대사 장애에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "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.
본 출원의 상기 요산산화효소-알부민 컨쥬게이트를 포함하는 조성물은 상기 성분에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다.The 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.
본 출원의 약학적 조성물은 상기 요산산화효소-알부민 컨쥬게이트를 유효 성분으로 포함하는 것 이외에 약학적으로 허용 가능한 담체를 추가로 포함할 수 있다.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.
본 출원에서 사용될 수 있는 담체의 종류는 특별히 제한되지 아니하며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로는, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 말토덱스트린, 글리세롤, 에탄올 등을 들 수 있다. 이들은 단독으로 사용되거나 2 종 이상을 혼합하여 사용될 수 있다.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.
또한, 본 출원의 약학적 조성물은 필요한 경우, 부형제, 희석제, 항산화제, 완충액 또는 정균제 등 기타 약학적으로 허용 가능한 첨가제들을 첨가하여 사용할 수 있으며, 충진제, 증량제, 습윤제, 붕해제, 분산제, 계면 활성제, 결합제 또는 윤활제 등을 부가적으로 첨가하여 사용할 수 있다.In addition, if necessary, 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.
본 출원의 약학적 조성물에 있어서, 상기 요산산화효소-알부민 컨쥬게이트는 약학적 조성물의 전체의 중량을 기준으로 0.00001중량％ 내지 99.99중량％ 로 포함될 수 있으며, 바람직하게는 0.1중량% 내지 90중량%, 보다 바람직하게는 0.1중량% 내지 70중량%, 더욱 바람직하게는 0.1중량% 내지 50중량%로 포함될 수 있으나, 이에 한정되지 않으며 투여 대상의 상태, 구체적인 병증의 종류, 진행 정도 등에 따라 다양하게 변경될 수 있다. 필요한 경우, 약학적 조성물의 전체 함량으로도 포함될 수 있다.In the pharmaceutical composition of the present application, 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.
본 출원에서 사용된 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 출원의 약학 조성물을 도입하는 것을 의미하며, 본 출원의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.As used herein, 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.
본 출원의 약학적 조성물은 경구 투여 또는 비경구 투여를 위한 적합하고 다양한 제형으로 제제화되어 사용될 수 있다.The pharmaceutical composition of the present application may be formulated and used in various dosage forms suitable for oral or parenteral administration.
본 출원의 약학적 조성물을 이용한 경구 투여용 제제의 비제한적인 예로는, 트로키제 (troches), 로젠지 (lozenge), 정제, 수용성 현탁액, 유성 현탁액, 조제 분말, 과립, 에멀젼, 하드 캡슐, 소프트 캡슐, 시럽 또는 엘릭시르제 등을 들 수 있다.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.
본 출원의 약학적 조성물을 경구 투여용으로 제제화하기 위하여, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴 등과 같은 결합제; 디칼슘 포스페이트 등과 같은 부형제; 옥수수 전분 또는 고구마 전분 등과 같은 붕해제; 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌 글리콜 왁스 등과 같은 윤활유 등을 사용할 수 있으며, 감미제, 방향제, 시럽제 등도 사용할 수 있다. 나아가 캡슐제의 경우에는 상기 언급한 물질 외에도 지방유와 같은 액체 담체 등을 추가로 사용할 수 있다.In order to formulate the pharmaceutical composition of the present application for oral administration, 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. Furthermore, in the case of capsules, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.
본 출원의 약학적 조성물을 이용한 비경구용 제제의 비제한적인 예로는, 주사액, 좌제, 호흡기 흡입용 분말, 스프레이용 에어로졸제, 연고, 도포용 파우더, 오일, 크림 등을 들 수 있다.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.
본 출원의 약학적 조성물을 비경구 투여용으로 제제화하기 위하여, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조 제제, 외용제 등을 사용할 수 있으며, 상기 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.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.
본 출원의 약학적 조성물을 주사액으로 제제화하는 경우, 본 출원의 약학적 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고 이를 앰플 (ampoule) 또는 바이알 (vial)의 단위 투여용으로 제제화할 수 있다.When 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.
본 출원의 약학적 조성물을 에어로졸제로 제제화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합할 수 있다.When the pharmaceutical composition of the present application is formulated as an aerosol, a propellant or the like may be blended with additives so that the water-dispersed concentrate or wet powder is dispersed.
본 출원의 약학적 조성물을 연고, 크림, 도포용 파우더, 오일, 피부 외용제 등으로 제제화하는 경우에는, 동물성 유, 식물성 유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화 아연 등을 담체로 사용하여 제제화할 수 있다.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.
본 출원의 약학적 조성물의 약학적 유효량, 유효 투여량은 약학적 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로 등에 의해 다양해질 수 있으며, 약학 조성물의 투여로 달성하고자 하는 반응의 종류와 정도, 투여 대상이 되는 개체의 종류, 연령, 체중, 일반적인 건강 상태, 질병의 증세나 정도, 성별, 식이, 배설, 해당 개체에 동시 또는 이시에 함께 사용되는 약물 기타 조성물의 성분 등을 비롯한 여러 인자 및 의약 분야에서 잘 알려진 유사 인자에 따라 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자는 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다. 예를 들어, 본 출원의 약학적 조성물의 일일 투여량은 0.01 내지 1000mg/kg이고, 바람직하게는 0.1 내지 100mg/kg이며, 하루 일회 내지 수회에 나누어 투여할 수 있다.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. For example, 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.
본 출원의 약학적 조성물의 투여는 하루에 1회 투여될 수 있고, 수회에 나누어 투여될 수도 있다. 본 출원의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양으로 투여할 수 있으며, 이는 당업자에 의해 용이하게 결정될 수 있다.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.
본 출원의 약학적 조성물의 투여 경로 및 투여 방식은 각각 독립적일 수 있으며, 목적하는 해당 부위에 상기 약학적 조성물이 도달할 수 있는 한, 특별한 제한없이 임의의 투여 경로 및 투여 방식에 따를 수 있다. 상기 약학적 조성물은 경구 투여 또는 비경구 투여 방식으로 투여할 수 있다.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.
본 출원의 약학적 조성물의 비경구 투여 방법으로는, 정맥 내 투여, 복강 내 투여, 근육 내 투여, 경피 투여 또는 피하 투여 등을 이용할 수 있으며, 상기 조성물을 질환 부위에 도포하거나 분무, 흡입하는 방법 또한 이용할 수 있으나 이에 제한되지 않는다.As a parenteral administration method of the pharmaceutical composition of the present application, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, etc. may be used, and a method of applying, spraying, or inhaling the composition to the diseased area It can also be used, but is not limited thereto.
본 출원의 약학적 조성물은 통풍, 고요산혈증, 또는 고요산혈증 관련 대사 장애를 예방 또는 치료하기 위하여 추가적으로 호르몬 치료, 약물 치료 등의 다양한 방법들과 병용하여 사용될 수 있다.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.
2) 식품 조성물2) Food composition
본 출원은 상기 방법에 의하여 생산된 요산산화효소-알부민 컨쥬게이트를 유효성분으로 포함하는 고요산혈증, 통풍, 관절내 요산염 결정의 침착, 요산염 결정의 침착으로 인한 급성 통풍성 관절염, 요로 결석증, 신결석증 및 통풍성 신병증으로 이루어진 군으로부터 선택된 1종 이상의 질환의 예방 또는 개선용 식품 조성물을 제공한다.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.
본 출원의 용어, "개선"이란, 본 출원의 조성물의 투여로 질환이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "improvement" refers to all activities that improve or beneficially change a disease by administering the composition of the present application.
본 출원의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 "건강기능식품"이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.The food composition of the present application can be used as a health functional food. The term “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.
본 출원의 식품 조성물은 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin), 비오틴(biotin), 폴레이트(folate), 판토텐산(panthotenic acid) 등을 포함할 수 있다. 또한, 아연(Zn), 철(Fe), 칼슘(Ca), 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu) 등의 미네랄을 포함할 수 있다. 또한, 라이신, 트립토판, 시스테인, 발린 등의 아미노산을 포함할 수 있다. 또한, 방부제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 디히드로초산나트륨 등), 살균제(표백분과 고도표백분, 차아염소산나트륨 등), 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨루엔(BHT) 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 조미료(MSG 글루타민산나트륨 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(food additives)을 첨가할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.The food composition of the present application may include additional ingredients that are commonly used and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included. In addition, 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. can be added. The additive may be selected according to the type of food and used in an appropriate amount.
본 출원의 식품 조성물을 식품 첨가물로 사용할 경우, 이를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다.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.
본 출원의 식품 조성물에 있어서, 요산산화효소-알부민 컨쥬게이트의 함량은 특별히 제한되지 않으며, 투여 대상의 상태, 구체적인 병증의 종류, 진행 정도 등에 따라 다양하게 변경될 수 있다. 필요한 경우, 식품의 전체 함량으로도 포함될 수 있다.In the food composition of the present application, 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.
Ⅲ. 실시예III. Example
1. BCN 링커를 이용한 요산산화효소-알부민 컨쥬게이트 생산 프로토콜1. Uric acid oxidase-albumin conjugate production protocol using BCN linker
1) 위치특이적으로 비천연아미노산으로 치환된 요산산화효소 생산을 위한 발현 벡터의 제작 및 형질전환1) Construction and transformation of an expression vector for the production of uric acid oxidase site-specifically substituted with a non-natural amino acid
p-Azido-L-phenylalanine(AzF)는 Chem-Impex International(Wood Dale, IL)로부터 구입하였으며, 0.2 M NaOH에 용해하여 100 mM 스톡(stock solution)을 만들어 사용하였다. pQE80 플라스미드는 Qiagen(Valencia, CA)으로부터 구입하였다.p-Azido-L-phenylalanine (AzF) 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).
위치 특이적으로 AzF가 포함된 Uox 변이체 제작을 위한 발현벡터의 제작Construction of expression vector for site-specific AzF-containing Uox mutant construction
Methanococcus jannaschii로부터 기원한 tyrosyl-tRNA synthetase와 amber suppressor tRNA로 이루어진 AzF 특이적인 engineered pair를 포함하고 있는 pEVOL-pAzF 플라스미드(Plasmid ID: 31186)를 Addgene(Cambridge, MA)로부터 구입하여 추가적인 수정 없이 사용하였다. Aspergillus flavus로부터 기원한 재조합 요소산화효소(Uox 변이체)를 발현하기 위한 박테리아용 발현벡터를 제작하기 위하여, Uox 서브유닛의 코딩서열(서열번호 1)을 합성한 후, pQE80에 클로닝하여 pQE80-Uox를 만들었다. 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. To construct 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.
Uox의 174번 위치의 트립토판을 amber 코돈(UAG)으로 대체하기 위하여 상기 pQE80-Uox를 템플레이트로 Site-directed mutagenic PCR을 수행하였다. 174번 위치에 amber 코돈을 도입하기 위해 5-CACTGAAGGAGACTTAGGATAGAATCCTG-3(서열번호 16)및 5-CAGGATTCTATCCTAAGTCTCCTTCAGTG-3(서열번호 17)프라이머를 사용하였다.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). In order to introduce an amber codon at position 174, 5-CACTGAAGGAGACTTAGGATAGAATCCTG-3 (SEQ ID NO: 16) and 5-CAGGATTCTATCCTAAGTCTCCTTCAGTG-3 (SEQ ID NO: 17) primers were used.
모든 DNA 클로닝은 제한효소를 사용하지 않는 클로닝기술을 이용하였다.All DNA cloning was performed using cloning technology that does not use restriction enzymes.
2) 배양2) culture
1차 종배양Primary seed culture
아래 조성으로 1차 종배양을 위한 배지 150mL를 준비하고 멸균하였다.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, KH2PO4 (덕산, Cat no. 432): 2.3g/L(별도 멸균 후 첨가), K2HPO4 (덕산, Cat no. 562): 12.53 g/mL(별도 멸균 후 첨가), Kanamycin(Sigma, Cat no. 60615) : 35μg/mL(제균 여과 후 첨가), Chloramphenicol(WAKO, Cat no. 036-10571) : 35μg/mL(제균 여과 후 첨가).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 μg/mL (added after sterilization filtration), Chloramphenicol (WAKO, Cat no. 036-10571): 35 μg/mL (added after sterilization filtration).
AzF가 포함된 Uox 변이체(Uox-W174AzF)를 발현하기 위하여 E. coli C321.△A.exp(Addgene, ID: 49018)를 사용하였으며, pEVOL-pAzF와 pQE80-UoxW174amb로 동시에 형질전환시켜 C321.△A.exp [Uox-W174amb]로 명명하여 사용하였다.To express the AzF-containing Uox variant (Uox-W174AzF), E. coli C321.ΔA.exp (Addgene, ID: 49018) was used, and pEVOL-pAzF and pQE80-UoxW174amb were simultaneously transformed to C321.Δ It was named and used as A.exp [Uox-W174amb].
150mL 종배양 배지/500mL baffled flask에 상기 균주 0.5%를 접종하고 아래 조건으로 종배양을 진행하였다.150mL seed culture medium/500mL baffled flask was inoculated with 0.5% of the strain, and seed culture was performed under the following conditions.
온도 : 37℃, RPM : 220rpm , 시간 : 14시간.Temperature: 37°C, RPM: 220 rpm, Time: 14 hours.
2차 종배양Secondary seed culture
아래 조성으로 2차 종배양을 위한 배지 2L를 준비하고 멸균하였다.2L of medium for secondary seed culture was prepared with the following composition and sterilized.
Soy peptone : 12g/L , Yeast extract : 24g/L, KH2PO4 : 2.3g/L(별도 멸균 후 첨가), K2HPO4 : 12.53 g/mL(별도 멸균 후 첨가), Kanamycin : 35μg/mL(제균 여과 후 첨가), Chloramphenicol : 35μg/mL(제균 여과 후 첨가).Soy peptone : 12g/L , Yeast extract : 24g/L, KH 2 PO 4 : 2.3g/L (added after separate sterilization), K 2 HPO 4 : 12.53 g/mL (added after separate sterilization), Kanamycin : 35μg/L mL (added after sterilization filtration), Chloramphenicol: 35 μg/mL (added after sterilization filtration).
1차 종배양액 25mL을 500mL 2차 종배양 배지/2L baffled flask에 접종하고 아래 조건으로 2차 종배양을 진행하였다.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.
온도 : 37℃, RPM : 200rpm , 시간 : 5시간.Temperature: 37°C, RPM: 200 rpm, Time: 5 hours.
본배양main culture
아래 조성으로 본배양을 위한 별도 멸균용 배지를 준비하였다.A separate sterilization medium for main culture was prepared with the following composition.
Glucose(대정, Cat no. 3020-4405) : 20g/L(별도 멸균 후 첨가), MgSO4(대정, Cat no. 5513-4405): 1.2g/L(별도 멸균 후 첨가), Kanamycin : 35μg/mL(제균 여과 후 첨가), chloramphenicol : 35μg/mL(제균 여과 후 첨가), KH2PO4 : 2.3g/L(별도 멸균 후 첨가), K2HPO4 : 12.53g/L(별도 멸균 후 첨가).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: 35μg/L mL (added after disinfection filtration), chloramphenicol : 35 μg/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) ).
FeedingFeeding
아래 조성으로 Feeding 용 C-source, N-source 제조하고 별도 멸균하였다.C-source and N-source for feeding were prepared with the following composition and sterilized separately.
Glucose : 300g/L(C-source), MgSO4:1.0g/L(C-source), Yeast extract : 211g/L(N-source), (NH4)2SO4 (대정, Cat no. 1082-1405)-: 1.5g/L(N-source).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).
Base 및 antifoamBase and antifoam
5N NaOH 와 50% antifoam(Sigma, Cat no. A6426) 용액을 준비하여 별도 멸균하였다.A 5N NaOH and 50% antifoam (Sigma, Cat no. A6426) solution was prepared and separately sterilized.
농축배지concentrated medium
아래 조성으로 본배양을 위한 농축 배지 5L를 준비하여 50L 발효기의 시료주입구에 넣고 정제수로 vessel 무게를 초기 배양 부피에 맞추어 멸균하였다. 농축배지의 멸균이 끝난 뒤 연동식 펌프를 이용하여 준비한 별도 멸균용 배지를 발효기 내부로 주입하였다.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 : 12g/L , Yeast extract : 24g/L, Thiamine-HCl(Alfa Aesar, Cat no. A19560) :0.1g/L, Antifoam : 0.1g/L.Soy peptone: 12 g/L, Yeast extract: 24 g/L, Thiamine-HCl (Alfa Aesar, Cat no. A19560): 0.1 g/L, Antifoam: 0.1 g/L.
OD 3.0~5.0의 2차 종배양액 5%를 균주 접종용 병에 옮겨 담았다. 50L 발효기 내부로 균주를 접종하고 배양을 시작하였다.5% of the secondary seed culture with an OD of 3.0 to 5.0 was transferred to a strain inoculation bottle. The strain was inoculated into the 50L fermentor and culture was started.
배양 조건 : Air 1vvm, pH 7.0, DO(%) 30, 온도 30℃, RPM 360, 내압 100mbar.Culture conditions: Air 1vvm, pH 7.0, DO (%) 30, temperature 30℃, RPM 360, internal pressure 100mbar.
50L 발효기 배양 28시간 후 최종 OD는 56을 기록하였고 wet cell 104.6g/L를 확보하였다. 20시간 이후 균주 성장이 지속되지 않았다.After 28 hours of cultivation in the 50L fermentor, the final OD was 56, and 104.6 g/L of wet cells were obtained. Strain growth did not continue after 20 hours.
배양 조건 변경 : RPM 600, 내압 400mbar Change of culture conditions: RPM 600, internal pressure 400mbar
교반속도와 발효기 내압을 변경한 결과 34시간 배양 후 최종 OD는 96을 기록하였고 wet cell은 161.2g/L를 확보하였다. 배양 20시간 까지는 배양 조건 변경 전과 비슷한 균주 생장 곡선을 보였으나, 그 이후에는 성장 지연이 나타나지 않고, 균주 성장이 계속되어 더 높은 배양 수율을 얻을 수 있었다(도 1 참고).As a result of changing the agitation speed and the internal pressure of the fermenter, the final OD after 34 hours of culture was recorded as 96, and the wet cell obtained 161.2 g / L. Up to 20 hours of culture, a strain growth curve similar to that before the culture condition change was shown, but after that, no growth delay was observed, and a higher culture yield was obtained as the strain continued to grow (see FIG. 1).
[표 2] 배양 조건[Table 2] Culture conditions
Figure PCTKR2022009319-appb-I000050
Figure PCTKR2022009319-appb-I000050
조건 추가 변경Add condition change
아래의 조건으로 위치특이적으로 비천연아미노산이 포함된 요산산화효소 생산 균주를 배양한다. 배양 수율을 높이기 위해 추가적으로 일부 조건이 변경되었다.Under the following conditions, a site-specific strain producing urate oxidase containing unnatural amino acids is cultured. Some conditions were additionally changed to increase the culture yield.
최종 배양 조건Final culture conditions
Air : 1.5vvm, pH 7.0, DO(%) 30, Temp:30℃, RPM:600, 내압 400mbar.Air: 1.5vvm, pH 7.0, DO (%) 30, Temp:30℃, RPM:600, internal pressure 400mbar.
Induction : 제균 여과한 AzF, arabinose(Alfa Aesar, Cat no. A11921), IPTG(바이오세상, Cat no. IF1006-005-00)를 각각 최종 농도 2mM, 0.2%, 1mM이 되도록 첨가하였다. 배양 시작 후 흡광도 값이 OD 40에 도달하면 연동식 펌프를 이용하여 inducer를 주입하고 Uox-AzF (Uox 변이체)단백질 발현을 유도하였다.Induction: AzF filtered for eradication, arabinose (Alfa Aesar, Cat no. A11921), and IPTG (Biosesang, Cat no. IF1006-005-00) were added to final concentrations of 2 mM, 0.2%, and 1 mM, respectively. After the start of the culture, when the absorbance value reached OD 40, the inducer was injected using a peristaltic pump, and expression of Uox-AzF (Uox variant) protein was induced.
배양 결과, 최종 배양시간은 42시간이며, 최종 OD는 150.8을 기록하였다. 최종 wet-cell의 무게는 11.8kg으로 나타났으며, 221.8g/L의 배양 수율을 보였다(표 3 및 도 2 참고).As a result of the culture, 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).
[표 3] 최종배양결과[Table 3] Final culture results
Figure PCTKR2022009319-appb-I000051
Figure PCTKR2022009319-appb-I000051
단백질 농도 및 효소 활성 측정Measurement of protein concentration and enzyme activity
일정한 시간 간격으로 배양액을 취하여 원심분리로 wet cell을 회수하고, 초음파 분쇄기로 파쇄한 뒤 총 단백질 농도와 Uox 효소 활성을 측정한 결과, 배양을 지속할수록 단백질 농도가 계속 증가하여 배양 37시간 이후 35mg/mL이상의 단백질 농도를 확인하였다.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.
또한, 효소 활성의 경우 inducer 첨가 전에는 0.01U/mL로 활성을 보이지 않았으나, inducer 첨가 후에는 단백질 농도 6mg/mL 샘플 기준 약 0.1U/mL의 활성을 유지하는 것을 확인하였다.In addition, in the case of enzyme activity, it was confirmed that the activity of about 0.01 U / mL was not shown before the addition of the inducer, but the activity of about 0.1 U / mL based on the protein concentration of 6 mg / mL sample was maintained after the addition of the inducer.
[표 4] 단백질 농도 및 효소 활성 분석 결과 [Table 4] Protein concentration and enzyme activity analysis results
Figure PCTKR2022009319-appb-I000052
Figure PCTKR2022009319-appb-I000052
단백질 농도 6mg/mL 기준 효소 활성 측정 결과Enzyme activity measurement result based on protein concentration of 6mg/mL
SDS-PAGE 분석으로 Uox-AzF 단백질이 발현되었음을 확인하였고, 발현된 단백질이 Uox 효소 활성을 갖는 것을 uric acid를 이용하여 확인하였다(표 4 및 도 3 참고). It was confirmed by SDS-PAGE analysis that the Uox-AzF protein was expressed, and that the expressed protein had Uox enzyme activity was confirmed using uric acid (see Table 4 and FIG. 3).
3) 세포 파쇄3) cell disruption
아래의 파쇄 조건으로 세포 파쇄기(Avestin Emulsiflex D20)를 이용하여 세포를 파쇄하였다. 100g의 Uox-AzF wet cell을 10배 부피인 1L 파쇄 buffer로 2회 세척 후 파쇄하고, 원심분리한 상등액을 이용하여 파쇄 효율을 비교하였다. 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.
파쇄 조건crushing conditions
파쇄 buffer : 20mM Tris-HCl(pH9.0), 파쇄 압력 : 20,000psi, 파쇄 cycle : 1~4, wet cell(g) to buffer(mL) ration : 1:10.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.
[표 5] Uox-AzF 세포 파쇄 결과[Table 5] Uox-AzF cell disruption results
Figure PCTKR2022009319-appb-I000053
Figure PCTKR2022009319-appb-I000053
Wet cell 1g 당 10mL의 파쇄 buffer로 20,000psi 파쇄 압력에서 1cycle 파쇄를 진행하였을 때, 가장 높은 파쇄 효율을 얻을 수 있었다. 그러나 1 cycle 파쇄 후에는 파쇄액 점성이 높은 상태로 다음 정제 공정에 적용하기 어려워 2cycle 세포 파쇄 후 크로마토그래피 공정을 진행하였다.When 1 cycle disruption was performed at 20,000 psi disruption pressure with 10 mL of disruption buffer per 1 g of wet cell, the highest disruption efficiency was obtained. However, after 1 cycle disruption, it was difficult to apply to the next purification process due to the high viscosity of the disruption solution, so a chromatography process was performed after 2 cycle cell disruption.
4) 분리 정제4) Separation and purification
(1)1차 음이온 크로마토그래피 정제(1) Primary anion chromatography purification
세포 파쇄 후 원심분리와 제균 여과한 상등액을 Anion exchange membrane column(sartobind Q nano 3mL(8mm))을 이용하여 Sartobind Q 800mL로 AKTA Pilot 600에서 정제하였다. Binding buffer는 20mM sodium carbonate pH 9.5, elution buffer는 20mM sodium phosphate pH 6.0 + 0.1 M NaCl을 사용하였다. After cell disruption, 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, and elution buffer was 20 mM sodium phosphate pH 6.0 + 0.1 M NaCl.
(2)2차 양이온 크로마토그래피 정제(2) Secondary cation chromatography purification
1차 음이온 크로마토그래피에서 분리한 sample에 2차 binding buffer를 5배 희석하여 전도도 값을 3mS/cm 이하로 낮춘 뒤 양이온 membrane column(Sartobind S nano 1mL(4mm))을 이용하였다. Binding buffer는 20mM sodium carbonate pH 6.0을 사용하였다. Elution buffer는 20mM sodium phosphate pH 8.5를 사용하였다.After diluting the secondary binding buffer 5 times in the sample separated from the primary anion chromatography to lower the conductivity value to 3 mS/cm or less, 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.
5) 알부민 링커 결합5) albumin linker binding
Uox-rHSA 결합을 위해 BCN-링커와 rHSA(Albumedix, Recombumin Elite) (서열번호 4)를 아래 표와 같은 비율로 혼합한 뒤 차광 처리한 인큐베이터(23℃, 130rpm)에서 2시간 동안 결합반응을 유도하였다.For Uox-rHSA binding, 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℃, 130 rpm). did
사용된 BCN-링커의 구조는 다음과 같다:The structure of the BCN-linker used is as follows:
Figure PCTKR2022009319-appb-I000054
.
Figure PCTKR2022009319-appb-I000054
.
[표 6] BCN-링커와 HSA 반응 조건[Table 6] BCN-linker and HSA reaction conditions
Figure PCTKR2022009319-appb-I000055
Figure PCTKR2022009319-appb-I000055
6) Uox-HSA 결합6) Uox-HSA binding
정제된 Uox-AzF는 10배 농축하여 buffer change(PBS pH 7.4)하였고, 결합 반응물이 rHSA-링커는 Uox-rHSA 결합을 위해 1,200mL에서 400mL 로 약 3배 농축한 뒤 아래 표와 같은 비율로 반응을 유도하였다. 총 반응 용량은 1,200mL으로 차광 처리한 인큐베이터(23℃, 130rpm)에서 15시간 동안 처리하였다.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.
[표 7] Uox-AzF와 HSA 반응 조건[Table 7] Uox-AzF and HSA reaction conditions
Figure PCTKR2022009319-appb-I000056
Figure PCTKR2022009319-appb-I000056
반응물의 순도시험을 위해 HPLC를 이용하여 측정하였으며, HPLC column은 TSKgel G3000SWXL, 7.8mm I.D * 30cm을 이용하여 flow rate 0.6mL/min으로 측정하였다. Mobile phase는 20mM sodium phosphate pH 7.0을 사용하였으며 220nm 파장으로 결합 반응물의 순도 시험을 진행하였다. Uox-AzF와 rHSA-링커의 결합 반응물의 순도는 total area 기준 12.24%임을 확인하였다(도 4 참고). For the purity test of the reaction, it was measured using HPLC, and the HPLC column was measured at a flow rate of 0.6mL/min using TSKgel G3000SW XL , 7.8mm ID * 30cm. For the mobile phase, 20mM sodium phosphate pH 7.0 was used, and the purity test of the coupling reaction was performed at a wavelength of 220nm. It was confirmed that the purity of the coupling reaction between Uox-AzF and rHSA-linker was 12.24% based on the total area (see FIG. 4).
7) Uox-rHSA 정제 7) Uox-rHSA purification
(1) 1차 양이온 크로마토그래피 정제(1) First cation chromatography purification
Uox-rHSA 결합 반응 원심분리와 제균 여과한 상등액을 XK 50/30 column에 SP-Sepharose high performance를 후정제 용출된 peak를 측정하였다. 정제 조건으로 flow rate는 100cm/h(33mL/min), binding buffer는 20mM sodium phosphate pH6.0, elution buffer는 20mM sodium phosphate pH 6.0 + 0.5 M NaCl을 사용하였다. 1차 양이온 크로마토그래피 정제 결과 elution 1~4에서 Uox-rHSA가 확인되었고, 결합 반응하지 않은 잔존물들이 정제 과정 중 일부 제거되었음을 알 수 있었다(도 5 내지 도 7 참고).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. As purification conditions, the flow rate was 100 cm/h (33 mL/min), the binding buffer was 20 mM sodium phosphate pH6.0, and the elution buffer was 20 mM sodium phosphate pH 6.0 + 0.5 M NaCl. As a result of the first cation chromatography purification, 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).
(2) 2차 음이온 크로마토그래피(2) Secondary anion chromatography
1차 양이온 크로마토그래피 정제 후 고순도 Uox-rHSA를 획득하기 위해 2차 음이온 크로마토그래피를 수행하였다. 1차 정제물은 20mM bis-tris pH 6.5로 buffer change하여 제균 여과한 상등액을 Hi-scale 50/20 column에 Q-Sepharose high performance를 200mL 충진 후 정제 용출된 peak를 측정하였다. 정제 조건으로 flow rate 100cm/h(33mL/min), binding buffer는 20mM bis-tris pH 6.5, elution buffer는 20mM bis-tris pH6.5 + 0.3M NaCl을 사용하였다. 2차 음이온 크로마토그래피 정제 결과 elution 7~8에서 고순도의 rUox-rHSA를 확인하였고, 그 순도는 Uox-tetra rHSA 기준으로 elution 7에서 84.93%, elution 8에서 87.67%를 보였다. 단백질 농도는 elution 7에서 0.61mg/mL로 200mL을 회수하였고 효소활성은 38.56U/mg을 보였다. Elution 8에서는 0.74mg/mL의 단백질 농도로 450mL를 회수하였고 효소활성은 39.24U/mg임을 확인하였다(표 8, 도 8 및 도 9 참고).After purification by primary cation chromatography, secondary anion chromatography was performed to obtain high-purity Uox-rHSA. The first purified water was buffer changed to 20mM bis-tris pH 6.5, and the filtered supernatant was filled with 200mL of Q-Sepharose high performance in a Hi-scale 50/20 column, and then the purified eluted peak was measured. As purification conditions, a flow rate of 100 cm/h (33 mL/min), a binding buffer of 20 mM bis-tris pH 6.5, and an elution buffer of 20 mM bis-tris pH 6.5 + 0.3 M NaCl were used. As a result of secondary anion chromatography purification, high purity rUox-rHSA was confirmed in elution 7-8, and the purity was 84.93% in elution 7 and 87.67% in elution 8 based on Uox-tetra rHSA. The protein concentration was 0.61mg/mL in elution 7, and 200mL was recovered, and the enzyme activity was 38.56U/mg. In Elution 8, 450mL was recovered at a protein concentration of 0.74mg/mL, and it was confirmed that the enzyme activity was 39.24U/mg (see Table 8, Figs. 8 and 9).
[표 8] 2차 크로마토그래피 정제물 특성[Table 8] Characteristics of second chromatography purified product
Figure PCTKR2022009319-appb-I000057
Figure PCTKR2022009319-appb-I000057
2. BCN과 DBCO 비교 실험2. BCN and DBCO comparison experiment
1) 결합반응1) Combination reaction
요산산화화효소의 174번 위치에 도입된 AzF와 알부민 결합 수율 비교를 위해 BCN-PEG3-MAL(endo) (CAS NO. : 2141976-33-0)와 DBCO-PEG4-MAL(CAS NO. : 1480516-75-3) 링커를 이용하여 수율 비교를 진행하였다. BCN-PEG3-MAL (endo) (CAS NO.: 2141976-33-0) and DBCO-PEG4-MAL (CAS NO.: 1480516 -75-3) Yield comparison was performed using the linker.
사용된 BCN-PEG3-MAL (endo) 링커의 구조는 다음과 같다: The structure of the BCN-PEG3-MAL (endo) linker used is as follows:
Figure PCTKR2022009319-appb-I000058
.
Figure PCTKR2022009319-appb-I000058
.
알부민과 링커(BCN-PEG3-MAL 또는 DBCO-PEG4-MAL)을 1:4 몰비율로 상온에서 2시간 conjugation을 진행하였다. 각각의 수율은 DBCO-PEG4-HSA는 97.6%, BCN-PEG3-HSA는 96.6%의 수율을 보였다. 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.
174번 위치에 AzF가 도입된 요산산화효소와 알부민-링커 컨쥬게이트(BCN-PEG3-HSA 또는 DBCO-PEG4-HSA)를 1:8 몰비율로 상온에서 15시간 conjugation을 진행하였다. 이후 양이온교환 크로마토그래피(SP-HP)를 이용하여 분리정제하고, 크기배제 크로마토그래피(SEC)를 이용하여 각 분획을 SEC-HPLC로 분석하였다. 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).
2) 결합 알부민의 수2) the number of bound albumin
상기 기재된 반응 조건에서 DBCO-PEG4-HSA 또는 BCN-PEG3-HSA와 Uox-AzF의 conjugation 결과를 비교하였다. 그 결과, BCN-PEG3-HSA의 경우 DBCO-PEG4-HSA 링커보다 알부민이 3~4개 결합된 형태인 Uox-multi-HSA conjugate가 1.5배 이상의 수율을 보였으며, 알부민이 2개 결합된 형태인 Uox-di-HSA conjugate는 약 2.2배 높은 수율을 보였다 (도 11 및 도 12 참고). 이는 DBCO group보다 BCN group이 Azide group과의 반응속도가 빠른 것에서 기인한 것으로 생각된다. 따라서, 컨쥬게이트를 제작할 때 BCN을 이용하는 것이 더 유리하다. Conjugation results of DBCO-PEG4-HSA or BCN-PEG3-HSA and Uox-AzF were compared under the reaction conditions described above. As a result, in the case of BCN-PEG3-HSA, the yield of Uox-multi-HSA conjugate, in which 3 to 4 albumins were combined, was more than 1.5 times higher than that of the DBCO-PEG4-HSA linker. Uox-di-HSA conjugate showed a yield about 2.2 times higher (see FIGS. 11 and 12). This is thought to be due to the fact that the reaction rate of the BCN group with the azide group is faster than that of the DBCO group. Therefore, it is more advantageous to use BCN when preparing the conjugate.
3. APN 링커 실험3. APN linker experiments
알부민 34번 위치의 free-cysteine과 링커의 반응에 있어서, 다양한 작용기 중 말레이미드(Maleimide, MAL)와 3-아릴프로피올로니트릴(3-Arylpropiolonitriles, APN)의 반응성 및 안정성에 관하여 실험하였다.In the reaction between albumin 34-position free-cysteine and linker, the reactivity and stability of maleimide (MAL) and 3-arylpropiolonitrile (APN) among various functional groups were tested.
알부민과 MAL의 결합은 체내에서 25%이상 분해된다는 연구논문 보고(Bioconjugate Chem. 2014, 25, 202-206)에 따라 체내 안정성이 높은 APN 링커를 이용하여 실험을 진행하였다. 알부민(Albumedix, Recombumin Elite)과 링커(DBCO-PEG4-APN 또는 DBCO-PEG4-MAL)의 반응 후 SDS-PAGE를 분석하였다. 그 결과는 도 12에 나타나있다.According to a research thesis report (Bioconjugate Chem. 2014, 25, 202-206) that the binding of albumin and MAL is degraded by more than 25% in the body, the experiment was conducted using an APN linker with high stability in the body. After reaction of albumin (Albumedix, Recombumin Elite) and linker (DBCO-PEG4-APN or DBCO-PEG4-MAL), SDS-PAGE was analyzed. The results are shown in FIG. 12 .
그 결과 기존 MAL 반응기을 이용한 알부민 conjugation과 유사한 반응을 보였으며, APN 반응기 사용시 체내 안정성을 확보할 수 있을 것으로 확인되었다.As a result, it showed a similar reaction to albumin conjugation using the existing MAL reactor, and it was confirmed that in vivo stability could be secured when using the APN reactor.
또한, 체내에서 Maleimide의 안정성을 저해하는 요인인 Glutathione와 혈액 내에 존재하는 free 알부민을 첨가하여 in vitro 조건에서 Uox-HSA의 안정성을 확인한 결과 Maleimide로 결합된 컨쥬게이션은 역반응이 일어나 결과적으로 Uox 서브유닛이 SDS-PAGE상에 확인되었다. 그 결과는 도 13에 나타나있다. In addition, the stability of Uox-HSA was confirmed under in vitro conditions by adding glutathione, a factor that inhibits the stability of maleimide in the body, and free albumin present in the blood. This was confirmed on SDS-PAGE. The results are shown in Figure 13.
실험 결과를 종합하면, APN 반응기를 사용하는 경우 체내 안정성이 증가하는 효과를 가질 수 있다.Summarizing the experimental results, the use of an APN reactor may have an effect of increasing stability in the body.

Claims (24)

  1. 다음을 포함하는 요산산화효소-알부민 컨쥬게이트의 제조 방법:A method for preparing a uric acid oxidase-albumin conjugate comprising:
    (a) 알부민 및 링커를 접촉하여 알부민-링커 컨쥬게이트를 제조함, (a) contacting albumin and a linker to prepare an albumin-linker conjugate;
    이때 상기 링커는 일 말단에 제1 클릭화학작용기를 포함하고 다른 말단에 티올 반응성 그룹을 포함하고, 상기 제1 클릭화학작용기는 비시클로노닌(bicyclononyne) 그룹이며, In this case, 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,
    이때 상기 링커의 상기 티올 반응성 그룹과 상기 알부민의 시스테인의 티올기 간의 반응을 통해, 알부민-링커 컨쥬게이트가 제조됨, At this time, through the reaction between the thiol reactive group of the linker and the thiol group of the cysteine of the albumin, an albumin-linker conjugate is prepared,
    (b) 상기 알부민-링커 컨쥬게이트와 요산산화효소 변이체를 접촉함, (b) contacting the albumin-linker conjugate with a urate oxidase variant;
    이때 상기 요산산화효소 변이체는 하나 이상의 비천연아미노산을 포함하고, 상기 비천연아미노산은 상기 제1 클릭화학작용기와 클릭화학반응을 할 수 있는 제2 클릭화학작용기를 포함하고, In this case, the uric acid oxidase variant includes at least one non-natural amino acid, and 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,
    이때 상기 알부민-링커 컨쥬게이트의 일 말단의 상기 제1 클릭화학작용기와 상기 비천연아미노산에 포함된 상기 제2 클릭화학작용기의 반응을 통해, 요산산화효소-알부민 컨쥬게이트가 제조됨. At this time, 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.
  2. 제1항에 있어서, 상기 제2 클릭화학작용기는 아자이드 그룹인, The method of claim 1, wherein the second click chemical functional group is an azide group,
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  3. 제1항에 있어서, 상기 비천연아미노산은 p-Azido-L-phenylalanine인, The method of claim 1, wherein the non-natural amino acid is p-Azido-L-phenylalanine.
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  4. 제1항에 있어서, 상기 요산산화효소 변이체는 The method of claim 1, wherein the uric acid oxidase variant
    3개의 요산산화효소 변이체 서브유닛 및 1개의 야생형 요산산화효소 서브유닛으로 구성되거나, consists of three uric acid oxidase variant subunits and one wild-type uric acid oxidase subunit;
    4개의 요산산화효소 변이체 서브유닛으로 구성되는,Consisting of four uric acid oxidase variant subunits,
    사량체 형태인 것을 특징으로 하는, Characterized in that it is in the form of a tetramer,
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  5. 제4항에 있어서, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 것을 특징으로 하는, The method of claim 4, wherein 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 , position 87 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, 204 At least one amino acid selected from the group consisting of position phenylalanine, position 208 tryptophan, position 219 phenylalanine, position 233 tyrosine, position 251 tyrosine, position 258 tyrosine, position 259 phenylalanine, position 265 tryptophan and position 279 phenylalanine is p-Azido-L- Characterized in that it is substituted with phenylalanine (AzF),
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  6. 제4항에 있어서, 상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 160번째 트립토판, 또는 174번째 트립토판 중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 것을 특징으로 하는, The method of claim 4, wherein the urate oxidase variant subunit is obtained by replacing one or more amino acids selected from tryptophan at position 160 and tryptophan at position 174 of the amino acid sequence of SEQ ID NO: 1 with p-Azido-L-phenylalanine (AzF). characterized by,
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  7. 제4항에 있어서, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 것을 특징으로 하는, The method of claim 4, wherein the uric acid oxidase variant subunit is 10th tyrosine, 163rd tyrosine, 17th phenylalanine, 45th phenylalanine, 59th tyrosine of the amino acid sequence of SEQ ID NO: 2, 77 tryptophan, 82 phenylalanine, 90 phenylalanine, 94 tyrosine, 109 tryptophan, 112 tyrosine, 123 phenylalanine, 136 tyrosine, 137 tyrosine, 143 Tyrosine, position 162 phenylalanine, position 163 tyrosine, position 165 tyrosine, position 170 phenylalanine, position 189 tryptophan, position 191 tryptophan, position 200 tyrosine, position 211 phenylalanine, position 215 tyrosine, position 226 phenylalanine, position 239 phenylalanine, position 253 tyrosine, Characterized in that one or more amino acids selected from 257th tyrosine, 264th tyrosine, 265th phenylalanine, 271st tryptophan, 281st phenylalanine, and 282nd tyrosine are substituted with p-Azido-L-phenylalanine (AzF),
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  8. 제4항에 있어서, 상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 163번째 타이로신(tyrosine), 170번째 페닐알라닌 (phenylalanine), 200번째 타이로신, 및 271번째 트립토판(tryptophan) 중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 것을 특징으로 하는, The method of claim 4, wherein the urate oxidase variant subunit is 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. Characterized in that the above amino acids are substituted with p-Azido-L-phenylalanine (AzF),
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  9. 제1항에 있어서,According to claim 1,
    상기 링커는 하기의 화학식 1의 구조를 갖는, 요산산화효소-알부민 컨쥬게이트의 제조 방법:Method for preparing a urate oxidase-albumin conjugate, wherein the linker has a structure represented by Formula 1 below:
    [화학식 1][Formula 1]
    F1 - L - F2,F 1 - L - F 2 ,
    이때, At this time,
    F1은 상기 제1 클릭화학작용기를 포함하는 제1 반응 작용기이고,F 1 is a first reactive functional group including the first click chemical functional group;
    F2는 상기 티올 반응성 그룹을 포함하는 제2 반응 작용기이고, 이때 상기 티올 반응성 그룹은 말레이미드 그룹 또는 3-아릴프로피올니트릴(3-Arylpropiolonitriles) 그룹이고, 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;
    L은 치환 또는 비치환된 C1-50 알킬렌, 치환 또는 비치환된 C1-50 헤테로알킬렌, 치환 또는 비치환된 C2-50 알케닐렌, 치환 또는 비치환된 C2-50 헤테로알케닐렌, 치환 또는 비치환된 C2-50 알키닐렌, 치환 또는 비치환된 C2-50 헤테로알키닐렌이고, 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,
    이때 상기 헤테로알킬렌, 헤테로알케닐렌, 및 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함하고, 이때 상기 헤테로원자는, 각각 독립적으로, O, S, 및 N 중에서 선택되고, wherein the heteroalkylene, heteroalkenylene, and heteroalkynylene each independently include one or more heteroatoms, wherein the heteroatoms are each independently selected from O, S, and N;
    이때 상기 치환된 알킬렌, 치환된 헤테로알킬렌, 치환된 알케닐렌, 치환된 헤테로알케닐렌, 치환된 알키닐렌, 및 치환된 헤테로알키닐렌은, 각각 독립적으로, 하나 이상의 치환기를 포함하고, 이때 상기 치환기는, 각각 독립적으로 할로겐, C1-3알킬, -NH2, =O, =S, -OH, 및 -SH 중에서 선택됨.In this case, the substituted alkylene, substituted heteroalkylene, substituted alkenylene, substituted heteroalkenylene, substituted alkynylene, and substituted heteroalkynylene each independently include one or more substituents, wherein the Substituents are each independently selected from halogen, C 1-3 alkyl, -NH 2 , =O, =S, -OH, and -SH.
  10. 제1항에 있어서, 상기 링커는 하기의 화학식 1-2의 구조를 갖는, 요산산화효소-알부민 컨쥬게이트의 제조 방법:The method of claim 1, wherein the linker has a structure represented by Formula 1-2 below:
    [화학식 1-2][Formula 1-2]
    Figure PCTKR2022009319-appb-I000059
    ,
    Figure PCTKR2022009319-appb-I000059
    ,
    이때 np는 1 이상 6 이하의 정수이고, In this case, np is an integer of 1 or more and 6 or less,
    이때 L1은 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌이고, In this case, L 1 is a bond, or an unsubstituted C 1-3 alkylene or an unsubstituted C 1-3 heteroalkylene;
    이때 L3는 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌임. In this case, L 3 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene.
  11. 제1항에 있어서, 상기 링커는 하기의 화학식 1-3의 구조를 갖는, 요산산화효소-알부민 컨쥬게이트의 제조 방법:The method of claim 1, wherein the linker has a structure represented by Formula 1-3 below:
    [화학식 1-3][Formula 1-3]
    Figure PCTKR2022009319-appb-I000060
    ,
    Figure PCTKR2022009319-appb-I000060
    ,
    이때 np는 1 이상 6 이하의 정수임. In this case, np is an integer of 1 or more and 6 or less.
  12. 제1항에 있어서, 상기 알부민은 인간 혈청 알부민 또는 이의 변이체인, 요산산화효소-알부민 컨쥬게이트의 제조 방법. The method of claim 1, wherein the albumin is human serum albumin or a variant thereof.
  13. 제1항에 있어서, 상기 알부민은 서열번호 4 내지 15 중에서 선택된 어느 하나의 아미노산 서열을 포함하는, 요산산화효소-알부민 컨쥬게이트의 제조 방법.The method of claim 1, wherein the albumin comprises any one amino acid sequence selected from SEQ ID NOs: 4 to 15.
  14. 제1항에 있어서, 상기 알부민은 인간 혈청 알부민 또는 이의 변이체이고, The method of claim 1, wherein the albumin is human serum albumin or a variant thereof,
    이때 상기 (a)에서, 상기 티올 반응성 그룹과 반응하는 상기 알부민의 시스테인의 티올기는 34번 시스테인의 티올기인 것을 특징으로 하는, In this case, in (a), the thiol group of the cysteine of the albumin reacting with the thiol-reactive group is characterized in that the thiol group of cysteine 34,
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  15. 제1항에 있어서, According to claim 1,
    상기 요산산화효소-알부민 컨쥬게이트는 다음을 포함하는 것을 특징으로 하는, 요산산화효소-알부민 컨쥬게이트의 제조 방법:A method for preparing a uric acid oxidase-albumin conjugate, characterized in that the uric acid oxidase-albumin conjugate comprises the following:
    1개의 서브유닛-알부민 컨쥬게이트, 및 3개의 요산산화효소 변이체 서브유닛;1 subunit-albumin conjugate, and 3 uric acid oxidase variant subunits;
    2개의 서브유닛-알부민 컨쥬게이트, 및 2개의 요산산화효소 변이체 서브유닛; two subunits-albumin conjugate, and two urate oxidase variant subunits;
    3개의 서브유닛-알부민 컨쥬게이트, 및 1개의 요산산화효소 변이체 서브유닛; 또는 3 subunits - albumin conjugate, and 1 uric acid oxidase variant subunit; or
    4개의 서브유닛-알부민 컨쥬게이트,4 subunit-albumin conjugates,
    이때 상기 서브유닛-알부민 컨쥬게이트는 하나의 알부민이 하나의 요산산화효소 변이체 서브유닛에 접합된 것임. In this case, the subunit-albumin conjugate is one albumin conjugated to one urate oxidase variant subunit.
  16. 하나 이상의 알부민이 요산산화효소 변이체에 접합된, 하기의 화학식 3-2의 구조를 갖는 요산산화효소-알부민 컨쥬게이트:A uric acid oxidase-albumin conjugate having the structure of Formula 3-2, wherein at least one albumin is conjugated to a uric acid oxidase variant:
    [화학식 3-2][Formula 3-2]
    Figure PCTKR2022009319-appb-I000061
    ,
    Figure PCTKR2022009319-appb-I000061
    ,
    이때,At this time,
    n은 1 이상 4 이하의 정수이고,n is an integer of 1 or more and 4 or less;
    Uoxv는 요산산화효소 변이체이고, 이때 상기 요산산화효소 변이체는 하나 이상의 비천연아미노산을 포함하고, 이때 상기 비천연아미노산은 아자이드 그룹을 포함하고, 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;
    A는 알부민이고,A is albumin;
    X1은 상기 비천연아미노산에 포함된 상기 아자이드 그룹과 비시클로노닌(bicyclononyne) 그룹이 반응하여 형성된 하기의 구조를 포함하고, 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,
    Figure PCTKR2022009319-appb-I000062
    ,
    Figure PCTKR2022009319-appb-I000062
    ,
    X2는 알부민에 포함된 시스테인의 티올기와 티올 반응성 작용기가 반응하여 형성된 하기의 구조 중 어느 하나를 포함하고, 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;
    Figure PCTKR2022009319-appb-I000063
    , 및
    Figure PCTKR2022009319-appb-I000064
    ,
    Figure PCTKR2022009319-appb-I000063
    , and
    Figure PCTKR2022009319-appb-I000064
    ,
    이때 S는 상기 알부민의 시스테인의 티올기로부터 유래되며, At this time, S is derived from the thiol group of the cysteine of the albumin,
    np는 1 이상 6 이하의 정수이며, np is an integer of 1 or more and 6 or less,
    L1은 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌이고, L 1 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene;
    L3는 결합(bond)이거나, 비치환된 C1-3알킬렌 또는 비치환된 C1-3 헤테로알킬렌이며, L 3 is a bond, or unsubstituted C 1-3 alkylene or unsubstituted C 1-3 heteroalkylene;
    이때 상기 헤테로알킬렌은, 각각 독립적으로, 하나 이상의 헤테로원자를 포함하고, 이때 상기 헤테로원자는, 각각 독립적으로, O, S, 또는 N임. wherein the heteroalkylene, each independently, includes one or more heteroatoms, wherein the heteroatoms are, each independently, O, S, or N.
  17. 제16항에 있어서, 상기 요산산화효소 변이체는 4개의 요산산화효소 변이체 서브유닛으로 구성되는 사량체 형태인, 17. The method of claim 16, wherein the uric acid oxidase variant is in the form of a tetramer consisting of four uric acid oxidase variant subunits,
    요산산화효소-알부민 컨쥬게이트. Uric acid oxidase-albumin conjugate.
  18. 제17항에 있어서, According to claim 17,
    상기 요산산화효소 변이체 서브유닛은 서열번호 1의 아미노산 서열의 8번째 타이로신(tyrosine), 16번째 타이로신, 30번째 타이로신, 46번째 타이로신, 65번째 타이로신, 79번째 페닐알라닌(phenylalanine), 87번째 페닐알라닌, 91번째 타이로신, 106번째 트립토판, 120번째 페닐알라닌, 159번째 페닐알라닌, 160번째 트립토판, 162번째 페닐알라닌, 167번째 타이로신, 174번째 트립토판, 186번째 트립토판, 188번째 트립토판, 191번째 페닐알라닌, 204번째 페닐알라닌, 208번째 트립토판, 219번째 페닐알라닌, 233번째 타이로신, 251번째 타이로신, 258번째 타이로신, 259번째 페닐알라닌, 265번째 트립토판 및 279번째 페닐알라닌으로 구성된 군으로부터 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 것을 특징으로 하는, The uric acid oxidase variant subunit is 8th tyrosine, 16th tyrosine, 30th tyrosine, 46th tyrosine, 65th tyrosine, 79th phenylalanine, 87th phenylalanine, 91st tyrosine of the amino acid sequence of SEQ ID NO: 1. position 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, position 208 tryptophan , At least one amino acid selected from the group consisting of phenylalanine at position 219, tyrosine at position 233, tyrosine at position 251, tyrosine at position 258, phenylalanine at position 259, tryptophan at position 265 and phenylalanine at position 279 is substituted with p-Azido-L-phenylalanine (AzF) characterized in that it has become
    요산산화효소-알부민 컨쥬게이트.Uric acid oxidase-albumin conjugate.
  19. 제18항에 있어서,According to claim 18,
    상기 요산산화효소 변이체 서브유닛은 서열번호 2의 아미노산 서열의 10번째 타이로신(tyrosine), 163번째 타이로신(tyrosine), 17번째 페닐알라닌(phenylalanine), 45번째 페닐알라닌, 59번째 타이로신, 77번째 트립토판(tryptophan), 82번째 페닐알라닌 (phenylalanine), 90번째 페닐알라닌(phenylalanine), 94번째 타이로신, 109번째 트립토판(tryptophan), 112번째 타이로신, 123번째 페닐알라닌, 136번째 타이로신, 137번째 타이로신, 143번째 타이로신, 162번째 페닐알라닌, 163번째 타이로신, 165번째 타이로신, 170번째 페닐알라닌, 189번째 트립토판, 191번째 트립토판, 200번째 타이로신, 211번째 페닐알라닌,215번째 타이로신, 226번째 페닐알라닌, 239번째 페닐알라닌, 253번째 타이로신, 257번째 타이로신, 264번째 타이로신, 265번째 페닐알라닌, 271번째 트립토판, 281번째 페닐알라닌, 및 282번째 타이로신중 선택되는 하나 이상의 아미노산이 p-Azido-L-phenylalanine(AzF)으로 치환된 것을 특징으로 하는, 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 , 82nd phenylalanine, 90th phenylalanine, 94th tyrosine, 109th tryptophan, 112th tyrosine, 123rd phenylalanine, 136th tyrosine, 137th tyrosine, 143rd tyrosine, 162nd phenylalanine, Tyrosine position 163, tyrosine position 165, phenylalanine position 170, tryptophan position 189, tryptophan position 191, tyrosine position 200, phenylalanine position 211, tyrosine position 215, phenylalanine position 226, phenylalanine position 239, tyrosine position 253, tyrosine position 257, phenylalanine position 264 Characterized in that one or more amino acids selected from tyrosine, 265th phenylalanine, 271st tryptophan, 281st phenylalanine, and 282nd tyrosine are substituted with p-Azido-L-phenylalanine (AzF),
    요산산화효소-알부민 컨쥬게이트.Uric acid oxidase-albumin conjugate.
  20. 제18항에 있어서,According to claim 18,
    상기 알부민은 인간 혈청 알부민 또는 이의 변이체인, The albumin is human serum albumin or a variant thereof,
    요산산화효소-알부민 컨쥬게이트.Uric acid oxidase-albumin conjugate.
  21. 제18항에 있어서,According to claim 18,
    상기 알부민은 서열번호 4 내지 15 중에서 선택된 어느 하나의 아미노산 서열을 포함하는,The albumin comprises any one amino acid sequence selected from SEQ ID NOs: 4 to 15,
    요산산화효소-알부민 컨쥬게이트.Uric acid oxidase-albumin conjugate.
  22. 제16항에 있어서,According to claim 16,
    상기 알부민은 인간 혈청 알부민 또는 이의 변이체이고, The albumin is human serum albumin or a variant thereof,
    X2의 S는 상기 알부민의 34번 시스테인의 티올기로부터 유래된 것을 특징으로 하는, X 2 S is characterized in that derived from the thiol group of cysteine 34 of the albumin,
    요산산화효소-알부민 컨쥬게이트의 제조 방법. A method for preparing a uric acid oxidase-albumin conjugate.
  23. 제16항에 있어서, According to claim 16,
    상기 요산산화효소-알부민 컨쥬게이트는 다음 중 어느 하나로 구성되는, 요산산화효소-알부민 컨쥬게이트:The uric acid oxidase-albumin conjugate, wherein the uric acid oxidase-albumin conjugate consists of any of the following:
    1개의 서브유닛-알부민 컨쥬게이트 및 3개의 요산산화효소 변이체 서브유닛의 복합체;a complex of one subunit-albumin conjugate and three urate oxidase variant subunits;
    2개의 서브유닛-알부민 컨쥬게이트 및 2개의 요산산화효소 변이체 서브유닛의 복합체; a complex of two subunit-albumin conjugates and two urate oxidase variant subunits;
    3개의 서브유닛-알부민 컨쥬게이트 및 1개의 요산산화효소 변이체 서브유닛의 복합체; 및 a complex of 3 subunit-albumin conjugates and 1 uric acid oxidase variant subunit; and
    4개의 서브유닛-알부민 컨쥬게이트의 복합체,a complex of four subunit-albumin conjugates,
    이때 상기 서브유닛-알부민 컨쥬게이트는 하나의 알부민이 하나의 요산산화효소 변이체 서브유닛에 접합된 것임. In this case, the subunit-albumin conjugate is one albumin conjugated to one urate oxidase variant subunit.
  24. 제16항의 요산산화효소-알부민 컨쥬게이트를 포함하는 종양용해증후군(tumor lysis syndrome, TLS), 고요산혈증, 통풍, 관절내 요산염 결정의 침착, 요산염 결정의 침착으로 인한 급성 통풍성 관절염, 요로 결석증, 신결석증 및 통풍성 신병증으로 이루어진 군으로부터 선택된 1종 이상의 질환의 예방 또는 치료용 약학적 조성물.Tumor lysis syndrome (TLS) containing the uric acid oxidase-albumin conjugate of claim 16, hyperuricemia, gout, deposition of urate crystals in joints, acute gouty arthritis due to deposition of urate crystals, urolithiasis A pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of nephrolithiasis and gouty nephropathy.
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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 (en) * 2015-04-24 2016-07-07 광주과학기술원 Site-Specifically Albumin Conjugated Urate Oxidase and The Method for site-specifically conjugating albumin to Protein

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