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WO2023068736A1 - Variant d'arginine décarboxylase et conjugué fonctionnel d'albumine-variant de polypeptide préparé à l'aide de celui-ci - Google Patents

Variant d'arginine décarboxylase et conjugué fonctionnel d'albumine-variant de polypeptide préparé à l'aide de celui-ci Download PDF

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WO2023068736A1
WO2023068736A1 PCT/KR2022/015832 KR2022015832W WO2023068736A1 WO 2023068736 A1 WO2023068736 A1 WO 2023068736A1 KR 2022015832 W KR2022015832 W KR 2022015832W WO 2023068736 A1 WO2023068736 A1 WO 2023068736A1
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group
variant
arginine decarboxylase
adc
amino acid
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Korean (ko)
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권인찬
김승균
임수진
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Proabtech Co Ltd
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Proabtech Co Ltd
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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
    • A61K38/51—Lyases (4)
    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • ADC variant an arginine decarboxylase variant comprising one or more unnatural amino acids.
  • One embodiment of the present application relates to arginine decarboxylase variant-albumin conjugates.
  • Arginine decarboxylase is an enzyme that catalyzes the conversion of L-arginine to agmatine and carbon dioxide. The conversion process consumes protons in the decarboxylation reaction and similar to other enzymes involved in amino acid metabolism such as ornithine decarboxylase and glutamine decarboxylase, pyridoxal-5'-phosphate ; PLP) using a cofactor.
  • arginine decarboxylase which catalyzes the conversion of arginine to agmatine and carbon dioxide, can be used as an anticancer agent (Philip, R., E. Campbell, and D. N. Wheatley. “Arginine deprivation, growth inhibition and tumor cell death. : 2. Enzymatic degradation of arginine in normal and malignant cell cultures.” British journal of cancer 88.4 (2003): 613-623.). Arginine decarboxylases can interfere with the growth and differentiation of cancer cells by depleting arginine required for metabolic activity in arginine auxotrophy tumor cells. Philip, R et al., pegylated human arginine decarboxylase and disclose that the pegylated arginine decarboxylase was stable but reduced in efficacy by 40%.
  • One object of the present application is to provide an arginine decarboxylase variant having increased stability and/or plasma half-life or a conjugate using the same.
  • the enzymatic activity of the arginine decarboxylase variant provided by the present application or a conjugate using the same is characterized by a small or higher difference when compared to wild-type arginine decarboxylase.
  • FPV is a functional polypeptide variant unit, said functional polypeptide variant unit is an arginine decarboxylase variant unit, said arginine decarboxylase variant unit is derived from an arginine decarboxylase variant, said arginine decarboxylase variant contains one or more unnatural amino acids;
  • the non-natural amino acid includes a first click chemical functional group, wherein the first click chemical functional group can undergo a click chemical reaction with a second click chemical functional group, wherein the first click chemical functional group is a terminal alkyne ) group, azide group, strained alkyne group, diene group, dienophile group, trans-cyclooctene group, alkene group, Including any one group selected from a thiol group, a tetrazine group, a triazine group, a dibenzocyclooctyne (DBCO) and a bicyclononyne group,
  • DBCO dibenzocyclooctyne
  • J 1 is a first bonding unit, and the first bonding unit has a structure formed by a click chemical reaction between the first click chemical functional group and the second click chemical functional group;
  • a 2 is a second anchor moiety, said second anchor unit being a substituted hydrocarbon chain containing one or more heteroatoms;
  • J 2 is a second junction unit, wherein the second junction unit has a structure formed by a reaction between a thiol-reactive group and a thiol group, wherein the thiol-reactive group is a maleimide group or an APN group;
  • P 1 is an albumin unit, said albumin unit being derived from albumin;
  • a is an integer greater than or equal to 1 and less than or equal to 10;
  • the arginine decarboxylase variant is a decamer of 10 arginine decarboxylase subunit variants, wherein the arginine decarboxylase subunit variant comprises one or more non-natural amino acids.
  • the arginine decarboxylase subunit variant is 39th threonine, 85th asparagine, 245th asparagine, 312th lysine of the amino acid sequence of SEQ ID NO: 01 ), an amino acid having at least 90% sequence identity with a sequence in which any one or more residues selected from glutamine at position 488, lysine at position 522, and glycine at position 657 are substituted with non-natural amino acids can have a sequence.
  • the arginine decarboxylase subunit variant may have an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 02 to SEQ ID NOs: 08 and amino acid sequences having 90% or more sequence identity thereto. .
  • the first click chemofunctional group may include a tetrazine group or an azide group.
  • the unnatural amino acid can be frTet or AzF.
  • the second click chemical functional group may include any one group selected from a trans cyclooctyne (TCO) group, a DBCO group, and a bicyclononine group.
  • TCO trans cyclooctyne
  • DBCO DBCO
  • bicyclononine group any one group selected from a trans cyclooctyne (TCO) group, a DBCO group, and a bicyclononine group.
  • the second click chemofunctional group may include a trans cyclooctyne (TCO) group.
  • the second bonding unit may have the following structure:
  • the S atom is derived from albumin.
  • the second bonding unit may have the following structure:
  • the S atom is derived from albumin.
  • said second anchor unit is -A 21 -A 22 -A 23 -;
  • a 22 is a bond, substituted or unsubstituted C 1-12 alkylene, substituted or unsubstituted C 1-12 heteroalkylene, -substituted or unsubstituted C 1-12 alkylene-[EG] n -, -Substituted or unsubstituted C 1-12- Heteroalkylene-[EG] n -, -Substituted or unsubstituted C 1-12 Alkylene-[EG] n -Substituted or unsubstituted C 1-12 Alkylene-, -substituted or unsubstituted C 1-12 heteroalkylene-[EG] n -substituted or unsubstituted C 1-12 alkylene-, and -substituted or unsubstituted C 1-12 heteroalkylene -[EG] any one selected from n -substituted or unsubstituted C 1-12 heteroalkylene-
  • EG is an ethylene glycol unit
  • the ethylene glycol unit has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -, where n is an integer of 2 or more and 6 or less,
  • heteroalkylene is each independently selected from N, O, and S,
  • a 21 A case in which both A 22 and A 23 are simultaneously bonded may not exist.
  • the second anchor unit may have any one of the following structures:
  • n is an integer of 2 or more and 8 or less
  • 3' is an attachment site with the first bonding unit
  • 4' is an attachment site with the second bonding unit
  • the albumin may have an amino acid sequence of any one of the amino acid sequences of SEQ ID NO: 09 to SEQ ID NO: 20.
  • a pharmaceutical composition for treating arginine auxotrophic tumors comprising a compound having the structure of Formula 1, is provided.
  • the arginine auxotrophic tumor is melanoma, liver cancer, hepatocellular carcinoma (HCC), prostate cancer, pancreatic cancer, breast cancer cancer), mammary gland cancer, lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (Glioblastoma multiforme; GBM), acute myeloid leukemia (AML), and primary and relapsed lymphomas.
  • HCC hepatocellular carcinoma
  • GBM glioblastoma multiforme
  • AML acute myeloid leukemia
  • Threonine at position 39 Asparagine at position 85, Asparagine at position 245, Lysine at position 312, Glutamine at position 488 of the amino acid sequence of SEQ ID NO: 01 ), a sequence in which any one or more residues selected from lysine at position 522, and glycine at position 657 are substituted with a non-natural amino acid, or an arginine decarboxylase sub having a sequence having at least 90% sequence identity therewith Unit variants are provided.
  • the arginine decarboxylase subunit variant may have a sequence in which the 39th threonine of the amino acid sequence of SEQ ID NO: 01 is substituted with a non-natural amino acid, or a sequence having 90% or more sequence identity therewith. there is.
  • the arginine decarboxylase subunit variant may have a sequence in which the 312th lysine of the amino acid sequence of SEQ ID NO: 01 is substituted with a non-natural amino acid, or a sequence having 90% or more sequence identity therewith. there is.
  • the non-natural amino acid can be frTet.
  • the non-natural amino acid can be AzF.
  • the arginine decarboxylase subunit variant may have any one of the amino acid sequence of SEQ ID NO: 02 to SEQ ID NO: 08 and an amino acid sequence having at least 90% sequence identity therewith. there is.
  • a pharmaceutical composition for treating arginine auxotrophic tumors comprising the arginine decarboxylase subunit variant of the present application.
  • the arginine auxotrophic tumor is melanoma, liver cancer, hepatocellular carcinoma (HCC), prostate cancer, pancreatic cancer, breast cancer cancer), mammary gland cancer, lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (Glioblastoma multiforme; GBM), acute myeloid leukemia (AML), and primary and relapsed lymphomas.
  • HCC hepatocellular carcinoma
  • GBM glioblastoma multiforme
  • AML acute myeloid leukemia
  • a method of treating arginine auxotrophic tumor in a subject comprising: administering to the subject a compound having the structure of Formula 1 or a composition comprising an arginine decarboxylase variant. .
  • composition comprising a compound having the structure of Formula 1 or an arginine decarboxylase subunit variant for treating arginine auxotrophic tumor in a subject.
  • the functional polypeptide variant-albumin conjugate prepared from the arginine decarboxylase variant according to one embodiment of the present application has improved plasma half-life and immunogenicity than wild-type arginine decarboxylase, and the enzyme activity is higher than that of wild-type arginine decarboxylase. Compared to raises, there is no significant difference or a higher advantage.
  • the arginine decarboxylase variant and/or the arginine decarboxylase subunit variant according to one embodiment of the present application includes an unnatural amino acid, and has no significant difference or no significant difference in enzymatic activity compared to wild-type arginine decarboxylase. has high advantages.
  • ADC_WT wild-type E. coli-derived arginine decarboxylase
  • FIG. 4 shows the metabolic activity measurement results for each cell line according to ADC_WT treatment and treatment concentration. Specifically, FIG. 4 is a result of breast cancer and/or mammary cancer-related cell lines.
  • FIG. 5 shows the metabolic activity measurement results for each cell line according to ADC_WT treatment and treatment concentration. Specifically, FIG. 5 is a result of lung cancer-related cell lines.
  • FIG. 5 is a result of pancreatic cancer-related cell lines.
  • ADC_Q488AzF and ADC_K522AzF show the results of performing PAGE after expressing ADC variants. (a) shows the results before purification and (b) after purification.
  • FIG. 13 shows PAGE results for ADC variants (ADC_T39frTet, ADC_N85frTet, and ADC_N245frTet). PAGE was performed after ADC_WT and ADC variant expression and purification.
  • ADC_K312frTet shows PAGE results for ADC variants (ADC_K312frTet, ADC_Q488frTet, ADC_K522frTet, ADC_G657frTet). SDS-PAGE was performed after ADC variant expression and purification.
  • ADC_K312frTet ADC variant (K312frTet) and ADC (K312frTet)-HSA conjugate.
  • the term "about” means approximately as close to a quantity as, relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length, such as 30, 25, 20, means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%.
  • polypeptide is used to mean a compound formed by consecutively linking a plurality of amino acids.
  • polypeptide is used to include both peptides and proteins.
  • a polypeptide can include, for example, but is not limited to, three or more amino acid residues.
  • a polypeptide can include, but is not limited to, 15 or more amino acid residues. In other instances, it may include, but is not limited to, 50 or more amino acid residues. In other instances, a polypeptide may include, but is not limited to, 200 or more amino acid residues.
  • 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 at least one heteroatom.
  • heteroatom is an atom other than carbon or hydrogen, including, for example, B, Si, N, P, O, S, F, Cl, Br, I and Se.
  • polyvalent elements such as N, O, and S or monovalent elements such as F, Cl, Br, and I are included, but are not limited thereto.
  • substituted means that one or more hydrogen atoms on an atom are replaced with a substituent, wherein the valence of the atom is normal and the substituted compound is stable.
  • the substituents are each independently selected.
  • Substituents may include deuterium and hydrogen variants.
  • one substituent is a halogen (eg, Cl, F, Br, and I, etc.)
  • halogen eg, Cl, F, Br, and I, etc.
  • 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.
  • alkyl or “alkane” is used to mean a fully saturated chain or branched hydrocarbon group.
  • 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 structures.
  • C xy for example when used with the term alkyl, 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 containing x to y carbons in the chain; , and further 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 used to mean a chain or branched non-aromatic hydrocarbon group 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 used to mean a chain or branched non-aromatic hydrocarbon group 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 refers to 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 may be used 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 and 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 can contain one or more heteroatoms, and each heteroatom can be the same or different.
  • a heteroalkylene group may contain one or more heteroatoms at non-terminal positions of a chain or branch, and each heteroatom may be the same or different.
  • a heteroalkylene group can contain one or more heteroatoms at each or all ends of a chain or branch, and each heteroatom can be the same or different.
  • C xy heteroalkylene is used to include all substituted or unsubstituted heteroalkylenes having x to y 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 heteroalkenylene group can contain one or more heteroatoms, and each heteroatom can be the same or different.
  • a heteroalkenylene group may contain one or more heteroatoms at non-terminal positions of a chain or branch, and each heteroatom may be the same or different.
  • a heteroalkenylene 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.
  • 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 alkynylene 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. It is intended to A heteroalkynylene group can contain one or more heteroatoms, and each heteroatom can be the same or different.
  • a heteroalkynylene group may contain one or more heteroatoms at non-terminal positions of a chain or branch, and each heteroatom may be the same or different.
  • a heteroalkynylene group can contain one or more heteroatoms at each or all ends of a chain or branch, and each heteroatom can be the same or different.
  • Compounds herein 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, an indication related to an isomer in a formula or structure disclosed in this application (eg, *, , , and etc.), the formula or structure disclosed is meant to include all possible isomers.
  • 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 Octyne (trans-cyclooctene), alkene (alkene), thiol (thiol), tetrazine (tetrazine), triazine (triazine), dibenzocyclooctyne (DBCO) and bicyclononyne (bicyclo[6.1.0]non-4- Including yne), a pair having reactivity with each other reacts.
  • DBCO dibenzocyclooctyne
  • bicyclononyne bicyclo[6.1.0]non-4- Including yne
  • click chemistry reactions include Huisgen 1,3-dipolar cycloaddition (see Tornoe et al., Journal of Organic Chemistry (2002) 67: 3075-3064, etc.); Diels-Alder reaction; inverse-demand Diels-Alder reaction; Nucleophilic addition to small strained rings such as epoxides and aziridines; a nucleophilic addition reaction to an activated carbonyl group; and addition reactions to carbon-carbon double bonds or triple bonds.
  • 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. do. 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), L-homopropargylglycine (L-Homopropargylglycine; HPG), O-propargyl-L-tyrosine (oPa), p-propargyloxyphenylalanine (pPa), 2-amino-3-(4 -Azidophenyl)propanoic acid (2-amino-3-(4-azidophenyl)propanoic acid), 2-amino-4-(4-azidophenyl)butanoic acid (2-amino-4-(4-azidophenyl) butanoic acid), and 4-(1,2,4,5-tetrazin
  • 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 can be used to refer to both amino acids not bound to other amino acids and amino acid residues included in proteins or peptides bound to other amino acids, and the contents of the paragraph in which the amino acid term is used Or it may be appropriately interpreted according to the context.
  • amino acid may be used to include both natural amino acids and non-natural amino acids.
  • alanine may be used to refer to alanine and/or an alanine residue.
  • arginine can be used to refer to arginine and/or arginine residues.
  • amino acid may be used to include both L-type amino acids and D-type amino acids. In some embodiments, where there is no reference to the L or D form, it can be interpreted as an L-form amino acid.
  • amino acid residue refers to an amino acid contained in a compound, peptide, and/or protein that is covalently linked to another portion of the compound, peptide, and/or protein. structure derived from it. For example, when alanine, arginine, and glutamic acid are linked through an amide bond to form a peptide having an ARE sequence, the peptide includes three amino acid residues, wherein A is an alanine residue, R is an arginine residue, and E is may be referred to as glutamic acid residues.
  • the peptide may include three amino acids, and A is alanine, R is arginine, and E is glutamic acid.
  • A is alanine
  • R is arginine
  • E is glutamic acid.
  • the peptide when aspartic acid, phenylalanine, and lysine are linked through an amide bond to form a peptide having a DFK sequence, the peptide includes three amino acid residues, wherein D is an aspartic acid residue, F is a phenylalanine residue, and K may be referred to as a lysine residue.
  • the peptide may include three amino acids, and D may be referred to as aspartic acid, F may be phenylalanine, and K may be referred to as lysine.
  • amino acid sequences are described in the N-terminal to C-terminal direction using one-letter notation or three-letter notation when describing amino acid sequences in the present specification.
  • 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.
  • Sequences presented herein are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequences of the presented sequences, provided that the desired function is identical. Sequences with identity may be included.
  • treatment refers to an approach for obtaining a beneficial or desirable clinical result.
  • a beneficial or desirable clinical result is limited to alleviation of symptoms, reduction of disease extent, stabilization of the disease state. (i.e., not worsening), delay or slowing of disease progression, prevention of disease, amelioration or palliation of disease state, and alleviation (partial or total), detectable or undetectable.
  • Treatment refers to both therapeutic treatment and prophylactic or prophylactic measures.
  • the term "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. .
  • arginine auxotrophy A characteristic that requires arginine as a nutrient source is called arginine auxotrophy, and a tumor that has a characteristic that requires arginine as a nutrient source is called an arginine auxotrophic tumor.
  • Arginine auxotrophy is increasingly recognized as a frequent feature of human malignancies. Arginine auxotrophy has been found in various tumors in addition to tumors such as malignant melanoma and hepatocellular carcinoma (HCC).
  • Tumors for which arginine auxotrophy has been found include: prostate, pancreatic, breast, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell. head and neck squamous cell carcinoma, Glioblastoma multiforme (GBM), acute myeloid leukemia (AML) primary and relapsed lymphomas (Riess, Christin, et al. Arginine-depleting enzymes-An increasingly recognized treatment strategy for therapy-refractory malignancies.” Cellular Physiology and Biochemistry 51.2 (2016): 854-870.).
  • GBM Glioblastoma multiforme
  • AML acute myeloid leukemia
  • arginine auxotrophic tumors As described above, studies have been conducted to treat arginine auxotrophic tumors using arginine degrading enzymes. Drugs that treat tumors by degrading arginine in arginine auxotrophic tumors and the environment surrounding the tumors may be referred to as anticancer agents.
  • Arginine decarboxylase one of the cancer metabolites, is an enzyme that catalyzes the conversion of arginine into agmatine and carbon dioxide. It is mainly found in bacteria and viruses.
  • arginine decarboxylase is part of an enzyme system present in Escherichia coli ( E. coli ), Salmonella Typhimurium , and the methanogenic bacterium Methanococcus jannaschii , which protects these organisms from highly acidic environments.
  • arginine decarboxylase from Escherichia coli has been shown to have maximum enzymatic activity at about pH 5.2 and decrease at pH 7.0 (Blethen, Sandra L., ELIZABETH A. Boeker, and EE4870599 Snell. " Arginine Decarboxylase from Escherichia coli: I. PURIFICATION AND SPECIFICITY FOR SUBSTRATES AND COENZYME.” Journal of Biological Chemistry 243.8 (1968): 1671-1677.). Arginine decarboxylase is a multimeric form of protein subunits (arginine decarboxylase subunits).
  • arginine decarboxylase found in Escherichia coli is in the form of a decamer formed by 10 homogeneous subunits and has a molecular weight of about 800 kDa.
  • Arginine decarboxylase of the decamer is composed of 5 dimers of arginine decarboxylase subunits. That is, arginine decarboxylase is a pentamer form of arginine decarboxylase subunit dimer.
  • Arginine decarboxylase derived from Escherichia coli is a bacterial-derived polymeric protein, and has immunogenicity when administered to the human body and has a short half-life and disappears from the body quickly after injection. Because of these problems, arginine decarboxylase, when administered alone, is expected to be difficult to reach the target site of cancer or tumor formation, and accordingly, it is expected that repeated injections will be required. In addition, due to the immune response generated during the first injection and subsequent injections, it is expected that the drug introduced into the body will be eliminated more rapidly during repeated injections.
  • arginine decarboxylase As an anticancer agent for treating tumors, attempts have been made to increase in vivo stability such as plasma half-life. As described above, Philip, R et al. pegylated arginine decarboxylase and confirmed its high stability. However, Philip, R et al. disclose a 40% reduction in the enzymatic activity of arginine decarboxylase. Furthermore, PEGylation was still reported to have immunogenicity.
  • arginine decarboxylase variants or albumin conjugates thereof with no reduced enzymatic activity, improved immunogenicity, and increased in vivo stability (eg plasma half-life).
  • an arginine decarboxylase variant provided by the present application an arginine decarboxylase variant subunit, a dimer of the arginine decarboxylase variant subunit, and a functional polypeptide variant-albumin conjugate prepared using the same are disclosed. do.
  • the present application provides functional polypeptide variant-albumin conjugates.
  • the functional polypeptide variant-albumin conjugate of the present application has a form in which the functional polypeptide variant is covalently linked to albumin through a linker.
  • Functional polypeptide variants, albumin, and linkers are used to prepare functional polypeptide variant-albumin conjugates.
  • the functional polypeptide variant is any one of an arginine decarboxylase variant, an arginine decarboxylase subunit variant, and a dimer of an arginine decarboxylase subunit variant.
  • the functional polypeptide variant-albumin conjugate is prepared by site-specifically linking (i) functional polypeptide variant and (ii) albumin through (iii) a linker.
  • the functional polypeptide variant may be an arginine decarboxylase variant, an arginine decarboxylase subunit variant, or a dimer of an arginine decarboxylase subunit variant.
  • Functional polypeptide variants include non-natural amino acid residues.
  • the linker includes a thiol reactive group and a click chemofunctional group.
  • the linker includes a thiol-reactive group at one end and a click chemofunctional group at the other end.
  • the thiol-reactive group of the linker reacts with the thiol group of albumin
  • the click chemistry functional group of the linker reacts with another click chemistry functional group included in the non-natural amino acid residue of the functional polypeptide variant.
  • Functional polypeptide variants, albumins, and linkers are each described in detail in the relevant sections.
  • a functional polypeptide variant-albumin conjugate having the structure of Formula 1 is provided:
  • FPV is a functional polypeptide variant unit.
  • a functional polypeptide variant unit may be a conjugated functional polypeptide variant. That is, a functional polypeptide variant unit is derived from a functional polypeptide variant.
  • the functional polypeptide variants include an arginine decarboxylase variant, an arginine decarboxylase subunit variant, and a dimer of the arginine decarboxylase subunit variant. variant).
  • J 1 is a first junction unit.
  • the first conjugation unit has a structure formed by a click chemical reaction between a first click chemical functional group included in the functional polypeptide variant and a second click chemical functional group capable of performing a click chemical reaction with the first click chemical functional group.
  • a 2 is a second anchor unit.
  • the second anchor unit refers to a construct linking the functional polypeptide variant unit and the albumin unit.
  • the second anchor unit may serve to adjust the distance between the functional polypeptide variant unit and the albumin unit, and is not particularly limited as long as it is a structure commonly used for adjusting the distance in the art.
  • the second anchor unit is derived from a linker.
  • J 2 is a second junction unit.
  • the second conjugation unit has a structure formed by reaction of a thiol reactive group with a thiol group of albumin.
  • P 1 is an albumin unit.
  • the albumin unit may be conjugated albumin. That is, the albumin unit is derived from albumin.
  • a is an integer of 1 or more and 20 or less. In certain embodiments, a can be an integer greater than or equal to 1 and less than or equal to 10.
  • a when the functional polypeptide variant is an arginine decarboxylase subunit variant and the arginine decarboxylase subunit variant contains one non-natural amino acid, a may be 1.
  • a when the functional polypeptide variant is a dimer of the arginine decarboxylase subunit variant and the arginine decarboxylase subunit variant contains one non-natural amino acid, a may be an integer of 1 to 2.
  • a when the functional polypeptide variant is an arginine decarboxylase variant, a may be an integer from 1 to 10.
  • functional polypeptide variant-albumin conjugates are prepared using a functional polypeptide, a linker, and albumin. Furthermore, in the conjugate, the functional polypeptide variant unit is derived from the functional polypeptide variant, the albumin unit is derived from albumin, and the second anchor unit is derived from the linker.
  • the elements used in the preparation of functional polypeptide variant-albumin conjugates are described in detail.
  • the term functional polypeptide is used herein to include all peptides, polypeptides, and proteins having more than one function.
  • the function may be related to treatment and/or prevention of a certain disease.
  • the function may be related to diagnosis.
  • the function may be to act with other polypeptides to form complexes.
  • the function may be to act with a cognate polypeptide to form a complex.
  • the complex can be used to treat any disease.
  • a function may be related to manipulation of a gene.
  • the function may be related to binding to other molecules (eg, antigens and targets, etc.).
  • the functional polypeptide is any one of arginine decarboxylase, arginine decarboxylase subunit, and dimer of arginine decarboxylase subunit.
  • Functional polypeptide variants are functional polypeptides that have been modified to include non-natural amino acids.
  • functional polypeptide variants may be those in which one or more amino acid residues included in the functional polypeptide are substituted with non-natural amino acid residues. That is, functional polypeptide variants contain one or more non-natural amino acid residues.
  • the non-natural amino acid residue may include a click chemofunctional group.
  • Functional polypeptide variants may be described by reference sequences (sequences of functional polypeptides) or reference polypeptides (functional polypeptides).
  • functional polypeptide variants can be specified by describing substitution positions and/or amino acids to be substituted in the reference sequence.
  • the functional polypeptide variant is any one selected from arginine decarboxylase variants, arginine decarboxylase subunit variants, and dimers of arginine decarboxylase subunit variants.
  • FPV is a functional polypeptide variant unit.
  • a functional polypeptide variant unit may be a conjugated functional polypeptide variant.
  • a functional polypeptide variant unit may be derived from a functional polypeptide variant.
  • the term functional polypeptide variant may be used to include both functional polypeptide variants not conjugated with other compounds and functional polypeptide variants conjugated with other compounds.
  • the functional polypeptide variant may have any one amino acid sequence selected from SEQ ID NO: 02 to SEQ ID NO: 08.
  • the functional polypeptide variant unit FPV in Formula 1 may be referred to as a functional polypeptide variant.
  • non-natural amino acid may be used to refer to both non-natural amino acids not bound to other amino acids and non-natural amino acid residues included in proteins and/or peptides bound to other amino acids.
  • the natural amino acid term may be appropriately interpreted depending on the content or context of the paragraph in which it is used.
  • a functional polypeptide variant contains non-natural amino acid residues.
  • a functional polypeptide variant is one in which one or more natural amino acid residues contained in the functional polypeptide are substituted with non-natural amino acid residues.
  • Non-natural amino acids containing a first click chemical functional group are non-natural amino acids containing a first click chemical functional group
  • a non-natural amino acid according to one embodiment of the present application includes a click chemofunctional group.
  • the non-natural amino acid may include a first click chemofunctional group.
  • the first click chemistry functional group has a click chemistry functional group capable of performing a click chemistry reaction with the aforementioned second click chemistry functional group.
  • the first click chemistry functional group is a terminal alkyne, azide, strained alkyne, diene, dienophile, trans cyclooctyne (trans) -cyclooctene, alkene, thiol, tetrazine, triazine, methylcyclopropene, norbornene, cyclopentene, styrene ), (including dibenzocyclooctyne (DBCO) and bicyclononyne (bicyclo[6.1.0]non-4-yne)).
  • the first click chemistry functional group may be any one selected from a tetrazine group or an analog thereof, a triazine group or an analog thereof, and an azide group.
  • the non-natural amino acid may include a tetrazine group. In one embodiment, the non-natural amino acid may include a triazine group. In one embodiment, the non-natural amino acid may include an azide group.
  • the non-natural amino acid can have the structure of Formula 3:
  • a 1 is the first anchor moiety.
  • H 1 is a first click chemical functional group.
  • the first click chemistry functional group has a click chemistry functional group, and the click chemistry functional group is a terminal alkyne, an azide, a strained alkyne, a diene, a diene Dienophile, trans-cyclooctene, alkene, thiol, tetrazine, triazine, dibenzocyclooctyne (DBCO) and bicyclononyne, bicyclo[6.1 .0] non-4-yne)).
  • DBCO dibenzocyclooctyne
  • bicyclononyne bicyclo[6.1 .0] non-4-yne
  • the first click chemofunctional group can be represented by any one of the following structures:
  • the first anchor moiety can be a bond or -A 11 -A 12 -.
  • a 11 may be a bond or C 1-5 alkylene.
  • a 12 is a bond or [arylene]p, -[arylene]pC 1-5 alkylene-, -[arylene]pC 1-5 heteroalkylene-, -arylene-C 1- 5 Alkylene-arylene-, -arylene-C 1-5 Heteroalkylene-arylene-, -arylene-heteroarylene-, [heteroarylene]p, -[heteroarylene]pC 1-5 Alkylene-, -[heteroarylene]pC 1-5 Heteroalkylene-, -heteroarylene-C 1-5 Alkylene-arylene-, -heteroarylene-C 1-5 Alkylene-heteroarylene -, -heteroarylene-C 1-5 heteroalkylene-arylene-, and -heteroarylene-C 1-5 heteroalkylene-heteroarylene-.
  • p may be an integer of 0 or more and 3 or less.
  • the non-natural amino acid may include a tetrazine group. In one embodiment, the non-natural amino acid may include a triazine group. In one embodiment, the non-natural amino acid may include an azide 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), 2-amino-3-(4-azidophenyl)propanoic acid, 2-amino-4-(4-azidophenyl)butanoic acid, 4-(1,2,3,4-tetrazin-3-yl) phenylalanine (frTet), 4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenylalanine (Tet_v2.0), 4-(6-methyl -s- tetrazin-3-yl)phenylalanine, 3-(4- (1,2,4-triazin-6-yl)phenyl)
  • the non-natural amino acid may have a structure of any one of the following formulas:
  • the functional polypeptide variants of the present application may be arginine decarboxylase variants, arginine decarboxylase subunit variants, and dimers of arginine decarboxylase subunit variants.
  • arginine decarboxylase is a decamer formed by gathering 10 identical monomers.
  • the monomer may be referred to as an arginine decarboxylase subunit. That is, arginine decarboxylase is a decamer formed by gathering 10 arginine decarboxylase subunits.
  • This paragraph discloses a variant of the arginine decarboxylase subunit constituting arginine decarboxylase, that is, an arginine decarboxylase subunit variant.
  • the arginine decarboxylase subunit variant provided according to one embodiment of the present application is characterized in that a part of the sequence of the arginine decarboxylase subunit derived from a microorganism is modified.
  • the arginine decarboxylase variants contain one or more non-natural amino acids. Furthermore, it can be site-specifically conjugated to albumin via each non-natural amino acid residue.
  • an arginine decarboxylase subunit variant may contain one or more unnatural amino acids.
  • the arginine decarboxylase subunit that is the prototype of the arginine decarboxylase subunit variant may be derived from a microorganism.
  • the arginine decarboxylase subunit may be an arginine decarboxylase subunit derived from Escherichia coli ( E. coli ).
  • E. coli-derived arginine decarboxylase subunit refers to a monomer of E. coli-derived arginine decarboxylase (decamer).
  • the arginine decarboxylase subunit derived from E. coli may have an amino acid sequence represented by SEQ ID NO: 01:
  • Arginine decarboxylase subunit variants include one or more unnatural amino acids.
  • the non-natural amino acid includes a first click chemical functional group capable of performing a click chemical reaction with a second click chemical functional group.
  • the non-natural amino acids included in the arginine decarboxylase subunit variants are described in detail in the relevant sections including the section 'Unnatural Amino Acids'.
  • the arginine decarboxylase subunit variant may be one in which one or more amino acids of the amino acid sequence of SEQ ID NO: 01 are substituted with other amino acids.
  • the one or more amino acids may be substituted with non-natural amino acids.
  • the arginine decarboxylase subunit variant is 39th threonine, 85th asparagine, 245th asparagine, 312th lysine of the amino acid sequence of SEQ ID NO: 01 ), at least one residue selected from glutamine at position 488, lysine at position 522, and glycine at position 657 may be substituted with another amino acid.
  • the arginine decarboxylase subunit variant is 39th threonine, 85th asparagine, 245th asparagine, 312th lysine of the amino acid sequence of SEQ ID NO: 01 ), at least one residue selected from glutamine at position 488, lysine at position 522, and glycine at position 657 may be substituted with a non-natural amino acid.
  • the arginine decarboxylase subunit variant is 39th threonine, 85th asparagine, 245th asparagine, 312th lysine of the amino acid sequence of SEQ ID NO: 01 ), at least one residue selected from glutamine at position 488, lysine at position 522, and glycine at position 657 may be substituted with frTet.
  • the arginine decarboxylase subunit variant comprises Threonine at position 39, Asparagine at position 245, Lysine at position 312, and Glutamine at position 488 of the amino acid sequence of SEQ ID NO: 01.
  • any one or more residues selected from the 522nd lysine (Lysine) may be substituted with frTet.
  • the arginine decarboxylase subunit variant may be one in which at least one residue selected from glutamine at position 488 and lysine at position 522 of the amino acid sequence of SEQ ID NO: 01 is substituted with Azf.
  • the polypeptide having the amino acid sequence of SEQ ID NO: 01 is an arginine decarboxylase subunit derived from E. coli.
  • the arginine decarboxylase subunit variant can have any one of the following sequences:
  • X means a non-natural amino acid residue.
  • X may be a frTet moiety.
  • X may be an AzF moiety.
  • the functional polypeptide variant may be an arginine decarboxylase variant.
  • One embodiment of the present application provides arginine decarboxylase variants.
  • arginine decarboxylase variants are also decamer proteins containing 10 subunits.
  • the arginine decarboxylase variants include 1 to 10 arginine decarboxylase subunit variants, and the arginine decarboxylase subunit variants are derived from one or more wild-type arginine decarboxylase subunits. Characterized in that an amino acid is substituted with a non-natural amino acid.
  • the arginine decarboxylase variant may be an E. coli-derived arginine decarboxylase variant.
  • the E. coli-derived arginine decarboxylase variant includes 1 to 10 E. coli-derived arginine decarboxylase subunit variants.
  • an arginine decarboxylase variant may comprise any of the following:
  • the functional polypeptide variant may be a dimer of an arginine decarboxylase subunit variant.
  • One embodiment of the present application provides dimers of arginine decarboxylase subunit variants.
  • arginine decarboxylase is in the form of a complex of dimers of five arginine decarboxylase subunits.
  • Arginine decarboxylase variants may also be complexes of dimers of five arginine decarboxylase subunit variants.
  • the dimer of the arginine decarboxylase subunit variant includes one or two arginine decarboxylase subunit variants, and the arginine decarboxylase subunit variant is a wild-type arginine decarboxylase subunit that is the prototype. Characterized in that one or more amino acids in the unit are substituted with non-natural amino acids.
  • the dimer of the arginine decarboxylase subunit variant may be a dimer of an arginine decarboxylase subunit variant derived from E. coli.
  • the dimer of the E. coli-derived arginine decarboxylase subunit variant includes one or two E. coli-derived arginine decarboxylase subunit variants.
  • a dimer of an arginine decarboxylase subunit variant may comprise any of the following:
  • Linker contains click chemofunctional groups and thiol reactive groups
  • the linker of the present application includes a click chemofunctional group and a thiol reactive group. More specifically, the linker of the present application includes a first click chemical functional group, a second click chemical functional group capable of a click chemical reaction, and a thiol reactive group capable of reacting with a thiol group.
  • Functional polypeptide variants are linked to albumin via a linker.
  • an albumin-linker conjugate may be prepared by reacting albumin with a linker
  • a functional polypeptide variant-albumin conjugate may be prepared by reacting the albumin-linker conjugate with a functional polypeptide variant.
  • a functional polypeptide variant-linker conjugate is prepared by reacting the functional polypeptide variant with a linker
  • a functional polypeptide variant-albumin conjugate is prepared by reacting the functional polypeptide variant-linker conjugate with albumin.
  • the linker contains a thiol reactive group and a click chemofunctional group.
  • the thiol-reactive group reacts with the thiol group of albumin to form a structure included in the second conjugation unit.
  • the click chemical functional group reacts with other click chemical functional groups included in the non-natural amino acid of the functional polypeptide variant to form a structure included in the first conjugation unit.
  • a linker can include a thiol reactive group and a second click chemofunctional group.
  • the thiol-reactive group may react with a thiol group, and the second click chemical functional group may undergo a click chemical reaction with the first click chemical functional group.
  • Linkers of the present application include thiol reactive groups.
  • a thiol-reactive group is reactive with a thiol group.
  • the thiol-reactive group can be a maleimide group or a 3-Arylpropiolonitriles (APN) group.
  • a thiol reactive group included in a linker of the present application may have the following structure:
  • One embodiment of the present application provides a linker having the structure of Formula 2:
  • H 2 is a second click chemical functional group.
  • the second click chemistry functional group may have a click chemistry functional group.
  • the second click chemistry functional group may undergo a click chemistry reaction with the first click chemistry functional group.
  • B is a thiol reactive group.
  • the thiol reactive group can be a maleimide group or an APN group.
  • a 2 is a second anchor unit.
  • the second anchor unit serves to adjust the distance between the second click chemofunctional group and the thiol reactive group.
  • the second anchor unit serves to control the distance between the functional polypeptide variant unit and the albumin unit.
  • the second click chemistry functional group has a click chemistry functional group.
  • the second click chemistry functional group is a terminal alkyne, azide, strained alkyne, diene, dienophile, trans cyclooctyne (trans) -cyclooctene, alkene, thiol, tetrazine, triazine, methylcyclopropene, norbornene, cyclopentene, styrene ), (including dibenzocyclooctyne (DBCO) and bicyclononyne (bicyclo[6.1.0]non-4-yne)).
  • trans trans cyclooctyne
  • alkene alkene
  • thiol thiol
  • tetrazine triazine
  • methylcyclopropene norbornene
  • cyclopentene styrene
  • DBCO dibenzocyclooctyne
  • bicyclononyne bicyclo[6.1.0]non-4-
  • the second click chemistry functional group is a trans-bicyclo[6.1.0]nonene group, a trans-cyclooctene (TCO) group, a methylcyclopropene group (methylcyclopropene) group, bicyclo[6.1.0]nonyne group, cyclooctyne group, norbornene group, cyclopentene group, styrene group, and a dibenzocyclooctyne group, but is not limited thereto.
  • TCO trans-cyclooctene
  • TCO trans-cyclooctene
  • methylcyclopropene group methylcyclopropene
  • bicyclo[6.1.0]nonyne group cyclooctyne group
  • norbornene group norbornene group
  • cyclopentene group styrene group
  • dibenzocyclooctyne group but is not limited thereto.
  • the second click chemofunctional group can have any one of the following structures:
  • the wavy line represents an attachment site to another part of the linker.
  • a wavy line may indicate an attachment site with the second anchor unit.
  • the second click chemofunctional group can have the following structure:
  • the second click chemofunctional group can have the following structure:
  • the wavy line represents an attachment site to another part of the linker.
  • a wavy line may indicate an attachment site with the second anchor moiety.
  • a 2 is a second anchor unit.
  • the second anchor unit is a substituted hydrocarbon chain comprising one or more heteroatoms.
  • the hetero atom may be each independently selected from N, O, and S.
  • the second anchor unit may include a polyethylene glycol unit composed of a plurality of ethylene glycol units.
  • EG means an ethylene glycol unit and has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -. In this case, n may be an integer of 1 or more and 12 or less.
  • the second anchor unit can be -substituted C 1-6 heteroalkylene-[EG] n -substituted C 3-15 heteroalkylene-.
  • EG means an ethylene glycol unit and has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -.
  • n may be an integer of 1 or more and 6 or less.
  • the second anchor unit can be a substituted C 5-30 heteroalkylene.
  • the linker may have the structure of Formula 2-1:
  • H 2 is a second click chemical functional group, which is described in detail in the section 'Second click chemical functional group (H 2 )'.
  • -A 21 -A 22 -A 23 - is a second anchor unit.
  • a 22 is a bond, substituted or unsubstituted C 1-12 alkylene, substituted or unsubstituted C 1-12 heteroalkylene, -[EG] n -, -substituted or unsubstituted C 1- 12 alkylene-[EG] n -, -substituted or unsubstituted C 1-12- heteroalkylene-[EG] n -, -substituted or unsubstituted C 1-12 alkylene-[EG] n -substituted or unsubstituted C 1-12 alkylene-, -substituted or unsubstituted C 1-12 heteroalkylene-[EG] n -substituted or unsubstituted C 1-12 alkylene-, and -substituted or unsubstituted It may be any one selected from C 1-12 heteroalkylene-[EG] n -substituted or unsubstit
  • EG means an ethylene glycol unit and has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -. In this case, n may be an integer of 1 or more and 6 or less.
  • B is a group containing a thiol reactive group.
  • the thiol reactive group can be a maleimide group or an APN group.
  • the linker can have a structure of any one of the following formulas:
  • n may be an integer of 2 or more and 8 or less.
  • albumin is used in the preparation of functional polypeptide variant-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.
  • P 1 is an albumin unit.
  • the albumin unit may be conjugated albumin.
  • the albumin unit may be derived from albumin.
  • albumin may be used to include both albumin unconjugated with other compounds and albumin conjugated with other compounds.
  • albumin can be a protein having the sequence of SEQ ID NO: 09.
  • albumin unit P 1 in Formula 1 may be referred to as albumin.
  • albumin Specific examples include albumin
  • the albumin can be mammalian albumin, eg serum albumin.
  • the 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 (recombinant albumins).
  • 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
  • the albumin can be human serum albumin, wherein the human serum albumin can comprise the amino acid sequence below.
  • the albumin can be human serum albumin or a variant thereof, and the human serum albumin or variant thereof can comprise a sequence selected from any one of the following sequences:
  • a thiol residue of a cysteine included in human serum albumin or a variant thereof can react with a thiol-reactive group at one end of the linker.
  • a thiol residue of cysteine contained in human serum albumin or a variant thereof may react with a maleimide group or an APN group at one end of a linker.
  • 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.
  • the cysteine reacting with one end of the linker may be cysteine 34 (Cys 34).
  • the functional polypeptide variant-albumin conjugate of the present application has the structure of Formula 1:
  • FPV is a functional polypeptide variant unit.
  • Examples of functional polypeptide variant units are detailed in the section relating to functional polypeptide variants.
  • J 1 is a first junction unit.
  • the first conjugation unit has a structure formed by a click chemical reaction between a first click chemical functional group included in the functional polypeptide variant and a second click chemical functional group capable of performing a click chemical reaction with the first click chemical functional group.
  • Each of the first click chemofunctional group and the second click chemofunctional group is described in detail in the relevant section.
  • a 2 is a second anchor unit.
  • the second anchor unit refers to a construct linking the functional polypeptide variant unit and the albumin unit.
  • the second anchor unit serves to adjust the distance between the functional polypeptide variant unit and the albumin unit. If it is a structure commonly used for distance control in the art, it is not significantly limited.
  • the second anchor unit is derived from a linker.
  • the second anchor unit is described in detail in the section 'Linker: contains a click chemofunctional group and a thiol reactive group' and is also described below.
  • J 2 is a second junction unit.
  • the second conjugation unit has a structure formed by reaction of a thiol reactive group with a thiol group of albumin.
  • a is an integer of 1 or more and 20 or less. In certain embodiments, a is an integer greater than or equal to 1 and less than or equal to 10.
  • a 2 is a second anchor unit. As described above, the second anchor unit is derived from a linker.
  • the second anchor unit is a substituted hydrocarbon chain comprising one or more heteroatoms.
  • hydrocarbon chains may consist of 1 to 500 atoms.
  • the hetero atom may be each independently selected from N, O, and S.
  • the second anchor unit may include a polyethylene glycol unit composed of a plurality of ethylene glycol units.
  • EG means an ethylene glycol unit and has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -.
  • n may be an integer of 1 or more and 12 or less.
  • the second anchor unit can be a substituted -C 1-6 heteroalkylene-[EG] n -substituted C 3-15 heteroalkylene.
  • EG means an ethylene glycol unit and has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -.
  • n may be an integer of 1 or more and 6 or less.
  • the second anchor unit can be a substituted C 5-30 heteroalkylene.
  • -A 2 - can be -A 21 -A 22 -A 23 -.
  • EG means an ethylene glycol unit and has a structure of -CH 2 CH 2 O- or -CH 2 OCH 2 -. In this case, n may be an integer of 2 or more and 6 or less.
  • the second anchor unit can have any one of the following structures:
  • n may be an integer of 2 or more and 8 or less.
  • 3' may be an attachment site with the first bonding unit.
  • 4' may be an attachment site with the second bonding unit.
  • 3' may be an attachment site with the second bonding unit.
  • 4' may be an attachment site with the first bonding unit.
  • J 1 is a first junction unit.
  • the first bonding unit has a structure formed by a click chemical reaction between a first click chemical functional group and a second click chemical functional group.
  • the first conjugation unit may have a structure formed by a click chemical reaction between a first click chemical functional group included in a non-natural amino acid residue of a functional polypeptide variant and a second click chemical functional group at one end of a linker.
  • the first click chemistry functional group is a terminal alkyne, an azide, a strained alkyne, a diene, a dienophile, a trans-cyclooctyne (trans- cyclooctene, alkene, thiol, tetrazine, triazine, dibenzocyclooctyne (DBCO) and bicyclononyne (bicyclo[6.1.0]non-4-yne). ) may include any one group selected from among the groups.
  • the first click chemofunctional group may include any one of tetrazine, triazine, and azide groups.
  • the second click chemistry functional group is a terminal alkyne, azide, strained alkyne, diene, dienophile, trans-cyclooctyne (trans- cyclooctene, alkene, thiol, tetrazine, triazine, dibenzocyclooctyne (DBCO) and bicyclononyne (bicyclo[6.1.0]non-4-yne). ) may include any one group selected from among the groups.
  • the second click chemofunctional group may include any one of TCO, DBCO, and bicyclononine groups.
  • the first bonding unit can have any one of the following structures:
  • 1' may be an attachment site with a functional polypeptide variant. More specifically, 1' may be an attachment site with the first anchor unit. In this case, 2' may be an attachment site to another structure of the linker. More specifically, 2' may be an attachment site with the second anchor unit.
  • 1' may be an attachment site with another structure of the linker. More specifically, 1' may be an attachment site with the second anchor unit. In this case, 2' may be an attachment site with a functional polypeptide variant. More specifically, 2' may be an attachment site with the first anchor unit.
  • J 2 is a second junction unit.
  • the second junction unit has a structure formed by reaction of a thiol reactive group with a thiol group.
  • the second conjugation unit may have a structure formed by reaction of a thiol group of albumin with a thiol-reactive group at one end of a linker.
  • the second conjugation unit may have a structure formed by reaction of a maleimide group at one end of a linker with a thiol group of albumin.
  • the second conjugation unit may have a structure formed by the reaction of a thiol group of albumin with an APN group at one end of a linker.
  • the thiol group of albumin can be a Cys 34 thiol group.
  • the second bonding unit may have any one of the following structures:
  • the S atom may be derived from albumin. Specifically, the S atom may be derived from a cysteine residue of albumin. More specifically, the S atom may be derived from a thiol group of a cysteine residue of albumin. In certain embodiments, the S atom may be derived from the thiol group of cysteine 34 (Cys 34) of albumin.
  • FPV is a functional polypeptide variant unit.
  • a functional polypeptide unit may be derived from a functional polypeptide variant.
  • a functional polypeptide variant is one in which the functional polypeptide has been modified to include non-natural amino acids.
  • the functional polypeptide may be any one selected from arginine decarboxylase, arginine decarboxylase subunit, and dimer of arginine decarboxylase subunit, but is not limited thereto.
  • the functional polypeptide has an amino acid sequence having about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence represented by SEQ ID NO: 01 can have a sequence.
  • the functional polypeptide variant may be any one selected from arginine decarboxylase variants, arginine decarboxylase subunit variants, and dimers of arginine decarboxylase subunit variants. That is, the functional polypeptide variant unit may be any one selected from an arginine decarboxylase variant unit, an arginine decarboxylase subunit variant unit, and a dimer unit of an arginine decarboxylase subunit variant. Functional polypeptides and functional polypeptide variants are described in detail in the relevant paragraphs.
  • P 1 is an albumin unit.
  • the albumin unit may be derived from albumin.
  • the 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 (recombinant albumins).
  • the albumin is about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the amino acid sequence represented by any one of SEQ ID NOs: 09 to 20. It may have an amino acid sequence having sequence identity.
  • the S atom adjacent to the albumin unit is derived from albumin.
  • the S atom adjacent to the albumin unit is derived from a thiol group of albumin.
  • the S atom adjacent to the albumin unit may be derived from a thiol group of a cysteine residue of albumin.
  • the S atom adjacent to the albumin unit may be derived from the thiol group of cysteine 34 of human serum albumin.
  • n may be an integer of 1 or more and 12 or less. In certain embodiments, n may be an integer greater than or equal to 2 and less than or equal to 6.
  • a may be an integer of 1 or more and 20 or less. In certain embodiments, a can be an integer greater than or equal to 1 and less than or equal to 10.
  • the functional polypeptide variant unit is an arginine decarboxylase subunit variant unit
  • the functional polypeptide variant unit may be an arginine decarboxylase subunit variant.
  • a may be an integer of 1 or more and 4 or less.
  • an arginine decarboxylase subunit variant may comprise one unnatural amino acid, where a is 1.
  • an arginine decarboxylase subunit variant may comprise two unnatural amino acids, where a is 1 or 2.
  • an arginine decarboxylase subunit variant may comprise three unnatural amino acids, where a is an integer greater than or equal to 1 and less than or equal to 3.
  • an arginine decarboxylase subunit variant may comprise four unnatural amino acids, where a is an integer greater than or equal to 1 and less than or equal to 4.
  • the functional polypeptide variant unit is an arginine decarboxylase variant unit
  • arginine decarboxylase is in the form of a decamer formed by gathering 10 arginine decarboxylase subunits, and arginine decarboxylase variants are formed by gathering 10 arginine decarboxylase subunit variants, or arginine decarboxylase variants. It is a decamer form formed by gathering 10 reboxylase subunit variants and arginine decarboxylase subunits.
  • a may be an integer of 1 to 20 or less.
  • a is 1.
  • a is 6.
  • the arginine decarboxylase variants include 10 arginine decarboxylase subunit variants and one albumin is conjugated to each of the 10 arginine decarboxylase subunit variants
  • a is 10.
  • the arginine decarboxylase variant comprises nine arginine decarboxylase subunit variants and one arginine decarboxylase subunit variant, and each of the nine arginine decarboxylase subunit variants contains one albumin. In this conjugated case, a is 9.
  • a is 20 . That is, when the functional polypeptide variant unit is an arginine decarboxylase variant unit, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, It may be 17, 18, 19, or 20.
  • the functional polypeptide variant unit is a dimer unit of an arginine decarboxylase subunit variant
  • arginine decarboxylase subunits and/or arginine decarboxylase subunit variants may form dimers.
  • a may be an integer of 1 to 8 or less.
  • the dimer of the arginine decarboxylase subunit variant comprises two arginine decarboxylase subunit variants, and each of the two arginine decarboxylase subunit variants is conjugated with one albumin a is 2.
  • a is 1
  • the dimer of the arginine decarboxylase subunit variant contains two arginine decarboxylase subunit variants, and one albumin is conjugated to one arginine decarboxylase subunit variant
  • a is 4
  • the dimer of the arginine decarboxylase subunit variant comprises the arginine decarboxylase subunit variant and the arginine decarboxylase subunit, and one albumin is present in one arginine decarboxylase subunit variant.
  • a is 1.
  • a functional polypeptide variant-albumin conjugate can be prepared by reacting a functional polypeptide variant, albumin, and a linker.
  • the functional polypeptide variant-albumin conjugate can be prepared by (1) reacting albumin with a linker to prepare an albumin-linker conjugate, and then reacting the functional polypeptide variant with the prepared albumin-linker conjugate; or (2) a functional polypeptide variant-linker conjugate is prepared by reacting the linker with the functional polypeptide variant, and then reacted with albumin to prepare the functional polypeptide variant-linker conjugate.
  • the functional polypeptide variant-albumin conjugate can be prepared regardless of the reaction order of the three elements (linker, functional polypeptide variant, albumin).
  • a functional polypeptide variant is required to prepare a functional polypeptide variant-albumin conjugate.
  • a method for preparing a functional polypeptide variant will be described through an example of an arginine decarboxylase subunit variant.
  • substitution sites may be selected from those with high solvent accessibility.
  • the substitution site may be selected from amino acids constituting a random coil.
  • the substitution site may be selected from among amino acids that are not located at dimer/decamer forming sites.
  • the substitution site may be selected from positions in which there is no difference in score between the mutant and the wild-type arginine decarboxylase subunit when substituted in a rosetta design.
  • the location of substitution with a non-natural amino acid in the wild-type arginine decarboxylase subunit sequence can be determined by referring to molecular modeling simulation results.
  • the molecular modeling simulation result may be a scoring result of a Rosetta molecular modeling package.
  • 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: 01.
  • threonine at position 39, asparagine at position 85, asparagine at position 245, lysine at position 312, glutamine at position 488 of the amino acid sequence of SEQ ID NO: 01, Any one or more residues selected from Lysine at position 522 and Glycine at position 657 may be substituted with a non-natural amino acid.
  • the arginine decarboxylase subunit variant is 39th threonine, 85th asparagine, 245th asparagine, 312th lysine of the amino acid sequence of SEQ ID NO: 01 ), at least one residue selected from glutamine at position 488, lysine at position 522, and glycine at position 657 may be substituted with frTet.
  • the arginine decarboxylase subunit variant comprises Threonine at position 39, Asparagine at position 245, Lysine at position 312, and Glutamine at position 488 of the amino acid sequence of SEQ ID NO: 01.
  • the arginine decarboxylase subunit variant may be one in which at least one residue selected from glutamine at position 488 and lysine at position 522 of the amino acid sequence of SEQ ID NO: 01 is substituted with Azf.
  • it is an arginine decarboxylase subunit derived from E. coli having the amino acid sequence of SEQ ID NO: 01.
  • the method for preparing the arginine decarboxylase subunit variant involves the following components: a cell line to express the arginine decarboxylase subunit variant; foreign suppressor tRNA (foreign suppressor tRNA) recognizing a specific stop codon; foreign tRNA synthetase; and a vector encoding an arginine decarboxylase subunit variant encoding an unnatural amino acid using the stop codon.
  • the exogenous suppressor tRNA and the exogenous tRNA synthetase are introduced from the outside, not the suppressor tRNA and tRNA synthetase native to the expressing cell line.
  • the exogenous suppressor tRNA is characterized in that it does not react with the tRNA synthetase native to the expressing cell line.
  • the exogenous tRNA synthetase reacts i) only with the exogenous suppressor tRNA, and ii) shows activity only with the non-natural amino acid to be included in the arginine decarboxylase variant.
  • the non-natural amino acid can be introduced into the amino acid sequence by specifically linking the exogenous suppressor tRNA with the non-natural amino acid.
  • the method for producing a functional polypeptide variant includes: 1) in the cell line, 2) involving the exogenous suppressor tRNA and exogenous tRNA synthetase, 3) based on a vector encoding the functional polypeptide variant, 4) the functional polypeptide It is a method for expressing a variant.
  • the order of each process is not particularly limited as long as the functional polypeptide variant can be expressed in the cell line, and additional processes may be included as necessary.
  • the method for producing the arginine decarboxylase subunit variant is characterized in that the arginine decarboxylase subunit variant is obtained by expressing the arginine decarboxylase subunit variant in a cell line.
  • the cell line expressing the arginine decarboxylase subunit variant is not particularly limited as long as it can produce the arginine decarboxylase subunit variant.
  • the release facotr recognizing the stop codon in the cell line functions properly, the release factor competes with the foreign-derived tRNA, resulting in a low yield. Therefore, it is preferable to use a cell line in which the release factor that recognizes the stop codon is inactivated.
  • the cell line expressing the arginine decarboxylase subunit variant may be selected from the following:
  • genus Escherichia Erwinia genus; Serratia genus; Providencia genus; Corynebacterium genus; Pseudomonas ( Pseudomonas ) genus; genus Leptospira ; genus Salmonella ; Brevibacterium genus; Hypomonas ( Hyphomonas ) genus; Chromobacterium ( chromobactorium ) genus; genus norcardia ; Fungi ; and Yeast .
  • the cell line may be one in which a release factor that recognizes a stop codon to terminate translation is inactivated.
  • the stop codon is an amber codon (5'-UAG-3'), an ocher codon (5'-UAA-3'), and an opal codon (5'-UGA-3). ') may be selected.
  • the cell line expressing the arginine decarboxylase subunit variant may be the cell line used in the registration publication KR 1637010 B1. Specifically, the cell line may be E.Coli C321. ⁇ A.exp (Addgene, ID: 49018).
  • the exogenous suppressor tRNA is a tRNA that functions to recognize a specific stop codon, and does not react with the tRNA synthesizing enzyme native to the expressing cell line.
  • the exogenous suppressor tRNA specifically reacts with the exogenous tRNA synthetase, and the exogenous tRNA synthetase functions to link a non-natural amino acid to the exogenous suppressor tRNA.
  • the exogenous suppressor tRNA can recognize the specific stop codon and introduce the non-natural amino acid at the corresponding position.
  • the suppressor tRNA is an amber codon (5'-UAG-3'), an ocher codon (5'-UAA-3'), and an opal codon (5'-UGA). -3') can be recognized.
  • the suppressor tRNA may recognize an amber codon.
  • the suppressor tRNA may be a suppressor tRNA (MjtRNA Tyr CUA ) derived from Methanococcus jannaschii (Yang et.al, Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand DielsAlder Reaction, Bioconjugate Chemistry, 2020, 2456-2464).
  • the exogenous tRNA synthetase selectively reacts with a specific non-natural amino acid and serves to link the specific non-natural amino acid to the exogenous suppressor tRNA.
  • the exogenous tRNA synthetase does not react with the suppressor tRNA native to the expressing cell line, but specifically reacts only with the exogenous suppressor tRNA.
  • the tRNA synthetase may have a function of linking a non-natural amino acid including a tetrazine derivative and/or a triazine derivative to the exogenous suppressor tRNA.
  • the tRNA synthetase may have a function of linking AzF to the exogenous suppressor tRNA.
  • the tRNA synthetase may be tyrosyl-tRNA synthetase (MjTyrRS) derived from Methanococcus jannaschii (Yang et.al, Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels-Alder Reaction, Bioconjugate Chemistry, 2020, 2456-2464).
  • the tRNA synthetase may be the C11 variant of MjTyrRS.
  • an exogenous suppressor tRNA that specifically reacts only with an exogenous tRNA synthetase is collectively referred to as an orthogonal tRNA/synthetase pair.
  • an orthogonal tRNA/synthetase pair it is important to express the orthogonal tRNA/synthetase pair in the expression cell line, and if this purpose can be achieved, the method will greatly Not limited.
  • the method for producing the arginine decarboxylase subunit variant includes transforming a vector capable of expressing the orthogonal tRNA/synthetase pair into the cell line.
  • a vector capable of expressing the orthogonal tRNA/synthetase pair is Yang et.al (Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels-Alder Reaction, Bioconjugate Chemistry, 2020, 2456-2464) may be pDule_C11.
  • vectors encoding functional polypeptide variants will be described.
  • the present application discloses vectors encoding functional polypeptide variants.
  • the vector encoding the variant functional polypeptide is characterized in that a non-natural amino acid in the sequence of the variant functional polypeptide is encoded using a stop codon.
  • the standard amino acid is encoded by a codon corresponding to the standard amino acid found in nature, and the non-natural amino acid is encoded by a stop codon.
  • the stop codon is an amber codon (5'-UAG-3'), an ocher codon (5'-UAA-3'), and an opal codon (5'-UGA-3'). 3') may be selected.
  • the stop codon may be selected from among 5'-TAG-3', 5'-TAA-3', and 5'-TGA-3'.
  • the vector encoding the functional polypeptide variant may be codon-optimized for the expression cell line.
  • a vector encoding the functional polypeptide variant may be E. coli codon optimized.
  • functional polypeptide variant-albumin conjugates can be prepared through the following methods:
  • a functional polypeptide variant-albumin conjugate is prepared by reacting the albumin-linker conjugate with the functional polypeptide variant.
  • the linker may be connected through a specific amino acid residue of albumin.
  • an albumin-linker conjugate may be prepared by reacting a thiol group of a cysteine contained in human serum albumin (HSA) with a thiol reactive 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 of a linker.
  • a functional polypeptide variant-albumin conjugate in which functional polypeptide variants are linked through a linker, can be prepared.
  • a functional polypeptide variant-albumin conjugate may be prepared by reacting a click chemical functional group at one end of the albumin-linker conjugate with a click chemical functional group included in a non-natural amino acid of the functional polypeptide variant.
  • the functional polypeptide variant-albumin conjugate can be prepared by reacting the second click chemical functional group at one end of the albumin-linker conjugate with the first click chemical functional group of the functional polypeptide variant.
  • a functional polypeptide variant-albumin conjugate can be prepared by reacting the TCO at one end of the albumin-linker conjugate with the tetrazine of the functional polypeptide variant.
  • a functional polypeptide variant-albumin conjugate can be prepared by reacting DBCO at one end of the albumin-linker conjugate with the azide of the functional polypeptide variant.
  • functional polypeptide variant-albumin conjugates can be prepared via a method comprising:
  • the albumin-linker conjugate reacts with the functional polypeptide variant to produce a functional polypeptide variant-albumin conjugate.
  • functional polypeptide variant-albumin conjugates can be prepared through the following methods:
  • a functional polypeptide variant-linker conjugate reacts with albumin to prepare a functional polypeptide variant-albumin conjugate.
  • a functional polypeptide variant-linker conjugate can be prepared by a click chemistry reaction between a click chemistry functional group at one end of a linker and a click chemistry functional group of a non-natural amino acid of a functional polypeptide variant.
  • a functional polypeptide variant-linker conjugate may be prepared by reacting a second click chemical functional group at one end of a linker with a first click chemical functional group included in a functional polypeptide variant.
  • a functional polypeptide variant-albumin conjugate can be prepared by reacting a thiol reactive group of a functional polypeptide variant-linker conjugate with a thiol group of cysteine contained in human serum albumin (HSA).
  • HSA human serum albumin
  • a functional polypeptide variant-albumin conjugate is prepared by reacting a thiol reactive group of a functional polypeptide variant-linker conjugate with a thiol group of cysteine 34 (Cys 34) of human serum albumin (HSA).
  • Cys 34 cysteine 34
  • HSA human serum albumin
  • functional polypeptide variant-albumin conjugates can be prepared via a method comprising:
  • a functional polypeptide variant-linker conjugate reacts with albumin to produce a functional polypeptide variant-albumin conjugate.
  • an albumin-linker conjugate can be prepared through the reaction of a linker with albumin.
  • One embodiment of the present application provides an albumin-linker conjugate.
  • the albumin-linker conjugate can have the structure of Formula 4:
  • H 2 is a second click chemical functional group.
  • the second click chemistry functional group includes a click chemistry functional group.
  • the second click chemistry functional group may undergo a click chemistry reaction with the first click chemistry functional group capable of performing a click chemistry reaction with the second click chemistry functional group.
  • the first bonding unit is formed by a click chemical reaction between the second click chemical functional group and the first click chemical functional group.
  • the second click chemofunctional group is described in detail in a related section.
  • a 2 is a second anchor unit.
  • the second anchor unit may refer to a component connecting the second click chemical functional group and the second conjugation unit and/or the albumin unit. If it is a structure commonly used for distance control in the art, it is not significantly limited.
  • the second anchor unit is derived from a linker. The second anchor unit is described in detail in the section related to the second anchor unit including the section 'Linker: containing a click chemofunctional group and a thiol reactive group'.
  • J 2 is a second junction unit.
  • the second junction unit has a structure formed by reaction of a thiol reactive group with a thiol group.
  • the second conjugation unit may have a structure formed by reaction of a thiol group of albumin with a thiol-reactive group at one end of a linker.
  • the thiol group of albumin can be that of cysteine 34 (Cys 34).
  • the second bonding unit is described in detail in the section related to the second bonding unit including the section 'second bonding unit'.
  • P 1 is an albumin unit.
  • Albumin units are derived from albumin.
  • Albumin and albumin units are each described in detail in the sections relating to albumin units, including the section 'Albumin and albumin units'.
  • a functional polypeptide variant-linker conjugate can be prepared by reacting a linker with a functional polypeptide variant.
  • One embodiment of the present application provides functional polypeptide variant-linker conjugates.
  • the functional polypeptide variant-linker conjugate may have the structure of Formula 5:
  • FPV is a Functional Polypeptide Variant Unit.
  • a functional polypeptide variant unit is derived from a functional polypeptide variant.
  • Each of the Functional Polypeptide Variants and Functional Polypeptide Variant Units is described in detail in the relevant section.
  • J 1 is a first junction unit.
  • the first bonding unit is described in detail in the section related to the first bonding unit including the section 'first bonding unit'.
  • a 2 is a second anchor unit.
  • the second anchor unit is described in detail in the section related to the second anchor unit including the section 'Linker: containing a click chemofunctional group and a thiol reactive group'.
  • J 2 is a second junction unit.
  • the second bonding unit is described in detail in the section related to the second bonding unit including the section 'second bonding unit'.
  • B is a thiol reactive group.
  • the thiol reactive group can be a maleimide group.
  • a thiol reactive group can be an APN group.
  • Thiol-reactive groups are described in detail in the sections relating to thiol-reactive groups, including the section 'Linkers: Including click chemofunctional groups and thiol-reactive groups'.
  • a may be an integer of 1 or more and 20 or less. In certain embodiments, a can be an integer greater than or equal to 1 and less than or equal to 10.
  • One embodiment of the present application provides a composition for the treatment of a target disease comprising a functional polypeptide variant-albumin conjugate as an active ingredient.
  • One embodiment of the present application provides a pharmaceutical composition for treating a target disease comprising a functional polypeptide variant-albumin conjugate as an active ingredient.
  • the target disease may be a tumor. More specifically, the target disease may be an arginine auxotrophic tumor.
  • the target disease is melanoma (malignant melanoma), liver cancer (liver cancer), hepatocellular carcinoma (HCC), prostate cancer (prostate cancer), pancreatic cancer (pancreatic cancer), breast cancer (breast cancer), breast cancer ( mammary gland cancer, lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (GBM) ), acute myeloid leukemia (AML), and primary and relapsed lymphomas.
  • melanoma malignant melanoma
  • liver cancer liver cancer
  • liver cancer liver cancer
  • HCC hepatocellular carcinoma
  • prostate cancer prostate cancer
  • pancreatic cancer pancreatic cancer
  • breast cancer breast cancer
  • mammary gland cancer lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (GBM)
  • composition for diagnosis comprising a functional polypeptide variant-albumin conjugate as an active ingredient may be provided.
  • treatment refers to any activity that improves or beneficially transforms the symptoms of a target disease by the administration of the pharmaceutical composition according to the present application, and includes prevention of the target disease.
  • prevention refers to any action that suppresses a target disease or delays the onset of a target disease by administration of the pharmaceutical composition according to the present application.
  • the pharmaceutical composition of the present application may further include a pharmaceutically acceptable carrier in addition to containing the functional polypeptide variant-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 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 a target disease.
  • One embodiment of the present application provides a composition for the treatment of a target disease comprising a functional polypeptide variant as an active ingredient and its use.
  • One embodiment of the present application provides a pharmaceutical composition for treating a target disease comprising a functional polypeptide variant as an active ingredient.
  • Another embodiment of the present application provides a method for treating a target disease, eg, cancer, comprising administering a pharmaceutical composition comprising a functional polypeptide variant as an active ingredient.
  • One embodiment of the present application provides the use of a functional polypeptide variant of the present invention for the manufacture of a cancer therapeutic.
  • any one of the arginine decarboxylase variant, the arginine decarboxylase subunit variant, and the dimer of the arginine decarboxylase subunit variant can be used in the preparation of a cancer therapeutic agent.
  • the functional polypeptide variant may be any one of an arginine decarboxylase variant, an arginine decarboxylase subunit variant, and a dimer of an arginine decarboxylase subunit variant. Since the arginine decarboxylase variant of the present application shows similar or superior enzymatic activity compared to arginine decarboxylase, it can be used as a therapeutic agent for arginine auxotrophic tumors.
  • the target disease may be a tumor. More specifically, the target disease may be an arginine auxotrophic tumor.
  • the target disease is melanoma (malignant melanoma), liver cancer (liver cancer), hepatocellular carcinoma (HCC), prostate cancer (prostate cancer), pancreatic cancer (pancreatic cancer), breast cancer (breast cancer), breast cancer ( mammary gland cancer, lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (GBM) ), acute myeloid leukemia (AML), and primary and relapsed lymphomas.
  • melanoma malignant melanoma
  • liver cancer liver cancer
  • liver cancer liver cancer
  • HCC hepatocellular carcinoma
  • prostate cancer prostate cancer
  • pancreatic cancer pancreatic cancer
  • breast cancer breast cancer
  • mammary gland cancer lung cancer, small cell lung cancer, malignant pleural mesot
  • composition of the present application may further include a pharmaceutically acceptable carrier in addition to containing the functional polypeptide variant 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 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 a target disease.
  • Yeast extract, tryotone, and agar were purchased from BD Biosciences (San Jose, CA, USA).
  • Nickel charged nitrilotriacetic acid (Ni-NTA) resin was purchased from qiagen (Valencia, CA, USA).
  • frTet (4-(1,2,3,4-tetazin-3-yl)phenylalanine) was purchased from Aldlab Chemicals (Woburn, MA, USA).
  • TCO-Cy3 was purchased from AAT Bioquest (Sunnyvale, CA, USA).
  • TCO-PEG4-maleidime (TCO-PEG4-MAL) was purchased from FutureChem (Seoul, Korea).
  • Disposable PD-10 desalting column and Superdex 200 10/300 GL increase column were purchased from Cytiva (Uppsala, Sweden).
  • Vivaspin 6 centrifugal concentrators with a molecular weight cut-off (MWCO) 10 kDa and 100 kDa were purchased from Sartorius (Gottingen, Germany).
  • HSA and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • E. coli genomic DNA was extracted using a genomic DNA extraction kit. PCR amplification was performed to amplify the gene translating the ADC from the extracted gDNA.
  • a primer having the sequence of 5'-ATCGGGATCCATGAAAGTATTAATTGTTGAAAGCGAG-3' (SEQ ID NO: 21) was used as a forward primer, and 5'-ATCGACTAGTTTAATGGTGATGGTGATGGTGCGCTTTCACGCACATAAC-3' (SEQ ID NO: 22) was used as a reverse primer.
  • a primer was designed to fusion a hexahistidine tag at the C-terminus of ADC for purification by affinity chromatography.
  • the amino acid sequence of the C-terminal hexahistidine tagged ADC is set forth below:
  • the pQE80 vector containing the amplified ADC gene and SpeI was treated with SpeI and BamHI restriction enzymes at 37°C for 3 hours. After that, ligation was performed according to the manual using Takara ligation mix. After transforming the TOP10 E.coli strain according to the Mix & Go transformation manual, spread it on an agar plate containing 100 ⁇ g/mL ampicillin and spread it to E. coli for 12 hours at 37°C. raised Thereafter, a single colony was inoculated into fresh LB media and cultured. The plasmid gene was obtained from E. coli cultured through mini-prep, and the sequence of the wild-type ADC was confirmed through sequencing to construct pQE80-ADC WT (wild-type, wild-type) plasmid.
  • the pQE80-ADC WT gene was transformed into a TOP10 E. coli strain according to the Mix & Go transformation manual, followed by 12 hours at 37 degrees, containing 100 ⁇ g/mL ampicillin.
  • TOP10 [pQE80-ADC WT] was obtained by growth on an agar plate. Thereafter, a single colony was inoculated into fresh LB media containing 100 ⁇ g/mL ampicillin and grown overnight at 37°C. Thereafter, 2 mL of TOP10 [pQE80-ADC WT] incubated in 200 mL fresh LB media containing 100 ⁇ g/mL ampicillin was added, and shaking culture was performed at 37 ° C and 200 rpm.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • BI indicates before induction
  • AI indicates after induction
  • P indicates purified.
  • transcription and translation of pQE80-ADC WT through IPTG can be confirmed through a newly identified band near ⁇ 75 kDa in the AI lane, followed by purification obtained through affinity chromatography. It was confirmed that the purified ADC WT (purified ADC WT) was obtained with high purity.
  • ADC WT 10 mg/mL of ADC was added to 20 mM potassium phosphate buffer (pH 7.4). Thereafter, incubation was performed at 37° C., and ADC WT activity was measured at 0, 0.5, 1, 3, 6, 12, and 24 time points to confirm stability.
  • 0.1 mg/mL ADC WT was mixed with 1 mM arginine and 200 ⁇ M pyridoxal 5'-phosphate (PLP) in 0.2 M sodium acetate (pH 5.2) and incubated at 37 degrees for 1 hour. proceeded. After this, 'Goldschmidt et al.
  • HeLa cells were seeded in a 96-well plate at 10,000 cells/well.
  • a pH 6.4 medium (DMEM, 15% FBS, 2% AA) was used.
  • the ADC solution was filtered through a 0.2 ⁇ m cellulose acetate filter and mixed with the medium to adjust the ADC concentration.
  • Each concentration of ADC solution was treated with the cells and cultured for 3 days. After 3 days of culture, the MTT assay was performed to measure the metabolic activity of the cells. MTT assay shows the intensity according to the metabolism of mitochondria in cells, and cell growth is suppressed as arginine is removed from the media.
  • FIGS. 04 to 06 The measurement results of metabolic activity for each cell line according to the ADC_WT treatment are shown in FIGS. 04 to 06.
  • 04 shows the metabolic activity measurement results of breast cancer and/or mammary cancer-related cell lines (MCF7, T47D) according to ADC_WT treatment.
  • 05 shows the metabolic activity measurement results of lung cancer-related cell lines (A549, NCI-H1299, HCC-827) according to ADC_WT treatment.
  • 06 shows the metabolic activity measurement results of pancreatic cancer-related cell lines (AsPC-1, Capan-1, Capan-2, MIA-PaCa-2).
  • ADC_WT is confirmed to be effective in inhibiting the metabolic activity of breast cancer and/or mammary cancer-related cell lines, lung cancer-related cell lines, and pancreatic cancer-related cell lines.
  • U is an enzymatic activity unit, and represents an active unit of an ADC enzyme that degrades 1 umol of arginine.
  • Gemcitabine was administered once a week at a dose of 125mg/kg, and 200ul was administered by intraperitoneal injection. The tumor size of the mice was observed for 26 days after administration. Information on specific experimental groups and administered samples is provided below.
  • - control group 1 mouse; ADC(-); gemcitabine (-)
  • ADC group 2 mice; ADC (0.2 U (6 mg)); gemcitabine (-)
  • - GEM group 1 mouse; ADC (-); gemcitabine (125mg/kg)
  • ADC-GEM group 2 mice; ADC (0.2 U (6 mg)); gemcitabine (125mg/kg)
  • FIG. 07 The in-vivo anticancer activity confirmation results are disclosed in FIG. 07 . As shown in FIG. 07 , it can be confirmed that tumor growth is inhibited in ADC alone and ADC-GEM combined administration.
  • the location for substituting AzF in the ADC was selected as follows. First, the active site and PLP binding site showing the enzyme activity of ADC_WT were excluded. (Refer to Andrell et al. Biochemistry 48.18 (2009): 3915-3927.) After that, through the 3D structure of ADC_WT (PDB ID: 2VYC), amino acids where protein assembly occurs were excluded. Among the remaining amino acid residues, two amino acids (Q488, K522) with the highest solvent accessibility and forming a random coil were selected by comparing solvent accessibility and random coil. .
  • ADC_AzF expression PCR was performed using pQE80_ADC-WT as a template.
  • a mutant plasmid (pQE80_ADC variant) was prepared by performing quick change PCR with an amber stop codon (TAG) at Q488 or K522 in the pQE80_ADC WT plasmid.
  • TAG amber stop codon
  • ADC_Q488Amb_F GGTTTTGCCGGTCTATGGGTCGGTGACGACTTCTTTGT (SEQ ID NO: 24);
  • ADC_Q488Amb_R ACAAAGAAGTCGTCACCGACCCATAGACCGGCAAAACC (SEQ ID NO: 25);
  • ADC_K522Amb_F CCAGTTATCCGGAATATCCTAGAAGCCGTGCCAGCTTTC (SEQ ID NO: 26);
  • ADC_K522Amb_R GAAAGCTGGCACGGCTTCTAGGATATTCCGGATAACTGG (SEQ ID NO: 27).
  • each mutant plasmid was transformed into C321 ⁇ A.exp [pEVOL-pAzF] competent cells, followed by C321 ⁇ Aexp [pEVOL-pAzF] [pQE80_ADC variant] E.coli cells. created Here, each of the pQE80_ADC variants used is pQE80_ADC_Q488amb (a codon encoding the 488th glutamine amino acid is substituted with an amber codon) or pQE80_ADC_K522amb.
  • Transformants were cultured overnight at 37° C. in Luria broth medium containing ampicillin (100 ⁇ g/mL) and chloroamphenicol (25 ⁇ g/mL). The pre-cultured E. coli cells were inoculated into the same fresh medium. To induce protein expression, a final concentration of 1 mM, AzF, 1 mM IPTG, and 0.4% arabinose were each added to the medium to reach an optical density of 0.5% (600 nm). After incubation at 18 °C for 18 h while shaking the culture medium, the product was obtained through centrifugation at 4 °C for 10 min at 5,000 rpm.
  • ADC variants containing AzF were prepared at 4 °C using polypropylene columns packed with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin according to the manufacturer's protocol (Qiagen). It was purified through immobilized-metal affinity chromatography. The purified ADC_AzF was desalted with PBS (pH 7.4) using a PD-10 column. The expression and purification process of ADC_WT followed a method similar to that of ADC_AzF, but chloroamphenicol and AzF were not added to the culture medium during the expression step.
  • Ni-NTA nickel-nitrilotriacetic acid
  • SDS-PAGE (run at 120V) using tris-glycine gels (5% acrylamide stacking and 12% acrylamide resolving gels) (running buffer: 25 mM Tris, 192 mM glycine, and 0.1 % SDS at pH8.3) was performed. At this time, a molecular weight standard marker (Bio-Rad Laboratories Inc. Berkeley, CA, USA) was used. 08 shows the SDS-PAGE results. Specifically, (a) shows the SDS page results before purification and (b) after purification. In FIG. 08, BI indicates before cell pellet processing (before induction cell pellet), and AI indicates after cell pellet processing (after induction cell pellet). -AzF represents a sample without AzF added. +AzF indicates a sample to which AzF was added.
  • ADC_WT and ADC_AzF were reacted with DBCO-Rhodamin (DBCO-PEG4-carboxyrhodamine dye) fluorescent dye at a molar ratio of 1:3 in PBS (pH 7.4) at room temperature. After 2 hours, the reaction mixture was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
  • DBCO-Rhodamin DBCO-PEG4-carboxyrhodamine dye
  • Fluorescence images of protein gels were obtained using a ChemiDoc XRS+ system (illumination at 302 nm, 510-610 nm filters, Bio-Rad Laboratories, Hercules, CA, USA). After fluorescence analysis, the protein gel was stained with Coomassie Brilliant Blue R-250 dye. Protein gel images were obtained using a ChemiDoc XRS+ system using white light illumination.
  • the fluorescence die labeling results for ADC_WT and ADC_AzF are shown in FIG. 09 .
  • the bands shown in the sample of FIG. 09 correspond to subunits and subunits labeled with fluorescent dyes.
  • Dye (-) represents the analysis result for the sample reacted without DBCO-Rhodamin.
  • Dye (+) represents the analysis result for the sample reacted with DBCO-Rhodamin. Bands in fluorescence were observed in ADC_Q488AzF Dye (+) and ADC_K522AzF Dye (+).
  • ADC_WT As a control.
  • ADC_WT and ADC_AzF variants at 0.1 mg/mL were reacted with 1 mM arginine and 200 ⁇ M PLP in 0.2 M sodium acetate (pH 5.2) buffer, respectively, at 37 °C for 1 hour. Thereafter, agmatine (an arginine degradation product by ADC) produced in each sample was compared.
  • ADC_AzF variants (ADC_Q488AzF and ADC_K522AzF) do not have lower enzymatic activity than ADC_WT.
  • DBCO-PEG4-Maleimide a linker used to prepare the arginine decarboxylase variant-albumin conjugate, was purchased from Future Chem, dissolved in DMSO at a concentration of 10 mM, and stored at -80 °C until use.
  • HSA-PEG4-DBCO An albumin-linker conjugate, HSA-PEG4-DBCO, was prepared by reacting 50 ⁇ M of albumin with 200 ⁇ M of a linker (DBCO-PEG4-Mal) in PBS (pH 7.0) for 2 hours at 23°C. Unreacted linkers were removed using a PD-10 desalting column. 1.7 mL was eluted to remove unreacted linker.
  • HSA-PEG4-DBCO has a molecular weight of about 66.5 kDa
  • ADC arginine decarboxylase subunit variant
  • arginine decarboxylase subunit variant-albumin conjugate has a molecular weight of about 148.5 kDa. has a molecular weight
  • the score value represents the sum of van der Waals forces, attractive forces, repulsive energies, Gaussian exclusion implicit solvation, and hydrogen bonds between atoms in different moieties (but long-range, backbone side chains and side chains) separated by distances.
  • the substitution site for frTet was selected from amino acids that have high solvent accessibility and form a random coil. At this time, an amino acid located at a site forming a dimer/decamer was tried to be excluded from the substitution site with frTet. A total of 30 loci were screened.
  • ADC_frTet PCR was performed using pQE80_ADC-WT as a template.
  • SpeI was introduced into the sfGFP coding region of the pBAD_sfGFP plasmid through PCR.
  • a mutant plasmid (pBAD_ADC variant) was prepared by performing quick change PCR with an amber stop codon (TAG) to introduce frTet into the previously selected site.
  • TAG amber stop codon
  • pBAD_sfGFP_spel_F TAAACAGTTCTTCACCTTTGCTAAACTAGTTTAATTCCTCCTGTTAGCCCAAAAAACGG (SEQ ID NO: 28)
  • pBAD_sfGFP_spel_R CCGTTTTTTGGGCTAACAGGAGGAATTAAACTAGTTTAGCAAAGGTGAAGAACTGTTTA (SEQ ID NO: 29)
  • pBAD_ADC_F AACTACTAGTATGAAAGTATTAATTGTTGAAAGCGAG (SEQ ID NO: 30)
  • pBAD_ADC_R AGATCTCGAGTTAATGGTGATGGTGATGGTG (SEQ ID NO: 31)
  • pBAD_ADC_T39Amb_F GAATGGCAAAACCATCATCAAAGGACTAGGATTTAATCACGGTAACATTTTGC (SEQ ID NO: 32)
  • pBAD_ADC_T39Amb_R GCAAAATGTTACCGTGATTAAATCCTAGTCCTTTGATGATGGTTTTGCCATTC (SEQ ID NO: 33)
  • pBAD_ADC_N85Amb_F GAAGACCGGCACCTATTGTTGGCGCTCATGAAGCTTA (SEQ ID NO: 34)
  • pBAD_ADC_N245Amb_F CAACGACCACGACATCCTAATCGGTCATGCAAGCC (SEQ ID NO: 36)
  • pBAD_ADC_K312Amb_F CCACGCAGTAAGACGGCTATTGCCCGGCTTTGTCTTTG (SEQ ID NO: 38)
  • pBAD_ADC_K312Amb_R CAAAGACAAAGCCGGGCAATAGCCGTCTTACTGCGTGG (SEQ ID NO: 39)
  • pBAD_ADC_Q488Amb_F GGTTTTGCCGGTCTATGGGTCGGTGACGACTTCTTTGT (SEQ ID NO: 40)
  • pBAD_ADC_Q488Amb_R ACAAAGAAGTCGTCACCGACCCATAGACCGGCAAAACC (SEQ ID NO: 41)
  • pBAD_ADC_K522Amb_F CCAGTTATCCGGAATATCCTAGAAGCCGTGCCAGCTTTC (SEQ ID NO: 42)
  • pBAD_ADC_G657Amb_F CTTCCGCCACCGGCAGCTAGGAATAGGCTTCGTTC (SEQ ID NO: 44)
  • ADC_frTet To express ADC_frTet, reference may be made to the method disclosed in Yang et.al, Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels-Alder Reaction, Bioconjugate Chemistry, 2020, 2456-2464. there is.
  • each mutant plasmid was transformed into C321 ⁇ A.exp [pDule C11RS] competent cells, generating C321 ⁇ Aexp [pDule C11RS] [pBAD_ADC variant] E.coli cells. did.
  • each pBAD_ADC variant used is pBAD_ADC_N85amb (codon encoding the 85th asparagine amino acid is substituted with an amber codon), pBAD_ADC_N245amb, pBAD_ADC_K312amb, pBAD_ADC_Q488amb, pBAD_ADC_K522amb, or pBAD_ADC_G657amb.
  • Transformants were cultured overnight at 37°C in Luria broth medium containing ampicillin (100 ⁇ g/mL) and tetracycline (10 ⁇ g/mL). The pre-cultured E. coli cells were inoculated into the same fresh medium. To induce protein expression, a final concentration of 1 mM and 0.4% of frTet and arabinose were added to the medium to reach an optical density of 0.5% (600 nm). After incubation at 18 °C for 18 h while shaking the culture medium, the product was obtained through centrifugation at 4 °C for 10 min at 5,000 rpm.
  • ADC variants containing frTet were prepared at 4 °C using a polypropylene column packed with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin according to the manufacturer's protocol (Qiagen). It was purified through immobilized-metal affinity chromatography. The purified ADC_frTet was desalted with PBS (pH 7.4) using a PD-10 column. The expression and purification process of ADC_WT followed a similar method to that of ADC_frTet, but tetracycline and frTet were not added to the culture medium during the expression step.
  • SDS-PAGE (run at 120V) using tris-glycine gels (5% acrylamide stacking and 12% acrylamide resolving gels) (running buffer: 25 mM Tris, 192 mM glycine, and 0.1 % SDS at pH8.3) was performed. At this time, a molecular weight standard marker (Bio-Rad Laboratories Inc., Berkeley, CA, USA) was used.
  • FIGS. 13 and 14 SDS-PAGE analysis results of ADC_WT and ADC variant (ADC_frTet) are shown in FIGS. 13 and 14 . More specifically, the band identified in the samples of FIGS. 13 and 14 corresponds to a subunit having a molecular weight of about 80 kDa. 13 and 14, BI means before induction, and AI means after induction.
  • ADC WT refers to the result of a sample expressing and purifying wild-type ADC.
  • ADC T39, ADC N85, ADC N245, ADC K312, ADC Q488, ADC K522, and ADC G657 refer to the results of samples in which ADC_T39frTet, ADC_N85frTet, ADC_N245frTet, ADC_K312frTet, ADC_Q488frTet, ADC_K522frTet, and ADCG_657frTet were expressed and refined, respectively.
  • frTet variants ADC_N245frTet and ADC_G657feTet were excluded from the candidate group due to molecular weight analysis that did not correspond to ADC variants during the expression purification process.
  • ADC_frTet Purified ADC_WT and its arginine decarboxylase variants, ADC_frTet (ADC_N85frTet, ADC_K312frTet, ADC_Q488frTet, and ADC_K522frTet, respectively) were reacted with TCO-Cy3 fluorescent dye at a 1:2 molar ratio in PBS (pH 7.4) at room temperature. After 2 hours, the reaction mixture was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Fluorescence images of protein gels were obtained using a ChemiDoc XRS+ system (illumination at 302 nm, 510-610 nm filters, Bio-Rad Laboratories, Hercules, CA, USA). After fluorescence analysis, the protein gel was stained with Coomassie Brilliant Blue R-250 dye. Protein gel images were obtained using a ChemiDoc XRS+ system using white light illumination.
  • FIG. 15 The fluorescence die labeling results for ADC_WT and ADC_frTet are shown in FIG. 15 . Specifically, the bands shown in the sample of FIG. 15 correspond to subunits and subunits labeled with fluorescent dyes.
  • Figure 15 (b) is the resulting data in fluorescence.
  • ADC_T39, ADC_N85, ADC_K312, ADC_Q488, and ADC_K522 represent ADC_T39frTet, ADC_N85frTet, ADC_K312frTet, ADC_Q488frTet, and ADC_K522frTet, respectively.
  • TCO-Cy3 represents the analysis result for the sample to which TCO-Cy3 was not added.
  • TCO-Cy3 (+) represents the analysis result of the reaction sample to which TCO-Cy3 was added.
  • ADC_WT As a control.
  • ADC_WT or ADC_frTet variant at 0.1 mg/mL was reacted with 1 mM arginine and 200 ⁇ M PLP in 0.2 M sodium acetate (pH 5.2) buffer for 1 hour at 37°C. Thereafter, the produced agmatine (an arginine degradation product by ADC) was compared.
  • ADC_T39, ADC_N85, ADC_K312, ADC_Q488, and ADC_K522 represent ADC_T39frTet, ADC_N85frTet, ADC_K312frTet, ADC_Q488frTet, and ADC_K522frTet, respectively.
  • ADC_frTet variants The inverse-electron-demand Diels-Alder (IEDDA) reaction rate of ADC_frTet variants was evaluated.
  • 25 ⁇ M ADC_WT or ADC_frTet (ADC_K312frTet, ADC_Q488frTet, ADC_K522frTet) was incubated in 100 ⁇ M HSA-PEG4-TCO and 0.2 M sodium acetate (pH 5.2) buffer at 23 °C for 5 hours. Thereafter, SDS-PAGE analysis was performed on each sample. As a result of the analysis, it was confirmed that the strength of the band corresponding to ADC disappeared the most in ADC_K312frTet.
  • ADC_K312frTet had the highest intensity in the band corresponding to the ADC-HSA conjugate (FIG. 17). Judging from the fact that the band corresponding to the ADC-HSA conjugate was not observed in ADC_WT, it can be assumed that the newly formed uppermost band is formed through the reaction between frTet and HSA-PEG4-TCO. Through the results, it can be seen that ADC_K312frTet has the highest IEDDA (Inverse electron-demand Diels-Alder) reaction rate.
  • IEDDA Inverse electron-demand Diels-Alder
  • TCO-PEG4-Maleimide linker was purchased from Future Chem, dissolved in DMSO at a concentration of 20 mM, and stored at -80°C until use.
  • the chemical formula of TCO-PEG4-Maleimide is:
  • Albumin-linker conjugate HSA-PEG4-TCO
  • PBS pH 7.0
  • Unreacted linkers were removed using a PD-10 desalting column. 1.7 mL was eluted to remove unreacted linker.
  • the half-life was analyzed through a two compartment model, a representative model for analyzing the disappearance of drugs from the blood. As a result of the analysis, it was confirmed that ADC_WT had a half-life of 4.1 hours, and the ADC(K312frTet)-HSA conjugate had a half-life of 23.1 hours (FIG. 21). It was confirmed that the arginine decarboxylase variant-albumin conjugate had an improved half-life of about 5.6-fold compared to the wild-type ADC.
  • TCO-PEG4-Maleimide linker was purchased from Future Chem, dissolved in DMSO at a concentration of 20 mM, and stored at -80°C until use.
  • HSA-PEG4-TCO was prepared by reacting 50 ⁇ M albumin and 200 ⁇ M linker (TCO-PEG4-Mal) in PBS (pH 7.0) for 2 hours at 23°C. Unreacted linkers were removed using a PD-10 desalting column. 1.7 mL was eluted to remove unreacted linker.
  • ADC(T39frTet)-HSA conjugate, ADC_WT, and ADC_T39frTet which are arginine decarboxylase variant-albumin conjugates obtained through size exclusion chromatography. Enzyme activity was confirmed under the same conditions as those described in "2. Stability confirmation of wild-type E. coli-derived ADC (ADC_WT)" of Experimental Example I.
  • Enzyme activity confirmation results are shown in FIG. 24 .

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Abstract

La présente demande concerne un variant d'arginine décarboxylase et un conjugué fonctionnel d'albumine-variant de polypeptide préparé à l'aide de celui-ci.
PCT/KR2022/015832 2021-10-18 2022-10-18 Variant d'arginine décarboxylase et conjugué fonctionnel d'albumine-variant de polypeptide préparé à l'aide de celui-ci Ceased WO2023068736A1 (fr)

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PCT/KR2022/015832 Ceased WO2023068736A1 (fr) 2021-10-18 2022-10-18 Variant d'arginine décarboxylase et conjugué fonctionnel d'albumine-variant de polypeptide préparé à l'aide de celui-ci

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200061688A (ko) * 2018-11-26 2020-06-03 광주과학기술원 알부민이 결합된 글루카곤 유사 펩타이드-1 유사체

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200061688A (ko) * 2018-11-26 2020-06-03 광주과학기술원 알부민이 결합된 글루카곤 유사 펩타이드-1 유사체

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDRÉLL JUNI, HICKS MATTHEW G., PALMER TRACY, CARPENTER ELISABETH P., IWATA SO, MAHER MEGAN J.: "Crystal Structure of the Acid-Induced Arginine Decarboxylase from Escherichia coli : Reversible Decamer Assembly Controls Enzyme Activity", BIOCHEMISTRY, vol. 48, no. 18, 12 May 2009 (2009-05-12), pages 3915 - 3927, XP093059503, ISSN: 0006-2960, DOI: 10.1021/bi900075d *
DATABASE Protein 16 December 2020 (2020-12-16), ANONYMOUS : "MULTISPECIES: arginine decarboxylase [Enterobacteriaceae] Identical Proteins FASTA Graphics", XP093059511, retrieved from ncbi Database accession no. WP_001381593 *
YANG BYUNGSEOP, KWON INCHAN: "Chemical Modification of Cysteine with 3-Arylpropriolonitrile Improves the In Vivo Stability of Albumin-Conjugated Urate Oxidase Therapeutic Protein", BIOMEDICINES, vol. 9, no. 10, 27 September 2021 (2021-09-27), pages 1334, XP093059502, DOI: 10.3390/biomedicines9101334 *
YANG BYUNGSEOP, KWON INCHAN: "Multivalent Albumin–Neonatal Fc Receptor Interactions Mediate a Prominent Extension of the Serum Half-Life of a Therapeutic Protein", MOLECULAR PHARMACEUTICS, AMERICAN CHEMICAL SOCIETY, US, vol. 18, no. 6, 7 June 2021 (2021-06-07), US , pages 2397 - 2405, XP093021165, ISSN: 1543-8384, DOI: 10.1021/acs.molpharmaceut.1c00231 *

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