WO2023068736A1

WO2023068736A1 - Arginine decarboxylase variant and functional polypeptide variant-albumin conjugate prepared using same

Info

Publication number: WO2023068736A1
Application number: PCT/KR2022/015832
Authority: WO
Inventors: 권인찬; 김승균; 임수진
Original assignee: 주식회사 프로앱텍
Priority date: 2021-10-18
Filing date: 2022-10-18
Publication date: 2023-04-27

Abstract

The present application relates to an arginine decarboxylase variant and a functional polypeptide variant-albumin conjugate prepared using same.

Description

Arginine decarboxylase variants and arginine decarboxylase variants-albumin conjugates prepared using the same

One embodiment of the present application relates to an arginine decarboxylase variant (ADC 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.

It has been found that 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.

In one embodiment of the present application, a compound having the structure of Formula 1 is provided:

[Formula 1]

FPV-[J ₁ -A ₂ -J ₂ -P ₁ ] _a ,

At this time:

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;

In this case, 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,

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;

In this case, the heteroatoms are each independently selected from N, O, and S, wherein the substitution is substituted with one or more non-hydrogen substituents, and the non-hydrogen substituents are, each independently, halogen, C _1-3 Any one selected from the group consisting of alkyl, -NH ₂ , =O, and =S,

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;

In certain embodiments, 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. can

In a specific embodiment, 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.

In a specific embodiment, 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. .

In a specific embodiment, the first click chemofunctional group may include a tetrazine group or an azide group.

In certain embodiments, the unnatural amino acid can be frTet or AzF.

In a specific embodiment, 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.

In certain embodiments, the second click chemofunctional group may include a trans cyclooctyne (TCO) group.

In a specific embodiment, the second bonding unit may have the following structure:

, or

,

wherein in the above structure, the S atom is derived from albumin.

,

wherein in the above structure, the S atom is derived from albumin.

In certain embodiments, said second anchor unit is -A ₂₁ -A ₂₂ -A ₂₃ -;

At this time:

A ₂₁ is a bond, -CH ₂ CH ₂ OC(=O)NH-, -CH ₂ CH ₂ C(=O)NH-, -CH ₂ CH ₂ C(=O)-, -CH ₂ OC (=O)NH-, -CH ₂ C(=O)NH-, -CH ₂ C(=O)-, -OC(=O)NH-, -C(=O)NH-, -NH- or -C(=O)-,

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-;

In this case, EG is an ethylene glycol unit, and 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,

In this case, the heteroalkylene is each independently selected from N, O, and S,

In this case, the substitution is substituted with one or more non-hydrogen substituents, and the non-hydrogen substituents are each independently any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S,

A ₂₃ is a bond, -CH ₂ CH ₂ OC(=O)NH-, -CH ₂ CH ₂ C(=O)NH-, -CH ₂ CH ₂ C(=O)-, -CH ₂ OC (=O)NH-, -CH ₂ C(=O)NH-, -CH ₂ C(=O)-, -OC(=O)NH-, -C(=O)NH-, -NH- or -C(=O)-,

At this time, A ₂₁ ,A case in which both A ₂₂ and A ₂₃ are simultaneously bonded may not exist.

In certain embodiments, the second anchor unit may have any one of the following structures:

,

In this case, n is an integer of 2 or more and 8 or less,

In this case, 3' is an attachment site with the first bonding unit, and 4' is an attachment site with the second bonding unit.

In a specific embodiment, 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.

In one embodiment of the present application, a pharmaceutical composition for treating arginine auxotrophic tumors, comprising a compound having the structure of Formula 1, is provided.

In a specific embodiment, 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.

In one embodiment of the present application, 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.

In a specific embodiment, 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.

In a specific embodiment, 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.

In certain embodiments, the non-natural amino acid can be frTet.

In certain embodiments, the non-natural amino acid can be AzF.

In a specific embodiment, 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.

In one embodiment of the present application, a pharmaceutical composition for treating arginine auxotrophic tumors is provided, comprising the arginine decarboxylase subunit variant of the present application.

In one embodiment of the present application, there is provided 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. .

In one embodiment of the present application, the use of a composition comprising a compound having the structure of Formula 1 or an arginine decarboxylase subunit variant for treating arginine auxotrophic tumor in a subject is provided.

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.

1 is a PAGE result of wild-type E. coli-derived arginine decarboxylase (ADC_WT). PAGE was performed after ADC_WT expression and purification.

2 is a result of confirming the stability of ADC_WT.

3 is a result of confirming the metabolic activity inhibitory effect of ADC_WT for each pH. Experiments were conducted by treating Hela cells with ADC_WT.

Figure 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.

Figure 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.

Figure 6 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 pancreatic cancer-related cell lines.

7 is a result of confirming the in vivo anticancer activity of ADC_WT.

8 shows the results of performing PAGE after expressing ADC variants (ADC_Q488AzF and ADC_K522AzF). (a) shows the results before purification and (b) after purification.

9 shows the fluorescence die labeling results for ADC variants.

10 shows the results of confirming the enzyme activity of ADC_WT and ADC variants.

11 is a result of confirming the preparation of an arginine decarboxylase variant-albumin conjugate through PAGE.

12 is a result of confirming the preparation of an arginine decarboxylase variant-albumin conjugate through PAGE.

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.

14 shows PAGE results for ADC variants (ADC_K312frTet, ADC_Q488frTet, ADC_K522frTet, ADC_G657frTet). SDS-PAGE was performed after ADC variant expression and purification.

15 shows the fluorescence die labeling results for ADC variants.

16 shows the results of confirming the enzyme activity of ADC_WT and ADC variants.

17 is a result of confirming the IEDDA reaction rate through PAGE when preparing an arginine decarboxylase variant-albumin conjugate.

18 shows the results of size exclusion chromatography after preparation of the ADC(K312frTet)-HSA conjugate.

19 shows the results of PAGE performed using three fractions (F1 to F3) obtained through size exclusion chromatography.

20 shows the result of confirming the enzyme activity of ADC variant (ADC_K312frTet) and ADC (K312frTet)-HSA conjugate.

21 is a result of confirming the PK of the ADC(K312frTet)-HSA conjugate.

22 shows the results of size exclusion chromatography after preparation of the ADC(T39frTet)-HSA conjugate.

23 shows the results of PAGE performed using three fractions (F1 to F3) obtained through size exclusion chromatography.

24 shows the result of confirming the enzyme activity of the ADC variant (ADC_T39frTet) and the ADC(T39frTet)-HSA conjugate.

용어의 정의Definition of Terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents and other references mentioned herein are incorporated by reference in their entirety.

As used herein, 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%.

As used herein, the term "polypeptide" is used to mean a compound formed by consecutively linking a plurality of amino acids. As used herein, the term "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. In another example, 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.

As used herein, the term "hetero" refers to a compound or group containing at least one heteroatom. The term "heteroatom" is an atom other than carbon or hydrogen, including, for example, B, Si, N, P, O, S, F, Cl, Br, I and Se. Preferably, 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.

As used herein, the term “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. In this case, the substituents are each independently selected. Substituents may include deuterium and hydrogen variants. When the substituent is oxygen (i.e. =O), it means that two hydrogen atoms are substituted. When one substituent is a halogen (eg, Cl, F, Br, and I, etc.), it means that one hydrogen atom has been replaced with a halogen. When two or more substituents are present in one group, the substituents present in the group may be the same or different. Unless otherwise specified, the type and number of substituents can be arbitrary, as long as they are chemically achievable. Illustratively, the substituent may be selected from halogen, C _1-6 alkyl, C _1-6 heteroalkyl, -NH ₂ , =O, =S, -OH, and -SH, but is not limited thereto. For example, substituted C _10-20 alkylene may mean that one or more hydrogen atoms linked to the main chain are substituted with substituents, and each substituent may be independently selected.

As used herein, the term "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. The term “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. For example, the term "C _xy alkyl" includes substituted or unsubstituted, chain-like alkyl groups, branched alkyl groups, or alkyl groups containing a cyclic structure 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. Examples of C _1-4 alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, iso-butyl, difluoromethyl, and 2,2,2-trifluoro roethyl and the like, but are not limited thereto.

As used herein, the term “heteroalkyl” refers to an alkyl containing one or more heteroatoms.

As used herein, the term "alkenyl" or "alkene" is used to mean a chain or branched non-aromatic hydrocarbon group containing one or more double bonds. For example, a chain-like or branched alkenyl group can have 2 to about 50, 2 to 20, or 2 to 10 carbon atoms. Alkenyl groups can include cyclic structures.

As used herein, the term “heteroalkene” refers to an alkene containing one or more heteroatoms.

As used herein, the term "alkynyl" or "alkyne" is used to mean a chain or branched non-aromatic hydrocarbon group containing one or more triple bonds. For example, a chain or branched alkynyl group can have 2 to about 50, 2 to 20, or 2 to 10 carbon atoms. An alkynyl group may contain one or more double bonds in addition to one or more triple bonds. Alkynyl groups can include cyclic structures.

As used herein, the term "heteroalkyn" refers to an alkyne containing one or more heteroatoms.

The term "alkylene" when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from an alkyl. The term "alkylene" may be used with the terms "substituted" or "unsubstituted", as appropriate. When the term "alkylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "alkylene" is intended to include both substituted and unsubstituted alkylenes. Alkylene may be exemplified by -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, and -CH ₂ CH ₂ CH ₂ CH ₂ -, but is not limited thereto. For example, alkylene may be used as C ₂ alkylene, which refers to an alkylene group containing two carbon atoms in the main chain. Illustratively, in this specification, "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.

The term “heteroalkylene” when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkyl. The term "heteroalkylene" may be used with the terms "substituted" or "unsubstituted" as appropriate. When the term "heteroalkylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "heteroalkylene" includes both substituted and unsubstituted heteroalkylenes. it is intended to Exemplary heteroalkylene groups include, but are not limited to —CH ₂ —CH ₂ —O—CH ₂ —CH ₂ —, and —CH ₂ —O—CH ₂ —CH ₂ —NH—CH ₂ —. Heteroalkylene groups can contain one or more heteroatoms, and each heteroatom can be the same or different. For example, 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. For example, 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. Illustratively, in this specification, "C _xy heteroalkylene" is used to include all substituted or unsubstituted heteroalkylenes having x to y carbon atoms in the main chain.

The term "alkenylene" when used as a molecule by itself or as part of a molecule refers to a divalent radical derived from an alkene. The term “alkenylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. When the term "alkenylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "alkenylene" is intended to include both substituted and unsubstituted alkenylene. Illustratively, the alkenylene group includes, but is not limited to, -CH=CH-, -CH ₂ CH=CHCH ₂ -, and -CH=CH-CH=CH-. Illustratively, in this specification, "C _xy alkenylene" is used to include all substituted or unsubstituted alkenylene having X to Y number of carbon atoms in the main chain.

The term "heteroalkenylene" when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkene. The term “heteroalkenylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. When the term "heteroalkenylene" is not used with the terms "substituted" or "unsubstituted", the term "heteroalkenylene" includes both substituted and unsubstituted heteroalkenylenes. it is intended to A heteroalkenylene group can contain one or more heteroatoms, and each heteroatom can be the same or different. For example, 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. For example, 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.

The term "alkynylene" when used as a molecule by itself or as part of a molecule refers to a divalent radical derived from an alkyne. The term “alkynylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. In cases where the term "alkynylene" is not used in conjunction with the terms "substituted" or "unsubstituted", the term "alkynylene" is intended to include both substituted and unsubstituted alkynylenes. For example, an alkynylene group includes, but is not limited to, -C≡C-, -CH ₂ C≡CCH ₂ -, and -C≡CC≡C-. Illustratively, in this specification, “C _xy alkynylene” is used to include all substituted or unsubstituted alkynylene having X to Y number of carbon atoms in the main chain.

The term "heteroalkynylene" when used as a molecule on its own or as part of a molecule refers to a divalent radical derived from a heteroalkyne. The term “heteroalkynylene” may be used with the terms “substituted” or “unsubstituted” as appropriate. When the term "heteroalkynylene" is not used with the terms "substituted" or "unsubstituted", the term "heteroalkynylene" includes both substituted and unsubstituted heteroalkynylene. it is intended to A heteroalkynylene group can contain one or more heteroatoms, and each heteroatom can be the same or different. For example, 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. For example, 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.

The term “click-chemistry” as used herein is defined by K. Barry Sharpless of the Scripps Research Institute to describe complementary chemical groups and chemical reactions designed to quickly and stably form a covalent bond between two molecules. It is a chemical concept introduced for Click chemistry in the present specification does not mean a specific reaction, but means a concept of a fast and stable reaction. In one embodiment, in order to form bonds between molecules by click chemistry, several conditions must be satisfied. The conditions are high yield, excellent selectivity for the reaction site, organic molecular bonding by operating in a modular manner, and rapid and accurate product production by proceeding in a thermodynamically stabilized direction. The click chemistry of the present specification is a click chemical functional group (eg, terminal alkyne, azide, strained alkyne, diene, dienophile, trans cyclo 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. Examples of 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.

As used herein, the term "natural amino acid" or "standard amino acid" refers to 20 types of amino acids synthesized through gene transcription and translation in the body of an organism. 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). 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.

The term “nonnatural amino acid” as used herein refers to an amino acid that is not synthesized in the body but artificially synthesized. The non-natural amino acids include, for example, p-Azido-L-phenylalanine (AzF), p-ethynyl-phenylalanine (pEthF), 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-3-yl) phenylalanine (frTet). Since the non-natural amino acid does not have a corresponding DNA codon and cannot be expressed in a general amino acid one-letter or three-letter notation, it is indicated using other characters and additionally supplemented. The subject referred to by the term non-natural amino acid should be appropriately interpreted according to the context, and includes all other meanings that can be recognized by those skilled in the art.

As used herein, the term "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. As used herein, the term "amino acid" may be used to include both natural amino acids and non-natural amino acids. For example, alanine may be used to refer to alanine and/or an alanine residue. For example, arginine can be used to refer to arginine and/or arginine residues. As used herein, the term "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.

As used herein, the term "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. Furthermore, as described above, in a peptide having an ARE sequence, the peptide may include three amino acids, and A is alanine, R is arginine, and E is glutamic acid. As another example, 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. Furthermore, as described above, in a peptide having a DFK sequence, 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.

Unless otherwise stated, 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. For example, when expressed as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected from the N-terminal to the C-terminal. For another example, when expressed as Thr-Leu-Lys, it means a peptide in which threonine, leucine, and lysine are sequentially connected from the N-terminal to the C-terminal. In the case of amino acids that cannot be expressed by the one-letter notation or the three-letter notation, other characters are used and additionally supplemented descriptions are provided. 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.

The term "treatment" refers to an approach for obtaining a beneficial or desirable clinical result. For the purposes of this invention, 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.

As used herein, 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. .

Hereinafter, specific details of the invention are disclosed.

아르기닌 영양요구성 종양(arginine auxotrophic tumor) 및 아르기닌 디카르복실레이즈(Arginine decarboxylase; ADC)를 이용한 종양 억제 방법 Arginine auxotrophic tumor and tumor suppression method using arginine decarboxylase (ADC)

An 'auxotrophic requirement' for arginine is a hallmark of some tumor types. 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 (2018): 854-870.).

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. A study was conducted to use arginine decarboxylase, an enzyme that degrades arginine, as an anticancer drug.

Arginine decarboxylase (ADC), 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. For example, 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. make it bearable For example, 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). For example, 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.

아르기닌 디카르복실레이즈의 PEGylation 및 이의 문제점PEGylation of arginine decarboxylase and its problems

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.

In order to solve these problems and use 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.

The inventors of the present application have developed arginine decarboxylase variants or albumin conjugates thereof with no reduced enzymatic activity, improved immunogenicity, and increased in vivo stability (eg plasma half-life). Hereinafter, 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.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트 개괄Overview of Functional Polypeptide Variants-Albumin Conjugates

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 제조에 사용되는 요소Elements Used in the Preparation of Functional Polypeptide Variant-Albumin Conjugates

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. Here, 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. Specifically, the functional polypeptide variant-albumin conjugate is prepared by site-specifically linking (i) functional polypeptide variant and (ii) albumin through (iii) a linker. Here, 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. Here, the linker includes a thiol reactive group and a click chemofunctional group. Specifically, 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, and 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.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 구조Structure of Functional Polypeptide Variant-Albumin Conjugates

In one embodiment of the present application, a functional polypeptide variant-albumin conjugate having the structure of Formula 1 is provided:

[Formula 1]

FPV-[J ₁ -A ₂ -J ₂ -P ₁ ] _a

In Formula 1, 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. Here, the functional polypeptide variants include an arginine decarboxylase variant, an arginine decarboxylase subunit variant, and a dimer of the arginine decarboxylase subunit variant. variant).

In Formula 1, 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.

In Formula 1, A ₂ is a second anchor unit. In a conjugate, 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.

In Formula 1, 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.

In Formula 1, P ₁ is an albumin unit. The albumin unit may be conjugated albumin. That is, the albumin unit is derived from albumin.

In Formula 1, 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. For example, 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. As another example, 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. As another example, when the functional polypeptide variant is an arginine decarboxylase variant, a may be an integer from 1 to 10.

As described above, 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. Hereinafter, the elements used in the preparation of functional polypeptide variant-albumin conjugates are described in detail.

기능성 폴리펩티드 변이체 및 기능성 폴리펩티드 변이체 유닛Functional Polypeptide Variants and Functional Polypeptide Variant Units

기능성 폴리펩티드 (functional polypeptide)functional polypeptide

The term functional polypeptide is used herein to include all peptides, polypeptides, and proteins having more than one function. For example, the function may be related to treatment and/or prevention of a certain disease. For example, the function may be related to diagnosis. For example, the function may be to act with other polypeptides to form complexes. For example, the function may be to act with a cognate polypeptide to form a complex. At this time, the complex can be used to treat any disease. For example, a function may be related to manipulation of a gene. For example, the function may be related to binding to other molecules (eg, antigens and targets, etc.). In the present specification, the functional polypeptide is any one of arginine decarboxylase, arginine decarboxylase subunit, and dimer of arginine decarboxylase subunit.

기능성 폴리펩티드 변이체 (functional polypeptide variant) 개괄: 비천연 아미노산을 포함함Overview of functional polypeptide variants: including non-natural amino acids

Functional polypeptide variants are functional polypeptides that have been modified to include non-natural amino acids. For example, 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. Here, 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). For example, functional polypeptide variants can be specified by describing substitution positions and/or amino acids to be substituted in the reference sequence.

In the present application, the functional polypeptide variant is any one selected from arginine decarboxylase variants, arginine decarboxylase subunit variants, and dimers of arginine decarboxylase subunit variants.

기능성 폴리펩티드 변이체 및 기능성 폴리펩티드 변이체 유닛Functional Polypeptide Variants and Functional Polypeptide Variant Units

In formula 1, 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.

As used herein, 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. For example, the functional polypeptide variant may have any one amino acid sequence selected from SEQ ID NO: 02 to SEQ ID NO: 08. For example, the functional polypeptide variant unit FPV in Formula 1 may be referred to as a functional polypeptide variant.

비천연 아미노산non-natural amino acids

비천연 아미노산 개괄Overview of Unnatural Amino Acids

As used herein, the term 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.

As noted above, functional polypeptide variants contain 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.

제1 클릭화학작용기를 포함하는 비천연 아미노산Non-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. In one embodiment, 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. In one embodiment, 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)). In a specific embodiment, 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.

In one embodiment, 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.

In one embodiment, the non-natural amino acid can have the structure of Formula 3:

[Formula 3]

.

Here, A ₁ is the first anchor moiety.

Here, H ₁ is a first click chemical functional group. In one embodiment, 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)).

In one embodiment, the first click chemofunctional group can be represented by any one of the following structures:

.

In this case, R ₁ is any one selected from H, halogen, C _1-3 alkyl, C _3-6 cycloalkyl, C _3-6 heterocycloalkyl, aryl, and heteroaryl, wherein the heterocycloalkyl, or Heteroaryl is a group of one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ -, or O , N, and may include one or more heteroatoms selected from the group consisting of S.

In one embodiment, the first anchor moiety can be a bond or -A ₁₁ -A ₁₂ -.

In this case, A ₁₁ may be a bond or C _1-5 alkylene.

In this case, 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-. In this case, p may be an integer of 0 or more and 3 or less. In this case, the heteroalkylene or the heteroarylene is selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - It may contain one or more heteroatom groups, or one or more heteroatoms selected from the group consisting of O, N, and S.

비천연 아미노산의 구체예Specific examples of non-natural amino acids

In one embodiment, 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)-2-aminopropanoic acid, 2-amino-3-(4-(2-(6-methyl-1,2,4,5-tetrazin-3- yl)ethyl)phenyl)propanoic acid, 2-amino-3-(4-(6-phenyl-1,2,4,5-tetrazin-3-yl)phenyl)propanoic acid, 3-(4-((1 ,2,4,5-tetrazin-3-yl)amino)phenyl)-2-aminopropanoic acid, 3-(4-(2-(1,2,4,5-tetrazin-3-yl)ethyl)phenyl) -2-aminopropanoic acid, 3-(4-((1,2,4,5-tetrazin-3-yl)thio)phenyl)-2-aminopropanoic acid, 2-amino-3-(4-((6- methyl-1,2,4,5-tetrazin-3-yl)thio)phenyl)propanoic acid, 3-(4-((1,2,4,5-tetrazin-3-yl)oxy)phenyl)-2 -aminopropanoic acid, 2-amino-3-(4-((6-methyl-1,2,4,5-tetrazin-3-yl)oxy)phenyl)propanoic acid, 3-(4'-(1,2 ,4,5-tetrazin-3-yl)-[1,1'-biphenyl]-4-yl)-2-aminopropanoic acid, 2-amino-3-(4'-(6-methyl-1,2, 4,5-tetrazin-3-yl)-[1,1'-biphenyl]-4-yl)propanoic acid, 2-amino-3-(6-(6-(pyridin-2-yl)-1,2 ,4,5-tetrazin-3-yl)pyridin-3-yl)propanoic acid, 3-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-2-aminopropanoic acid, and 2 -amino-3-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)propanoic acid.

In one embodiment, the non-natural amino acid may have a structure of any one of the following formulas:

[Formula 3-1]

,

[Formula 3-2]

,

[Formula 3-3]

,

[Formula 3-4]

,

[Formula 3-5]

,

[Formula 3-6]

,

[Formula 3-7]

,

[Formula 3-8]

,

[Formula 3-9]

,

[Formula 3-10]

,

[Formula 3-11]

, and

[Formula 3-12]

.

기능성 폴리펩티드 변이체 예시 (1) - 아르기닌 디카르복실레이즈 서브유닛 변이체 Examples of Functional Polypeptide Variants (1) - Arginine Decarboxylase Subunit Variants

아르기닌 디카르복실레이즈 서브유닛 변이체 개괄Overview of arginine decarboxylase subunit variants

The functional polypeptide variants of the present application may be arginine decarboxylase variants, arginine decarboxylase subunit variants, and dimers of arginine decarboxylase subunit variants.

As described above, 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.

One embodiment of the present application provides arginine decarboxylase subunit variants. 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. For example, an arginine decarboxylase subunit variant may contain one or more unnatural amino acids.

In one embodiment, the arginine decarboxylase subunit that is the prototype of the arginine decarboxylase subunit variant may be derived from a microorganism. In one embodiment, the arginine decarboxylase subunit may be an arginine decarboxylase subunit derived from Escherichia coli ( E. coli ). Here, the 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:

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAVDNNVLRQELVSIENLPGDKLSSPGEPYSLPGDKLSSPYN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 01).

아르기닌 디카르복실레이즈 서브유닛 변이체 구체예Arginine decarboxylase subunit variant specific examples

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'.

In one embodiment, 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. In one embodiment, the one or more amino acids may be substituted with non-natural amino acids.

In one embodiment, 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.

In one embodiment, 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.

In a specific embodiment, 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. In a specific embodiment, 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. , And any one or more residues selected from the 522nd lysine (Lysine) may be substituted with frTet. In a specific embodiment, 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. Here, the polypeptide having the amino acid sequence of SEQ ID NO: 01 is an arginine decarboxylase subunit derived from E. coli.

In one embodiment, the arginine decarboxylase subunit variant can have any one of the following sequences:

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSXSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAVDNNVLRQELVSIENLPGDKLSSPGEPYSLPGDKLSSPYN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 02);

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQXVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAVDNNVLRQELVSIENLPGDKLSSPGEPYSLPGDKLSSPYN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 03);

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLDHTGAFGESEKYARM DWS XDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSIDEARFLYVLTQAMKEVDARQFTA PWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPLRLSVSIENGPPNFGRIPANSQELVSIENGPPNFGDKYN SLQSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 04);

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQXPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAVDNNVLRQELVSIENLPGDKLSSPGEPYSLPGDKLSSPYN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 05);

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPXTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAIVDNNVLRIAANSQELVSIENGPGDKLSSPGEPYSLPGDKLSSPELN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 06);

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFXDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAIVDNNVLRQELVSIENLPGDKLSSPGEPYSLPGDRIAANSSPEL QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 07); and

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSXLPVAEVTPREAYNAVDNNVLRQELVSIENLPGDKLSSPGEPYSLPGDKLSSPGEN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKA (SEQ ID NO: 08).

In this case, X means a non-natural amino acid residue. In certain embodiments, X may be a frTet moiety. In certain embodiments, X may be an AzF moiety.

기능성 폴리펩티드 변이체 예시 (2) - 아르기닌 디카르복실레이즈 변이체Examples of Functional Polypeptide Variants (2) - Arginine Decarboxylase Variants

In one embodiment, the functional polypeptide variant may be an arginine decarboxylase variant.

One embodiment of the present application provides arginine decarboxylase variants.

Like arginine decarboxylase, 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. In one embodiment, the arginine decarboxylase variant may be an E. coli-derived arginine decarboxylase variant. In this case, the E. coli-derived arginine decarboxylase variant includes 1 to 10 E. coli-derived arginine decarboxylase subunit variants.

In one embodiment, an arginine decarboxylase variant may comprise any of the following:

10 arginine decarboxylase subunit variants;

9 arginine decarboxylase subunit variants and 1 wild type arginine decarboxylase subunit;

8 arginine decarboxylase subunit variants and 2 wild-type arginine decarboxylase subunits;

7 arginine decarboxylase subunit variants and 3 wild-type arginine decarboxylase subunits;

6 arginine decarboxylase subunit variants and 4 wild-type arginine decarboxylase subunits;

5 arginine decarboxylase subunit variants and 5 wild-type arginine decarboxylase subunits;

4 arginine decarboxylase subunit variants and 6 wild-type arginine decarboxylase subunits;

3 arginine decarboxylase subunit variants and 7 wild-type arginine decarboxylase subunits;

2 arginine decarboxylase subunit variants and 8 wild-type arginine decarboxylase subunits; and

One arginine decarboxylase subunit variant and nine wild-type arginine decarboxylase subunits.

기능성 폴리펩티드 변이체 예시 (3) - 아르기닌 디카르복실레이즈 서브유닛 변이체의 다이머 Exemplary Functional Polypeptide Variants (3) - Dimers of Arginine Decarboxylase Subunit Variants

In one embodiment, 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.

As described above, 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. In one embodiment, the dimer of the arginine decarboxylase subunit variant may be a dimer of an arginine decarboxylase subunit variant derived from E. coli. In this case, the dimer of the E. coli-derived arginine decarboxylase subunit variant includes one or two E. coli-derived arginine decarboxylase subunit variants.

In one embodiment, a dimer of an arginine decarboxylase subunit variant may comprise any of the following:

two arginine decarboxylase subunit variants; and

One arginine decarboxylase subunit variant and one wild-type arginine decarboxylase subunit.

링커: 클릭화학작용기 및 티올 반응성 그룹을 포함함Linker: contains click chemofunctional groups and thiol reactive groups

링커 개괄linker overview

This application provides linkers. 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. For example, an albumin-linker conjugate may be prepared by reacting albumin with a linker, and a functional polypeptide variant-albumin conjugate may be prepared by reacting the albumin-linker conjugate with a functional polypeptide variant. In another example, a functional polypeptide variant-linker conjugate is prepared by reacting the functional polypeptide variant with a linker, and 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. For example, 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.

티올 반응성 그룹thiol reactive group

Linkers of the present application include thiol reactive groups. A thiol-reactive group is reactive with a thiol group. In one embodiment, the thiol-reactive group can be a maleimide group or a 3-Arylpropiolonitriles (APN) group.

In one embodiment, a thiol reactive group included in a linker of the present application may have the following structure:

,

, and

.

링커의 구조 (화학식 2)Structure of the linker (Formula 2)

One embodiment of the present application provides a linker having the structure of Formula 2:

[Formula 2]

H ₂ -A ₂ -B.

In 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.

In Formula 2, B is a thiol reactive group. In one embodiment, the thiol reactive group can be a maleimide group or an APN group.

In Formula 2, A ₂ is a second anchor unit. In the linker, the second anchor unit serves to adjust the distance between the second click chemofunctional group and the thiol reactive group. In the conjugate, the second anchor unit serves to control the distance between the functional polypeptide variant unit and the albumin unit.

Second click chemical functional group (H ₂ )

As described above, the second click chemistry functional group has a click chemistry functional group.

In one embodiment, 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)).

In one embodiment, 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.

In one embodiment, the second click chemofunctional group can have any one of the following structures:

.

Here, the wavy line represents an attachment site to another part of the linker. For example, a wavy line may indicate an attachment site with the second anchor unit.

In certain embodiments, the second click chemofunctional group can have the following structure:

.

.

Here, the wavy line represents an attachment site to another part of the linker. For example, a wavy line may indicate an attachment site with the second anchor moiety.

Second Anchor Unit (A ₂ )

In Formula 2, A ₂ is a second anchor unit.

In one embodiment, the second anchor unit is a substituted hydrocarbon chain comprising one or more heteroatoms. In this case, the hetero atom may be each independently selected from N, O, and S. In this case, the substitution is substituted with one or more non-hydrogen substituents, and the non-hydrogen substituents are each independently any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S. can In one embodiment, the second anchor unit may include a polyethylene glycol unit composed of a plurality of ethylene glycol units.

In one embodiment, the second anchor unit is a substituted or unsubstituted C _1-50 alkylene, a substituted or unsubstituted C _1-50 heteroalkylene, -substituted or unsubstituted C _1-20 alkylene-[EG ] _n -, -substituted or unsubstituted C _1-20 heteroalkylene-[EG] _n -, -substituted or unsubstituted C _1-20 alkylene-[EG] _n -substituted or unsubstituted C _{1- 20} Alkylene-, -substituted or unsubstituted C _1-20 Alkylene-[EG] _n -substituted or unsubstituted C _1-20 heteroalkylene-, and -substituted or unsubstituted C _1-20 heteroalkyl [EG] _n -It may be any one selected from substituted or unsubstituted C _1-20 heteroalkylene-. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other. At this time, 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.

In certain embodiments, the second anchor unit can be -substituted C _1-6 heteroalkylene-[EG] _n -substituted C _3-15 heteroalkylene-. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other. At this time, 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.

In certain embodiments, the second anchor unit can be a substituted C _5-30 heteroalkylene. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other.

링커의 구체예Specific examples of linkers

In one embodiment, the linker may have the structure of Formula 2-1:

[Formula 2-1]

H ₂ -A ₂₁ -A ₂₂ -A ₂₃ -B.

In 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 )'.

In the structure of Formula 2-1, -A ₂₁ -A ₂₂ -A ₂₃ - is a second anchor unit.

In this case, A ₂₁ is a bond, -CH ₂ CH ₂ OC(=O)NH-, -CH ₂ CH ₂ C(=O)NH-, -CH ₂ CH ₂ C(=O)-, -CH ₂ OC(=O)NH-, -CH ₂ C(=O)NH-, -CH ₂ C(=O)-, -OC(=O)NH-, -C(=O)NH-, -NH- or -C(=O)-.

In this case, 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 unsubstituted C _1-12 heteroalkylene-. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other. At this time, 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.

In this case, A ₂₃ is a bond, -CH ₂ CH ₂ OC(=O)NH-, -CH ₂ CH ₂ C(=O)NH-, -CH ₂ CH ₂ C(=O)-, -CH ₂ OC(=O)NH-, -CH ₂ C(=O)NH-, -CH ₂ C(=O)-, -OC(=O)NH-, -C(=O)NH-, -NH- or -C(=O)-.

At this time A ₂₁ ,There is no case where both A ₂₂ and A ₂₃ are simultaneously bonded.

In the structure of Formula 2-1, B is a group containing a thiol reactive group. In one embodiment, the thiol reactive group can be a maleimide group or an APN group.

In one embodiment, the linker can have a structure of any one of the following formulas:

[Formula 2-2]

,

[Formula 2-3]

,

[Formula 2-4]

,

[Formula 2-5]

,

[Formula 2-6]

, and

[Formula 2-7]

.

In this case, n may be an integer of 2 or more and 8 or less.

알부민 및 알부민 유닛Albumin and Albumin Unit

알부민 및 알부민 유닛 개괄Overview of Albumin and Albumin Units

As noted above, 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.

In Formula 1, P ₁ is an albumin unit. The albumin unit may be conjugated albumin. The albumin unit may be derived from albumin.

As used herein, the term albumin may be used to include both albumin unconjugated with other compounds and albumin conjugated with other compounds. For example, albumin can be a protein having the sequence of SEQ ID NO: 09. For example, albumin unit P ₁ in Formula 1 may be referred to as albumin.

알부민의 구체예Specific examples of albumin

In one embodiment, the albumin can be mammalian albumin, eg serum albumin. In one embodiment, 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).

In one embodiment, the albumin can be wild type or recombinant human serum albumin. The human serum albumin has a long half-life of 2 weeks or more. This is because 1) it is not easily filtered in the glomerulus due to the electrostatic repulsion of the albumin molecule, and 2) it is degraded in the body due to the recycling action mediated by the neonatal Fc receptor (FcRn) of the endothelium. because it is long

In one embodiment, the albumin can be human serum albumin, wherein the human serum albumin can comprise the amino acid sequence below.

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCAEDCSLVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 09)

In another embodiment, 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:

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQMSTPTLVEVSRTPVNLGKVGVTCCCKHPEAKRMPCAEDYLSVDRLVLNRNHK FSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 10);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSAPTLVEVSRTPNLGKVGVTCCCKHPEAKRMPCAEDYLSVDRLVLNRNHK FSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 11);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCAEDCSLVNRRHH CFSALEVDETYVPKEFNARTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 12);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCAEDCSLVNRRHH CFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 13);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCAEDCSLVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 14);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCAEDCSLVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGYKLVAASQAALGL (SEQ ID NO: 15);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLIEVSRNLGKVGVTETCCCKHPEAKRMPCAEDYLSVDRLVLNRNHK FSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 16);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPDLGKVVTSKCCKHPEAKRMPCAEDSSLVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 17);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCVEKEDCLCCSLVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 18);

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRVNLGKVVTSKMPLCCSLVVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 19); and

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPECKLDELRDEGKASSAKQRLKKLVKLTSCASLQKFARKFSKAWAVRFHTP CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRTPNLGKVVTSKCCKHPEAKRMPCAEDCSLVNRRHH CFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFTAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 20).

In one embodiment, 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. For example, 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. More specifically, a thiol residue of cysteine included in human serum albumin or a variant thereof may react with a thiol-reactive group at one end of the linker. At this time, the cysteine reacting with one end of the linker may be cysteine 34 (Cys 34).

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 구조Structure of Functional Polypeptide Variant-Albumin Conjugates

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 구조 개괄Structural Overview of Functional Polypeptide Variant-Albumin Conjugates

As mentioned above, the functional polypeptide variant-albumin conjugate of the present application has the structure of Formula 1:

[Formula 1]

FPV-[J ₁ -A ₂ -J ₂ -P ₁ ] _a

In 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.

In Formula 1, 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.

In Formula 1, A ₂ is a second anchor unit. In a conjugate, 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.

In Formula 1, 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.

Hereinafter, each of the second anchor unit, the first bonding unit, and the second bonding unit will be described in detail.

제2 앵커 유닛Second Anchor Unit

In Formula 1, A ₂ is a second anchor unit. As described above, the second anchor unit is derived from a linker.

In one embodiment, the second anchor unit is a substituted hydrocarbon chain comprising one or more heteroatoms. In certain embodiments, hydrocarbon chains may consist of 1 to 500 atoms. In this case, the hetero atom may be each independently selected from N, O, and S. In this case, the substitution is substituted with one or more non-hydrogen substituents, and the non-hydrogen substituents are each independently any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S. can In one embodiment, the second anchor unit may include a polyethylene glycol unit composed of a plurality of ethylene glycol units.

In one embodiment, the second anchor unit is a substituted or unsubstituted C _1-50 alkylene, a substituted or unsubstituted C _1-50 heteroalkylene, -substituted or unsubstituted C _1-20 alkylene-[EG ] _n -, -substituted or unsubstituted C _1-20 heteroalkylene-[EG] _n -, -substituted or unsubstituted C _1-20 alkylene-[EG] _n -substituted or unsubstituted C _{1- 20} Alkylene-, -substituted or unsubstituted C _1-20 Alkylene-[EG] _n -substituted or unsubstituted C _1-20 heteroalkylene-, and -substituted or unsubstituted C _1-20 heteroalkyl [EG] _n -It may be any one selected from substituted or unsubstituted C _1-20 heteroalkylene-. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other. In this case, 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.

In certain embodiments, the second anchor unit can be a substituted -C _1-6 heteroalkylene-[EG] _n -substituted C _3-15 heteroalkylene. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other. At this time, 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.

In one embodiment, -A ₂ - can be -A ₂₁ -A ₂₂ -A ₂₃ -.

In this case, 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 heteroalkyl [EG] _n -It may be any one selected from substituted or unsubstituted C _1-12 heteroalkylene-. At this time, the heteroalkylene is one or more heteroatoms selected from the group consisting of -NH-, -O-, -S-, -ON=, -S(=O)-, and -S(=O) ₂ - group, or one or more heteroatoms selected from the group consisting of O, N, and S, and each heteroatom group or heteroatom may be independently selected. At this time, the substitution is substituted with one or more non-hydrogen substituents, wherein the non-hydrogen substituent is any one selected from the group consisting of halogen, C _1-3 alkyl, -NH ₂ , =O, and =S, and the non-hydrogen substituent can be selected independently of each other. At this time, 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.

In one embodiment, the second anchor unit can have any one of the following structures:

In this case, n may be an integer of 2 or more and 8 or less.

At this time, in one embodiment, 3' may be an attachment site with the first bonding unit. In this case, 4' may be an attachment site with the second bonding unit.

At this time, in another embodiment, 3' may be an attachment site with the second bonding unit. In this case, 4' may be an attachment site with the first bonding unit.

제1 접합 유닛1st joining unit

In Formula 1, 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. For example, 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.

In one embodiment, 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. In certain embodiments, the first click chemofunctional group may include any one of tetrazine, triazine, and azide groups. In one embodiment, 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. In certain embodiments, the second click chemofunctional group may include any one of TCO, DBCO, and bicyclononine groups.

In one embodiment, the first bonding unit can have any one of the following structures:

In this case, in one embodiment, 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.

At this time, in another embodiment, 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.

제2 접합 유닛Second bonding unit

In Formula 1, 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. For example, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, the thiol group of albumin can be a Cys 34 thiol group.

In one embodiment, the second bonding unit may have any one of the following structures:

;

; and

.

In this case, 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.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 구체예Specific examples of functional polypeptide variant-albumin conjugates

In the following, specific examples of functional polypeptide variant-albumin conjugates are disclosed.

The functional polypeptide variant-albumin conjugate according to one embodiment of the present application may have a structure of any one of the following formulas:

[Formula 1-1]

,

[Formula 1-2]

, and

[Formula 1-3]

.

Here, FPV is a functional polypeptide variant unit. A functional polypeptide unit may be derived from a functional polypeptide variant. As noted above, a functional polypeptide variant is one in which the functional polypeptide has been modified to include non-natural amino acids. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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.

At this time, P ₁ is an albumin unit. The albumin unit may be derived from albumin. In one embodiment, 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). In one embodiment, 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.

At this time, the S atom adjacent to the albumin unit is derived from albumin. In one embodiment the S atom adjacent to the albumin unit is derived from a thiol group of albumin. In one embodiment, the S atom adjacent to the albumin unit may be derived from a thiol group of a cysteine residue of albumin. In certain embodiments, the S atom adjacent to the albumin unit may be derived from the thiol group of cysteine 34 of human serum albumin.

In this case, 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.

In this case, 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.

기능성 폴리펩티드 변이체 유닛이 아르기닌 디카르복실레이즈 서브유닛 변이체 유닛인 경우When the functional polypeptide variant unit is an arginine decarboxylase subunit variant unit

As described above, the functional polypeptide variant unit may be an arginine decarboxylase subunit variant. In this case, a may be an integer of 1 or more and 4 or less. In one embodiment, an arginine decarboxylase subunit variant may comprise one unnatural amino acid, where a is 1. In one embodiment, an arginine decarboxylase subunit variant may comprise two unnatural amino acids, where a is 1 or 2. In one embodiment, 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. In one embodiment, 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.

기능성 폴리펩티드 변이체 유닛이 아르기닌 디카르복실레이즈 변이체 유닛인 경우When the functional polypeptide variant unit is an arginine decarboxylase variant unit

As described above, 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.

When the functional polypeptide variant unit is an arginine decarboxylase variant unit, a may be an integer of 1 to 20 or less. For example, when the arginine decarboxylase variant includes 10 arginine decarboxylase subunit variants and albumin is conjugated to one arginine decarboxylase subunit variant, a is 1. As another example, when the arginine decarboxylase variant includes 10 arginine decarboxylase subunit variants and one albumin is conjugated to each of the six arginine decarboxylase subunit variants, a is 6. As another example, when 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. In another example, 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. As another example, when the arginine decarboxylase variant contains 10 arginine decarboxylase subunit variants, and two albumins are conjugated to each of the 10 arginine decarboxylase subunit variants, 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.

기능성 폴리펩티드 변이체 유닛이 아르기닌 디카르복실레이즈 서브유닛 변이체의 다이머 유닛인 경우When the functional polypeptide variant unit is a dimer unit of an arginine decarboxylase subunit variant

As described above, arginine decarboxylase subunits and/or arginine decarboxylase subunit variants may form dimers.

When the functional polypeptide variant unit is a dimer unit of an arginine decarboxylase subunit variant, a may be an integer of 1 to 8 or less. For example, when 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. As another example, when 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 is 1 As another example, when the dimer of the arginine decarboxylase subunit variant contains two arginine decarboxylase subunit variants, and two albumins are conjugated to each of the two arginine decarboxylase subunit variants, a is 4 In another example, 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. When conjugated, a is 1.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 제조Preparation of Functional Polypeptide Variant-Albumin Conjugates

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 제조 개괄Overview of Preparation of Functional Polypeptide Variant-Albumin Conjugates

In one embodiment of the present application, a method for producing a functional polypeptide variant-albumin conjugate is provided. 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. Alternatively, 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. Hereinafter, a method for preparing a functional polypeptide variant will be described through an example of an arginine decarboxylase subunit variant.

기능성 폴리펩티드 변이체의 제조 예시 - 아르기닌 디카르복실레이즈 서브유닛 변이체의 제조 Examples of Preparation of Functional Polypeptide Variants - Preparation of Arginine Decarboxylase Subunit Variants

To produce an arginine decarboxylase subunit variant comprising an unnatural amino acid, one or more residues in the amino acid sequence of the wild-type arginine decarboxylase subunit may be changed to an unnatural amino acid. To this end, it is necessary to determine an optimal position that does not possibly affect the structure and function of the native arginine decarboxylase subunit. For example, substitution sites may be selected from those with high solvent accessibility. As another example, the substitution site may be selected from amino acids constituting a random coil. As another example, the substitution site may be selected from among amino acids that are not located at dimer/decamer forming sites. As another example, 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. In one embodiment, in order to create an arginine decarboxylase subunit variant, 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. Specifically, the molecular modeling simulation result may be a scoring result of a Rosetta molecular modeling package.

In one embodiment, the change to the non-natural amino acid may be a substitution of one or more residues of the amino acid sequence of SEQ ID NO: 01. In a specific embodiment, 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. In a specific embodiment, 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. In a specific embodiment, 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. , And any one or more residues selected from the 522nd lysine (Lysine) may be substituted with frTet. In a specific embodiment, 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. Here, it is an arginine decarboxylase subunit derived from E. coli having the amino acid sequence of SEQ ID NO: 01.

Hereinafter, an example of a method for preparing an arginine decarboxylase subunit variant as an example of a functional polypeptide variant is disclosed.

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. Here, 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. As a result, when the exogenous tRNA synthetase is used, 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. In the method for preparing the functional polypeptide 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.

아르기닌 디카르복실레이즈 서브유닛 변이체의 제조 - 아르기닌 디카르복실레이즈 서브유닛 변이체 발현 세포주Preparation of arginine decarboxylase subunit variants - cell lines expressing arginine decarboxylase subunit variants

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. However, when 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.

In one embodiment, 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 .

In one embodiment, the cell line may be one in which a release factor that recognizes a stop codon to terminate translation is inactivated. Specifically, 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.

In one embodiment, 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.ΔA.exp (Addgene, ID: 49018).

아르기닌 디카르복실레이즈 서브유닛 변이체의 제조 - 외래 유래 suppressor tRNAPreparation of arginine decarboxylase subunit variants - exogenous suppressor tRNA

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. As a result, the exogenous suppressor tRNA can recognize the specific stop codon and introduce the non-natural amino acid at the corresponding position.

In one embodiment, 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. Preferably, the suppressor tRNA may recognize an amber codon. For example, 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).

기 아르기닌 디카르복실레이즈 서브유닛 변이체의 제조 - 외래 유래 tRNA 합성효소(synthetase)Preparation of arginine decarboxylase subunit variants - exogenous tRNA synthetase

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. In one embodiment, 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. In one embodiment, the tRNA synthetase may have a function of linking AzF to the exogenous suppressor tRNA. In one embodiment, 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). Preferably, the tRNA synthetase may be the C11 variant of MjTyrRS.

아르기닌 디카르복실레이즈 서브유닛 변이체의 제조 - 직교적 tRNA/합성효소 페어 (Orthogonal tRNA/syntetase pair)Preparation of arginine decarboxylase subunit variants - orthogonal tRNA/synthetase pair (Orthogonal tRNA/syntetase pair)

In the present specification, 1) an exogenous suppressor tRNA that specifically reacts only with an exogenous tRNA synthetase, and 2) the exogenous tRNA synthetase is collectively referred to as an orthogonal tRNA/synthetase pair. In the method for preparing the arginine decarboxylase subunit variant disclosed herein, 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. In one embodiment, 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. Specifically, 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. Hereinafter, vectors encoding functional polypeptide variants will be described.

기능성 폴리펩티드 변이체를 암호화하는 벡터Vectors encoding functional polypeptide variants

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. In one embodiment, in the vector encoding the functional polypeptide variant, among the functional polypeptide variant sequences, 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. there is. For example, 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. In another example, the stop codon may be selected from among 5'-TAG-3', 5'-TAA-3', and 5'-TGA-3'. In one embodiment, the vector encoding the functional polypeptide variant may be codon-optimized for the expression cell line. For example, a vector encoding the functional polypeptide variant may be E. coli codon optimized.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 제조 예시 (1) Example of Preparation of Functional Polypeptide Variant-Albumin Conjugates (1)

As described above, functional polypeptide variant-albumin conjugates can be prepared through the following methods:

1) the linker and albumin react to produce an albumin-linker conjugate; and

2) A functional polypeptide variant-albumin conjugate is prepared by reacting the albumin-linker conjugate with the functional polypeptide variant.

In the following, 1) the reaction between the linker and albumin is described.

The linker may be connected through a specific amino acid residue of albumin.

In one embodiment, it can be linked to albumin via a cysteine residue, which linkage can be formed by reaction of a thiol reactive group of a linker with a cysteine residue of albumin. In a specific embodiment, 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. In a specific example, an albumin-linker conjugate may be prepared by reacting a thiol group of cysteine 34 (Cys 34) of human serum albumin (HSA) with a thiol-reactive group of a linker.

Hereinafter, 2) the reaction between the albumin-linker conjugate and the functional polypeptide variant will be described.

From the above reaction, 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. In one embodiment, 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. In a specific embodiment, 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. In certain embodiments, 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.

In one embodiment, 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.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 제조 예시 (2) Example of Preparation of Functional Polypeptide Variant-Albumin Conjugates (2)

1) the linker and the functional polypeptide variant are reacted to produce a functional polypeptide variant-linker conjugate; and

2) A functional polypeptide variant-linker conjugate reacts with albumin to prepare a functional polypeptide variant-albumin conjugate.

In the following, 1) the reaction of the linker and the functional polypeptide will be described.

In one embodiment, 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. In one embodiment, 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.

Hereinafter, 2) the reaction between the functional polypeptide variant-linker conjugate and albumin will be described.

In one embodiment, it can be linked to albumin via a cysteine residue, which linkage can be formed by reaction of a thiol reactive group of a functional polypeptide variant-linker conjugate with a cysteine residue of albumin. In a specific example, 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). In a specific example, 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). can

A functional polypeptide variant-linker conjugate reacts with albumin to produce a functional polypeptide variant-albumin conjugate.

알부민-링커 컨쥬게이트Albumin-Linker Conjugates

As described above, 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.

In one embodiment, the albumin-linker conjugate can have the structure of Formula 4:

[Formula 4]

H ₂ -A ₂ -J ₂ -P ₁

In 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.

In Formula 4, A ₂ is a second anchor unit. In the albumin-linker conjugate, 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'.

In Formula 4, J ₂ is a second junction unit. As described above, the second junction unit has a structure formed by reaction of a thiol reactive group with a thiol group. For example, 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. In one embodiment, 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'.

In Formula 4, 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'.

기능성 폴리펩티드 변이체-링커 컨쥬게이트Functional Polypeptide Variant-Linker Conjugates

As described above, 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.

In one embodiment, the functional polypeptide variant-linker conjugate may have the structure of Formula 5:

[Formula 5]

FPV-[J ₁ -A ₂ -J ₂ -B] _a

In 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.

In Formula 5, 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'.

In Formula 5, 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'.

In Formula 5, 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'.

In Formula 5, B is a thiol reactive group. In one embodiment, the thiol reactive group can be a maleimide group. In one embodiment, 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'.

In Formula 5, 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.

기능성 폴리펩티드 변이체-알부민 컨쥬게이트의 용도Uses of Functional Polypeptide Variant-Albumin Conjugates

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. Here, the target disease may be a tumor. More specifically, the target disease may be an arginine auxotrophic tumor. For example, 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.

In another embodiment of the present application, a composition for diagnosis comprising a functional polypeptide variant-albumin conjugate as an active ingredient may be provided.

As used herein, the term "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.

As used herein, the term "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.

In addition, if necessary, the pharmaceutical composition of the present application may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , a binder or a lubricant may be additionally added and used.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present application to a patient by any suitable method, and the route of administration of the composition of the present application can be oral or parenteral as long as it can reach the target tissue. It can be administered via any route.

The pharmaceutical 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.

In order to formulate the pharmaceutical composition of the present application for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate and the like; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be used, and sweeteners, aromatics, syrups, and the like may also be used. Furthermore, in the case of capsules, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.

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.

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.

When the pharmaceutical composition of the present application is formulated as an injection solution, the pharmaceutical composition of the present application is mixed in water together with a stabilizer or buffer to prepare a solution or suspension, which is used for unit administration in an ampoule or vial can be formulated.

When the pharmaceutical composition of the present application is formulated as an aerosol, a propellant or the like may be blended with additives so that the water-dispersed concentrate or wet powder is dispersed.

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. For example, the daily dose of the pharmaceutical composition of the present application is 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and may be administered once or several times a day.

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.

As a parenteral administration method of the pharmaceutical composition of the present application, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, etc. may be used, and a method of applying, spraying, or inhaling the composition to the diseased area It can also be used, but is not limited thereto.

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.

기능성 폴리펩티드 변이체의 용도Uses of Functional Polypeptide Variants

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. In one embodiment, 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.

As described above, 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. For example, 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.

The pharmaceutical composition of the present application may further include a pharmaceutically acceptable carrier in addition to containing the functional polypeptide variant as an active ingredient.

Hereinafter, the invention provided by the present application will be described in more detail through experimental examples or examples. These experimental examples are only for exemplifying the contents disclosed by this application, and it is to those skilled in the art that the scope of the contents disclosed by this specification is not construed as being limited by these experimental examples. It will be self-evident.

실험예Experimental example

재료ingredient

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).

실험예 I. 야생형 아르기닌 디카르복실레이즈(Arginine decarboxylase; ADC) 효과 확인Experimental Example I. Wild-type arginine decarboxylase (Arginine decarboxylase; ADC) effect confirmation

1. E. coli 유래 ADC 제조 1. Preparation of E. coli-derived ADC

To prepare E. coli-derived ADC, first, E. coli genomic DNA (gDNA) 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:

MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRTDMGINERTSVRTIMGSLLLDHTGAFGESEKYAWARWSDN DVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPTVFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGNSGLSDKFTA WNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRHGIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNEAYSGLPVAEVTPREAYNAVDNNVLRQELVSIENGPGDKLSSPGEPYSLPGDKLSSPYN QSWDHHFPGFEHETEGTEIIDGIYHVMCVKAHHHHHH (SEQ ID NO: 23).

To introduce the PCR-amplified ADC gene into an expression vector, 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 μg/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.

To obtain wild-type ADC through E. coli, 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 μg/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 μg/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 μg/mL ampicillin was added, and shaking culture was performed at 37 ° C and 200 rpm. . When the optical density at 600 nm reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to enable transcription of ADC WT in transformed E. coli. Put in and proceed with 200 rpm shaking incubation for 5 hours at 37 ° C. After incubation, E. coli was centrifuged at 5,000 x g for 10 minutes at 4°C, and the LB medium was discarded and the cell pellet was stored at -80 degrees.

To obtain WT ADC produced in E. coli, 10 mL of lysis buffer (50 mM sodium phosphate, 0.3 M NaCl, 10 mM imidazole, pH 7.4) was added to 0.5 g of cell pellet, followed by resuspension. After that, sonication (1 sec on/2sec off, total 10 min, 32% amplitude, 4 °C) was performed. Centrifugation (15,000 x g, 30 min, 4 ℃) was performed on the obtained cell lysate, and the obtained supernatant was transferred to a new 50 mL conical tube. After adding 500 μL of Ni-NTA resin to the supernatant, shaking incubation was performed at 15 ° C. for 30 min while blocking light. After that, after pouring the cell lysate into a polypropylene column, 15 mL of washing buffer (50 mM sodium phosphate, 0.3 M NaCl, 20 mM imidazole, pH 7.4) was poured and the elution buffer ( 5 mL of elution buffer) (50 mM sodium phosphate, 0.3 M NaCl, 250 mM imidazole, pH 7.4) was flowed to receive the eluent sample passing through the column. For further analysis, buffer exchange was performed using a PD-10 desalting column filled with the buffer required for the experiment for Eluent. The prepared ADC WT was confirmed through SDS-PAGE (FIG. 01). In FIG. 01, BI indicates before induction, AI indicates after induction, and P indicates purified. As a result of the analysis, 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.

2. 야생형의 E. coli 유래 ADC (ADC_WT)의 안정성 확인 2. Stability confirmation of wild-type E. coli-derived ADC (ADC_WT)

To confirm the stability of 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. To measure the activity of ADC WT, 0.1 mg/mL ADC WT was mixed with 1 mM arginine and 200 μM 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. "Simplified rapid procedure for determination of agmatine and other guanidino-containing compounds." The activity of ADC WT was confirmed by partially modifying the method disclosed in Analytical Chemistry 43.11 (1971): 1475-1479'. After the reaction, a 10% KOH solution saturated with NaCl in the same volume as the reaction mixture for the enzymatic reaction of ADC WT was added. Thereafter, n-butanol was added by 1/3 x volume of the reaction mixture for the enzymatic reaction of ADC WT. After vortexing for 2 minutes, centrifugation was performed at 1,000 xg for 1 minute. After centrifugation, the upper layer of n-butanol was transferred to a clean tube. Then, diacetyl solutions A and B (solution A: 0.24% of 2,3-butanidiol in distilled water; solution B: 40 g/L 1-napthtol in solution containing 0.1M NaCl and 0.5 M NaOH) were prepared. After mixing Solution A and B at 2:3 (v/v), vortexing was performed for 2 minutes with the same volume as n-butanol previously obtained. Thereafter, by centrifugation, the color-changed n-butanol in the upper layer was transferred to a 96-well plate, and optical density 530nm (OD ₅₃₀ ) and 600nm (OD ₆₀₀ ) values were obtained. The activity was compared with the relative activity by OD ₅₃₀ -OD ₆₀₀ values. As a result of the analysis, it was confirmed that ADC WT maintained stability for 24 hours (FIG. 02). Culture conditions are potassium phosphate buffer (pH 7.4), 37°C, and analysis conditions are sodium acetate buffer (pH 5.2).

3. ADC_WT의 대사 억제 효과 확인 (3. Confirmation of metabolic inhibitory effect of ADC_WT ( in vitroin vitro ))

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 μm 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.

It was confirmed that the intensity of the MTT assay was low in the ADC-treated group (FIG. 03). Since the metabolism of mitochondria is lower than that of the group not treated with ADC, the intensity of the MTT assay is low, which can be interpreted as a decrease in metabolic activity.

4. 세포주 별 ADC_WT의 대사 억제 효과 확인 (4. Confirmation of metabolic inhibitory effect of ADC_WT by cell line ( in vitroin vitro ) )

Cells were seeded in a 96-well plate at 3000 cells/well. A pH 6.4 medium (DMEM, 15% FBS, 2% AA) was used. The ADC solution was filtered through a 0.2 μm 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, MTT assay was performed to measure the metabolic activity of each cell line. Metabolic activity was measured in a method similar to that described in "3. Confirmation of metabolic inhibitory effect of ADC_WT ( in vitro )".

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.

5. ADC_WT의 5. ADC_WT in vivoin vivo 항암 활성 확인 Confirmation of anticancer activity

The in vivo tumor suppression effect of ADC was confirmed. Mia-PaCa-2 tumor cells (a cell line derived from a pancreatic tumor) were subcutaneously injected into the legs of mice to prepare mice with tumors. Experiments were performed on a total of 6 mice. ADC and/or gemcitabine were administered to mice once a week at 0.2 U/mouse (0.03 U/mg - 30 mg/ml, U = concentration of enzyme that breaks down 1 μmol of arginine per minute at 37 °C), intravenously. It was administered by intravenous injection. Here, 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)

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.

실험예 II. 비천연아미노산 AzF를 포함하는 아르기닌 디카르복실레이즈 (Arginine decarboxylase; ADC) 변이체 및 이를 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트의 제조 및 효과 확인 Experimental Example II. Arginine decarboxylase (ADC) variants containing the non-natural amino acid AzF and preparation and effect confirmation of arginine decarboxylase variant-albumin conjugates prepared using the same

1. 비천연아미노산 (AzF) 치환 위치 선정 1. Selection of non-natural amino acid (AzF) substitution sites

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. .

2. 아르기닌 디카르복실레이즈 변이체(ADC_AzF) 제조 2. Preparation of arginine decarboxylase variant (ADC_AzF)

For 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. The primers used for this are as follows. Primers are initiated in the 5' to 3' direction.

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).

For region-specific insertion of AzF into the ADC, each mutant plasmid was transformed into C321ΔA.exp [pEVOL-pAzF] competent cells, followed by C321ΔAexp [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 μg/mL) and chloroamphenicol (25 μg/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 (ADC_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. For cultured cells and purified ADC variants, 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.

3. 아르기닌 디카르복실레이즈 변이체에의 위치 특이적 형광 다이 라벨링3. Site-Specific Fluorescent Dye Labeling of Arginine Decarboxylase Variants

Purified ADC_WT and ADC_AzF (ADC_Q488AzF and ADC_K522AzF, respectively) 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). 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 . Specifically, the bands shown in the sample of FIG. 09 correspond to subunits and subunits labeled with fluorescent dyes. In FIG. 09, 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 (+).

4. 아르기닌 디카르복실레이즈 변이체(ADC_AzF) 효소 활성 확인4. Confirmation of arginine decarboxylase variant (ADC_AzF) enzyme activity

In order to confirm whether the enzyme activity according to pH was affected by the introduction of AzF, the enzyme activity was confirmed using ADC_WT as a control. ADC_WT and ADC_AzF variants at 0.1 mg/mL were reacted with 1 mM arginine and 200 μM 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.

10 shows the result of confirming the enzyme activity of ADC_AzF. It can be seen that the ADC_AzF variants (ADC_Q488AzF and ADC_K522AzF) do not have lower enzymatic activity than ADC_WT.

5. 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트 (ADC-HSA 컨쥬게이트) 제조 및 확인 5. Preparation and confirmation of arginine decarboxylase variant-albumin conjugate (ADC-HSA conjugate)

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.

The structure of the DBCO-PEG4-Maleimide linker is as follows:

.

An albumin-linker conjugate, HSA-PEG4-DBCO, was prepared by reacting 50 μM of albumin with 200 μM 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.

25 μM of an AzF-substituted arginine decarboxylase variant (ADC_Q488AzF or ADC_K522AzF) was reacted with 100 μM of HSA-PEG4-DBCO in PBS (pH 7.4) for 5 hours at 23°C.

Afterwards, SDS-PAGE analysis was performed to confirm that the arginine decarboxylase variant-albumin conjugate was produced. Analysis results ADC(Q488AzF)-HSA conjugate (arginine decarboxylase variant prepared using ADC_Q488AzF-albumin conjugate) and ADC(K522AzF)-HSA conjugate (arginine decarboxylase variant prepared using ADC_K522AzF) -Albumin conjugate) were identified respectively (Fig. 11 to Fig. 12). HSA-PEG4-DBCO has a molecular weight of about 66.5 kDa, ADC (arginine decarboxylase subunit variant) has a molecular weight of about 82 kDa, and arginine decarboxylase subunit variant-albumin conjugate has a molecular weight of about 148.5 kDa. has a molecular weight

실험예 III. 비천연아미노산 frTet을 포함하는 아르기닌 디카르복실레이즈 (Arginine decarboxylase; ADC) 변이체 및 이를 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트의 제조 및 효과 확인Experimental Example III. Arginine decarboxylase (ADC) variant containing non-natural amino acid frTet and preparation and effect confirmation of arginine decarboxylase variant-albumin conjugate prepared using the same

1. 비천연아미노산 (frTet) 치환 위치 선정 1. Selection of substitution sites for non-natural amino acids (frTet)

Screening of frTet substitution sites was performed using the molecular modularization software PyRosetta (Python-based Rosetta molecular modeling package), which performs point mutagenesis and energy scoring functions based on E. coli-derived ADC structures. In the wild-type (WT) ADC, the amino acid residue at the desired position to be substituted was replaced with Y (tyrosine) or W (tryptophan), and then the total atomic energy of the protein was calculated to find candidates for the frTet substitution site. The energetic function of PyRosetta is based on Anfinsen's hypothesis that native protein conformations represent unique, low-energy thermodynamically stable conformations. 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.

Furthermore, 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.

Using Rosetta design (point mutation, energy score function), when substituted with Tyrosin and Tryptophan, which are natural amino acids most similar in structure/solubility to non-natural amino acids, the top 6 variants that did not differ from the wild-type protein score ( N85, N245, K312, Q488, K522, G657) were selected. In the case of T39, it is located on the proximal surface where dimer and/or decamer assembly occurs, but it was selected as a candidate group because the score difference with ADC_WT was small.

2. 아르기닌 디카르복실레이즈 변이체(ADC_frTet) 제조 2. Preparation of arginine decarboxylase variant (ADC_frTet)

E. coli 유래 아르기닌 디카르복실레이즈 변이체(ADC_frTet) 발현을 위한 플라스미드 제조Construction of plasmid for expression of E. coli-derived arginine decarboxylase variant (ADC_frTet)

To prepare ADC_frTet, PCR was performed using pQE80_ADC-WT as a template. First, SpeI was introduced into the sfGFP coding region of the pBAD_sfGFP plasmid through PCR. Thereafter, 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. The primers used for this are as follows. The primers below start in the 5' to 3' direction.

1) pBAD_sfGFP_spel_F: TAAACAGTTCTTCACCTTTGCTAAACTAGTTTAATTCCTCCTGTTAGCCCAAAAAACGG (SEQ ID NO: 28)

2) pBAD_sfGFP_spel_R: CCGTTTTTTGGGCTAACAGGAGGAATTAAACTAGTTTAGCAAAGGTGAAGAACTGTTTA (SEQ ID NO: 29)

3) pBAD_ADC_F: AACTACTAGTATGAAAGTATTAATTGTTGAAAGCGAG (SEQ ID NO: 30)

4) pBAD_ADC_R: AGATCTCGAGTTAATGGTGATGGTGATGGTG (SEQ ID NO: 31)

5) pBAD_ADC_T39Amb_F: GAATGGCAAAACCATCATCAAAGGACTAGGATTTAATCACGGTAACATTTTGC (SEQ ID NO: 32)

6) pBAD_ADC_T39Amb_R: GCAAAATGTTACCGTGATTAAATCCTAGTCCTTTGATGATGGTTTTGCCATTC (SEQ ID NO: 33)

7) pBAD_ADC_N85Amb_F: GAAGACCGGCACCTATTGTTGGCGCTCATGAAGCTTA (SEQ ID NO: 34)

8) pBAD_ADC_N85Amb_R: TAAGCTTCATGAGCGCCAACAATAGGTGCCGGTCTTC (SEQ ID NO: 35)

9) pBAD_ADC_N245Amb_F: CAACGACCACGACATCCTAATCGGTCATGCAAGCC (SEQ ID NO: 36)

10) pBAD_ADC_N245Amb_R: GGCTTGCATGACCGATTAGGATGTCGTGGTCGTTG (SEQ ID NO: 37)

11) pBAD_ADC_K312Amb_F: CCACGCAGTAAGACGGCTATTGCCCGGCTTTGTCTTTG (SEQ ID NO: 38)

12) pBAD_ADC_K312Amb_R: CAAAGACAAAGCCGGGCAATAGCCGTCTTACTGCGTGG (SEQ ID NO: 39)

13) pBAD_ADC_Q488Amb_F: GGTTTTGCCGGTCTATGGGTCGGTGACGACTTCTTTGT (SEQ ID NO: 40)

14) pBAD_ADC_Q488Amb_R: ACAAAGAAGTCGTCACCGACCCATAGACCGGCAAAACC (SEQ ID NO: 41)

15) pBAD_ADC_K522Amb_F: CCAGTTATCCGGAATATCCTAGAAGCCGTGCCAGCTTTC (SEQ ID NO: 42)

16) pBAD_ADC_K522Amb_R: GAAAGCTGGCACGGCTTCTAGGATATTCCGGATAACTGG (SEQ ID NO: 43)

17) pBAD_ADC_G657Amb_F: CTTCCGCCACCGGCAGCTAGGAATAGGCTTCGTTC (SEQ ID NO: 44)

18) pBAD_ADC_G657Amb_R: GAACGAAGCCTATTCCTAGCTGCCGGTGGCGGAAG (SEQ ID NO: 45)

ADC_frTet 발현 및 정제ADC_frTet expression and purification

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.

For region-specific insertion of frTet into the ADC, each mutant plasmid was transformed into C321ΔA.exp [pDule C11RS] competent cells, generating C321ΔAexp [pDule C11RS] [pBAD_ADC variant] E.coli cells. did. Here, 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 μg/mL) and tetracycline (10 μg/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 (ADC_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. For cultured cells and purified ADC variants, 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.

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. Among the 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.

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). 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_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. In FIG. 15, ADC_T39, ADC_N85, ADC_K312, ADC_Q488, and ADC_K522 represent ADC_T39frTet, ADC_N85frTet, ADC_K312frTet, ADC_Q488frTet, and ADC_K522frTet, respectively. In FIG. 15, 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.

4. 아르기닌 디카르복실레이즈 변이체의 효소 활성 확인4. Confirmation of enzyme activity of arginine decarboxylase variants

In order to confirm whether the enzyme activity according to pH was affected by the introduction of frTet, the enzyme activity was confirmed using ADC_WT as a control. ADC_WT or ADC_frTet variant at 0.1 mg/mL was reacted with 1 mM arginine and 200 μM 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.

16 shows the results of enzyme activity analysis of wild-type ADC and ADC variants. In FIG. 16, ADC_T39, ADC_N85, ADC_K312, ADC_Q488, and ADC_K522 represent ADC_T39frTet, ADC_N85frTet, ADC_K312frTet, ADC_Q488frTet, and ADC_K522frTet, respectively.

5. 아르기닌 디카르복실레이즈 변이체의 IEDDA rate 평가5. IEDDA rate evaluation of arginine decarboxylase variants

The inverse-electron-demand Diels-Alder (IEDDA) reaction rate of ADC_frTet variants was evaluated. For the experiment, 25 µM ADC_WT or ADC_frTet (ADC_K312frTet, ADC_Q488frTet, ADC_K522frTet) was incubated in 100 µM 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. In addition, it was confirmed that 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.

6. ADC_K312frTet을 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트 제조 및 확인6. Preparation and confirmation of arginine decarboxylase variant-albumin conjugate prepared using ADC_K312frTet

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, was prepared by reacting 50 μM albumin and 200 μM 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.

25 μM of an arginine decarboxylase variant in which K312 is substituted with frTet (ADC_K312frTet) was reacted with 100 μM of HSA-PEG4-TCO in sodium acetate buffer (0.2M, pH 5.2) at 23°C for 5 hours. The reaction mixture was subjected to vivaspin (MWCO 10 kDa) and size exclusion chromatography using a Superdex ^TM 200 Increase 10/300GL column to obtain an arginine decarboxylase variant-albumin conjugate, ADC(K312frTet)-HSA conjugate. was purified (Flow rate: 0.1mL/min; buffer: 0.2M sodium acetate (pH 5.2)).

Size exclusion chromatography results are shown in FIG. 18 . It is confirmed that ADC(K312frTet)-HSA conjugates were prepared in yields of 95.3% and 92.5%.

PAGE를 통한 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트 제조 확인Confirmation of arginine decarboxylase variant-albumin conjugate production through PAGE

Three fractions (F1 to F3) could be identified through size exclusion chromatography, and each sample was collected and analyzed by SDS-PAGE. As a result of the analysis, ADC(K312frTet)-HSA conjugate and protein bands corresponding to ADC_K312frTet were identified in F1 (FIG. 19). As a result of checking the relative ratio by dividing the intensity of the band by the molecular weight of the protein, it was analyzed that the ADC (K312frTet)-HSA conjugate was present at more than 80% compared to the total ADC. It is assumed that the HSA-TCO conjugation occurred quickly because the IEDDA reaction rate of the ADC_K312frTet mutant was fast.

7. ADC_K312frTet을 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트의 효소 활성 7. Enzyme activity of arginine decarboxylase variant-albumin conjugate prepared using ADC_K312frTet

7.1. ADCv(K312frTet)-HSA 컨쥬게이트의 효소 활성 확인7.1. Enzymatic activity confirmation of ADCv(K312frTet)-HSA conjugate

Enzymatic activities of ADC_K312HSA obtained through size exclusion chromatography, ADC_WT and ADC_K312frTet according to pH were compared. Enzyme activity was confirmed under the same conditions as those of the method described in "2. Confirmation of stability of wild-type E. coli-derived ADC (ADC_WT)" of Experimental Example I.

Experimental results for enzyme activity are shown in FIG. 20 . As a result of the experiment, ADC_K312frTet and ADC(K312frTet)-HSA conjugate showed similar enzymatic activity to ADC_WT. Specifically, ADC_K312frTet and ADC(K312frTet)-HSA conjugates showed 95.3% and 90.8% relative enzymatic activity compared to ADC_WT, respectively. Through this, it was confirmed that the enzyme activity was maintained even after HSA conjugation.

8. ADC_K312frTet을 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트의 약동학(pharmacokinetics) 분석 8. Analysis of pharmacokinetics of arginine decarboxylase variant-albumin conjugate prepared using ADC_K312frTet

For the PK analysis of ADC_WT and ADC_K312HSA, 8-week-old Balb/c (female) mice were prepared (n=5 for each group). Thereafter, 4 nmol of ADC_WT and ADC(K312frTet)-HSA conjugate based on monomeric ADC (ADC subunit) were injected intravenously. Thereafter, blood was collected at predetermined time points and centrifuged at 2,500 rpm for 5 minutes to obtain serum. The obtained serum was stored at -20 ° C until analysis. To compare the enzyme activity of ADC in serum, 10 μL of serum was mixed with 290 μL of a solution (1 mM arginine and 200 μM PLP in 0.2 M sodium acetate buffer (pH 5.2)) and the reaction was performed to confirm the enzyme activity of ADC. did The reaction proceeded at 37 °C for 30 min. Thereafter, the resulting agmatine (arginine degradation product by ADC) was quantified by the method described in "2. Confirmation of stability of wild-type E. coli-derived ADC (ADC_WT)" in the table of contents of Experimental Example I. After that, the enzyme activity according to each time was relatively compared. 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.

9. ADC_T39frTet을 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트 제조 및 확인9. Preparation and confirmation of arginine decarboxylase variant-albumin conjugate prepared using ADC_T39frTet

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 μM albumin and 200 μM 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.

25 μM of an arginine decarboxylase variant in which T39 is substituted with frTet (ADC_T39frTet) was prepared in 6.2. It was reacted at 23°C for 5 hours in sodium acetate buffer (0.2M, pH 5.2) with HSA-PEG4-TCO 100μM prepared by ADC(T39frTet)-HSA conjugate was purified from the reaction mixture by size exclusion chromatography using vivaspin (MWCO 10 kDa) and a Superdex ^TM 200 Increase 10/300GL column (Flow rate: 0.1 mL/min; buffer: 0.2M sodium acetate (pH 5.2)). Chromatography results are shown in FIG. 22 .

PAGE를 통한 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트 제조 확인 Confirmation of arginine decarboxylase variant-albumin conjugate production through PAGE

Three fractions (F1 to F3) could be identified through size exclusion chromatography, and each sample was collected and analyzed by SDS-PAGE. As a result of the analysis, ADC(T39frTet)-HSA conjugate and protein bands corresponding to ADC_T39frTet were confirmed in F1 (FIG. 23). As a result of checking the relative ratio by dividing the intensity of the band by the molecular weight of the protein, it was analyzed that the ADC(T39frTet)-HSA conjugate was present at about 17.3% compared to the total ADC.

10. ADC_T39frTet을 이용하여 제조된 아르기닌 디카르복실레이즈 변이체-알부민 컨쥬게이트의 효소 활성 10. Enzyme activity of arginine decarboxylase variant-albumin conjugate prepared using ADC_T39frTet

Enzymatic activities of ADC(T39frTet)-HSA conjugate, ADC_WT, and ADC_T39frTet, which are arginine decarboxylase variant-albumin conjugates obtained through size exclusion chromatography, were compared. 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 .

Claims

A compound having the structure of Formula 1 below:

[Formula 1]

FPV-[J 1 -A 2 -J 2 -P 1 ] a ,

At this time:

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;

In this case, 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,

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;

In this case, the heteroatoms are each independently selected from N, O, and S, wherein the substitution is substituted with one or more non-hydrogen substituents, and the non-hydrogen substituents are, each independently, halogen, C 1-3 Any one selected from the group consisting of alkyl, -NH 2 , =O, and =S,

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;
According to claim 1,

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,

compound.
According to claim 2,

The arginine decarboxylase subunit variant is 39th threonine, 85th asparagine, 245th asparagine, 312th lysine, 488th glutamine of the amino acid sequence of SEQ ID NO: 01 A compound having an amino acid sequence having at least 90% sequence identity with a sequence in which any one or more residues selected from (Glutamine), lysine at position 522, and glycine at position 657 are substituted with non-natural amino acids. .
According to claim 2,

The arginine decarboxylase subunit variant has an amino acid sequence of any one of the amino acid sequences of SEQ ID NO: 02 to SEQ ID NO: 08, and amino acid sequences having 90% or more sequence identity therewith.
According to claim 1,

Wherein the first click chemical functional group comprises a tetrazine group or an azide group.
According to claim 1,

Wherein the non-natural amino acid is frTet or AzF.
According to claim 1,

Wherein the second click chemical functional group comprises any one group selected from a trans cyclooctyne (TCO) group, a DBCO group, and a bicyclononine group.
According to claim 1,

Wherein the second click chemofunctional group comprises a trans cyclooctyne (TCO) group.
According to claim 1,

A compound wherein the second junction unit has the following structure:

, or
,

wherein in the above structure, the S atom is derived from albumin.
According to claim 1,

A compound wherein the second junction unit has the following structure:

,

wherein in the above structure, the S atom is derived from albumin.
According to claim 1,

The second anchor unit is -A 21 -A 22 -A 23 -,

At this time:

A 21 is a bond, -CH 2 CH 2 OC(=O)NH-, -CH 2 CH 2 C(=O)NH-, -CH 2 CH 2 C(=O)-, -CH 2 OC (=O)NH-, -CH 2 C(=O)NH-, -CH 2 C(=O)-, -OC(=O)NH-, -C(=O)NH-, -NH- or -C(=O)-,

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-;

In this case, EG is an ethylene glycol unit, and 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,

In this case, the heteroalkylene is each independently selected from N, O, and S,

In this case, the substitution is substituted with one or more non-hydrogen substituents, and the non-hydrogen substituents are each independently any one selected from the group consisting of halogen, C 1-3 alkyl, -NH 2 , =O, and =S,

A 23 is a bond, -CH 2 CH 2 OC(=O)NH-, -CH 2 CH 2 C(=O)NH-, -CH 2 CH 2 C(=O)-, -CH 2 OC (=O)NH-, -CH 2 C(=O)NH-, -CH 2 C(=O)-, -OC(=O)NH-, -C(=O)NH-, -NH- or -C(=O)-,

At this time, A 21 , A 22 , and A 23 When both bond (bond) at the same time does not exist,

compound.
According to claim 1,

A compound wherein the second anchor unit has any one of the following structures:

;

; and

,

In this case, n is an integer of 2 or more and 8 or less,

In this case, 3' is an attachment site with the first bonding unit, and 4' is an attachment site with the second bonding unit.
According to claim 1,

The albumin has an amino acid sequence of any one of the amino acid sequences of SEQ ID NO: 09 to SEQ ID NO: 20, a compound.
A pharmaceutical composition for treating arginine auxotrophic tumors, comprising the compound of claim 1 .
According to claim 14,

The arginine auxotrophic tumor is melanoma (malignant melanoma), liver cancer (liver cancer), hepatocellular carcinoma (HCC), prostate cancer (prostate cancer), pancreatic cancer (pancreatic cancer), breast cancer (breast cancer), mammary gland ( mammary gland cancer, lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (GBM) ), acute myeloid leukemia (acute myeloid leukemia; AML), and primary and relapsed lymphoma (primary and relapsed lymphomas) any one selected from, the pharmaceutical composition.
Threonine at position 39, asparagine at position 85, asparagine at position 245, lysine at position 312, glutamine at position 488, and lysine at position 522 of the amino acid sequence of SEQ ID NO: 01 , And an arginine decarboxylase subunit variant having a sequence in which any one or more residues selected from glycine at position 657 are substituted with a non-natural amino acid, or a sequence having 90% or more sequence identity therewith.
According to claim 16,

An arginine decarboxylase subunit mutant having a sequence in which threonine at position 39 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.
According to claim 16,

An arginine decarboxylase subunit variant having 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 thereto.
According to claim 16,

The unnatural amino acid is frTet, an arginine decarboxylase subunit variant.
According to claim 16,

The arginine decarboxylase subunit variant, wherein the unnatural amino acid is AzF.
According to claim 16,

An arginine decarboxylase subunit variant having any one of the amino acid sequence of any one of SEQ ID NOs: 02 to SEQ ID NO: 08 and an amino acid sequence having 90% or more sequence identity therewith.
A pharmaceutical composition for treating arginine auxotrophic tumors, comprising the arginine decarboxylase subunit variant of claim 16.
The method of claim 22,

The arginine auxotrophic tumor is melanoma (malignant melanoma), liver cancer (liver cancer), hepatocellular carcinoma (HCC), prostate cancer (prostate cancer), pancreatic cancer (pancreatic cancer), breast cancer (breast cancer), mammary gland ( mammary gland cancer, lung cancer, small cell lung cancer, malignant pleural mesothelioma, head and neck squamous cell carcinoma, glioblastoma multiforme (GBM) ), acute myeloid leukemia (acute myeloid leukemia; AML), and primary and relapsed lymphoma (primary and relapsed lymphomas) any one selected from, the pharmaceutical composition.
Methods of treating arginine auxotrophic tumors in a subject comprising:

Administering a composition comprising the compound of claim 1 or the arginine decarboxylase subunit variant of claim 16 to a subject.
Use of a composition comprising the compound of claim 1 or the arginine decarboxylase subunit variant of claim 16 for the treatment of an arginine auxotrophic tumor in a subject.