Arginine deiminase mutants, covalent dimers and complexes thereof and uses

Abstract

The present invention provides arginine deiminase mutants, covalent dimers, and complexes thereof, as well as uses thereof. Mutation of amino acid sites on the provided arginine deiminase mutant arginine deiminase results in the formation of covalent disulfide bonds between the arginine deiminase subunits. Covalent complexes and their use in the field of cancer treatment are also provided. Site-specific mutation of amino acid sites promotes the formation of covalent disulfide bonds between the subunits of homologous non-covalent dimeric arginine deiminase, making the structure of the formed dimer more stable. Furthermore, by reducing the exposure of antigen epitopes at the interaction interface, immunogenicity in the body can be reduced, resulting in better antitumor effects.

Description

[Technical Field]

[0001] The present disclosure relates to the field of biopharmaceuticals, in particular to arginine deiminase mutants, covalent dimers and conjugates thereof and uses thereof, and in particular to the use of conjugates, especially pegylated covalent dimer arginine deiminase mutants, in the preparation of medicaments for arginine depletion, thereby reducing arginine in the body, extending half-life in the body and reducing immunogenicity. [Background technology]

[0002] Amino acid deprivation therapy can be an effective treatment for some metabolic disorders and certain cancers. To date, well-known commercially available drugs for this treatment include the use of L-covalent dimeric arginine deiminase mutants to reduce blood asparagine levels and the use of phenylalanine lyase to reduce blood phenylalanine (Phe) levels in adult patients with phenylketonuria (PKU). Arginine deprivation therapy is another treatment that can be used to control certain tumors, such as liver cancer and melanoma. Certain tumor cells have metabolic patterns that differ from those of normal cells. Research has shown that arginine is a nonessential amino acid for humans (Rogers 1994), and normal cells do not require arginine for growth. Normal cells can synthesize their own arginine needs by converting citrate via argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) (Haines 2011). On the other hand, arginine is an essential amino acid for hepatocellular carcinoma, melanoma, and some other sarcomas. These tumor cells lack one or both of the above two enzymes, so they cannot synthesize the necessary arginine themselves, and are auxotrophic for arginine, and can only survive with exogenous supplies. In research into the use of arginine-degrading enzymes in the treatment of cancers such as hepatocellular carcinoma, melanoma, and renal cell carcinoma, the two enzymes currently most frequently studied are arginine deiminase and arginase.

[0003] Regarding arginase, human type 1 arginase is currently being used in clinical research. The main clinical challenges of this enzyme are its low catalytic activity in serum and its immunogenicity. To overcome these drawbacks, Aeglea Biotherapeutics has used cobalt ions instead of manganese ions to increase the enzymatic activity of arginase and improve its stability in serum. At the same time, PEGylation has been applied to reduce immunogenicity, and clinical studies are currently underway.

[0004] Arginine deiminase (ADI, EC3.5.3.6) is another microbial enzyme that can decompose arginine into citrate and ammonia. Arginine deiminases from different sources not only differ in structure but also in physiological activity. For example, arginine deiminase extracted from Pseudomonas putida can effectively kill cancer cells in vitro (Jones JB 1981), but it shows little activity at physiological pH, making it unsuitable for in vivo use. Recently, structural modifications have been reported to enable activity at physiological pH. On the other hand, arginine deiminase derived from mycoplasma, unlike that from Pseudomonas putida, exhibits high activity under physiological conditions (Takaku 1992).

[0005] As a therapeutic agent, arginine deiminase shares the disadvantages of conventional protein drugs. Its low stability, susceptibility to degradation, and short half-life in vivo (only about 4 hours in mice) are also significant. Furthermore, as an exogenous protein, arginine deiminase is highly immunogenic, potentially eliciting antibodies in the body that inactivate the enzyme or cause immune damage. Chemical modification of proteins (with PEG, fatty acids, polysaccharides, etc.) can effectively mask antigenic epitopes on the protein surface, reducing immunogenicity. At the same time, the molecular weight increase achieved by modification can significantly extend the half-life in vivo. Therefore, chemical modification of protein drugs with polyethylene glycol, dextran, and polypeptide repeat sequences (PAS) is currently an effective method for addressing the immunogenicity issue. Polyethylene glycol (PEG) is a linear or branched polyether produced by the polymerization of ethylene oxide. It is widely used to modify protein drugs due to its excellent hydrophilicity, biocompatibility, and biological inertness. Modification with PEG can effectively reduce the immunogenicity of protein drugs and extend their half-life in vivo. PEGylation of drugs has become one of the most effective methods for the development and use of protein drugs, and its clinical value has already been very well established. Covalently conjugating PEG with arginine deiminase masks surface antigen epitopes, reducing immunogenicity and extending their half-life in vivo. The most extensively studied currently is arginine deiminase derived from human mycoplasma modified with SS-PEG-20K (ADI-PEG-20), which has already entered clinical trials. However, due to the presence of an unstable ester bond in the drug structure, polyethylene glycol is easily shed in the blood, re-exposing the surface antigens of ADI, and the immunogenicity problem remains unresolved.Furthermore, arginine deiminase derived from human mycoplasma is a homodimer, and because the three-dimensional structure of this dimer is maintained by non-covalent bonds such as hydrogen bonds and hydrophobic interactions, it is structurally unstable and has poor stability in vivo. Modification with PEG further destabilizes the modified structure, potentially leading to dissociation in vivo. As a result, antigenic epitopes present at the dimer interaction interface are exposed, resulting in increased immunogenicity of the drug and reduced retention of enzyme activity.

[0006] Therefore, how to improve the stability of arginine deiminase and reduce its immunogenicity in the body remains a major technical challenge in this field. Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure aims to solve at least one of the technical problems in the related art to some extent. Therefore, one objective of the present disclosure is to propose a covalent dimeric arginine deiminase mutant, a complex thereof, and its use. By forming a covalent disulfide bond between the subunits of homologous non-covalent dimeric arginine deiminase through site-specific mutation of an amino acid site, not only can the structure of the formed dimer be more stable, but also the exposure of antigen epitopes at the interaction interface can be reduced, thereby reducing immunogenicity in the body. [Means for solving the problem]

[0008] Specifically, the present disclosure provides the following technical solutions: A first aspect of the present disclosure provides an arginine deiminase mutant, which forms a covalent disulfide bond between subunits of arginine deiminase by site-specific mutation of an amino acid site on arginine deiminase.

[0009] The above arginine deiminase mutant further includes the following technical features: According to the above arginine deiminase mutant, the mutant promotes the formation of a covalent dimer between subunits of homologous non-covalent dimeric arginine deiminase by site-specific mutation of an amino acid site on arginine deiminase.

[0010] According to the above-mentioned arginine deiminase mutant, based on a sequence derived from human mycoplasma or a sequence homologous thereto, the mutation site of the mutant is selected from amino acids at positions 46, 145, and 337 shown in SEQ ID NO:1 in which at least one amino acid is substituted with cysteine.

[0011] According to the above-mentioned arginine deiminase mutant, the sequence derived from human mycoplasma or a sequence homologous thereto refers to the sequence shown in SEQ ID NO:1 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence shown in SEQ ID NO:1.

[0012] According to the above-mentioned arginine deiminase mutant, the mutant is derived from human mycoplasma, and the amino acid mutation site is the mutation of the 46th amino acid in the sequence shown in SEQ ID NO:1 to cysteine, and the amino acid sequence of the arginine deiminase subunit of the mutant is shown in SEQ ID NO:2.

[0013] According to the above-mentioned arginine deiminase mutant, the mutant is derived from human mycoplasma, and the amino acid mutation site is a mutation of the amino acid at position 145 in the sequence shown in SEQ ID NO:1 to cysteine, and the amino acid sequence of the arginine deiminase subunit of the mutant is shown in SEQ ID NO:3.

[0014] According to the above-mentioned arginine deiminase mutant, the mutant is derived from human mycoplasma, and the amino acid mutation site is a mutation of the amino acid at position 337 in the sequence shown in SEQ ID NO:1 to cysteine, and the amino acid sequence of the arginine deiminase subunit of the mutant is shown in SEQ ID NO:4.

[0015] A second aspect of the present disclosure provides a covalent dimer, wherein the covalent dimer comprises the arginine deiminase mutant described above.

[0016] A third aspect of the present disclosure provides a covalent complex, wherein the covalent complex is formed by covalently binding a modifying group to the above-mentioned arginine deiminase mutant or the above-mentioned covalently bonded dimer, and the modifying group forms an amide bond with the ε-amino group of a lysine residue or the α-amino group of the N-terminal amino acid residue in the arginine deiminase mutant.

[0017] According to an embodiment of the present disclosure, the above covalent complex may further include the following technical features: According to an embodiment of the present disclosure, the modifying group is selected from at least one of polyethylene glycol, dextran, and polypeptide repeat sequences having an active group.

[0018] According to an embodiment of the present disclosure, the molecular weight of the modifying group is 2KDa-20KDa, preferably 5KDa.

[0019] According to an embodiment of the present disclosure, the number of the modifying groups is 9 to 20.

[0020] According to an embodiment of the present disclosure, the active group is selected from at least one of succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate, and preferably, the active group is succinimidyl propionate.

[0021] According to a preferred embodiment of the present disclosure, the modifying group is polyethylene glycol having an active group.

[0022] According to a preferred embodiment of the present disclosure, the polyethylene glycol is linear or branched, and preferably linear.

[0023] According to an embodiment of the present disclosure, the molecular weight of the polyethylene glycol is 2KDa-20KDa, preferably 5KDa.

[0024] According to an embodiment of the present disclosure, the active group is selected from at least one of succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate, and preferably, the active group is succinimidyl propionate.

[0025] According to embodiments of the present disclosure, the covalent complex has one of the following properties: (a) the complex retains 50% or more of the enzymatic activity of the covalently bound dimeric arginine deiminase mutant; (b) the conjugate has low immunogenicity and an extended half-life in the body; (c) The complex can reduce arginine in the body.

[0026] A fourth aspect of the present disclosure provides a pharmaceutical composition, comprising the arginine deiminase mutant, the covalent dimer, or the covalent complex, and a pharmaceutically acceptable carrier.

[0027] A fifth aspect of the present disclosure provides use of the arginine deiminase mutant according to the first aspect, or the covalent dimer according to the second aspect, or the covalent complex according to the third aspect, or the pharmaceutical composition according to the fourth aspect, in the preparation of a medicament for treating a tumor.

[0028] According to an embodiment of the present disclosure, the above use may further include the following technical features. According to an embodiment of the present disclosure, the tumor is an arginine-sensitive tumor.

[0029] According to an embodiment of the present disclosure, the tumor is an ASS expression-deficient tumor, a P53 wild-type tumor, or a combination thereof.

[0030] According to an embodiment of the present disclosure, the tumor is selected from at least one of hepatocellular carcinoma, melanoma, pancreatic cancer, colorectal cancer, acute myeloid leukemia, and small cell lung cancer.

[0031] A sixth aspect of the present disclosure provides use of the arginine deiminase mutant according to the first aspect, or the covalent dimer according to the second aspect, or the covalent complex according to the third aspect, or the pharmaceutical composition according to the fourth aspect, in the prevention or treatment of tumors.

[0032] According to an embodiment of the present disclosure, the tumor is an arginine-sensitive tumor.

[0033] According to an embodiment of the present disclosure, the tumor is an ASS expression-deficient tumor, a P53 wild-type tumor, or a combination thereof.

[0034] According to an embodiment of the present disclosure, the tumor is selected from at least one of hepatocellular carcinoma, melanoma, pancreatic cancer, colorectal cancer, acute myeloid leukemia, and small cell lung cancer.

[0035] A seventh aspect of the present disclosure provides a method for preventing or treating a tumor, the method comprising administering to a subject: an arginine deiminase mutant according to the first aspect; a covalent dimer according to the second aspect, A covalent complex according to the third aspect, The pharmaceutical composition according to the fourth aspect of the present invention comprises administering at least one of the following:

[0036] According to an embodiment of the present disclosure, the tumor is an arginine-sensitive tumor.

[0037] According to an embodiment of the present disclosure, the tumor is an ASS expression-deficient tumor, a P53 wild-type tumor, or a combination thereof.

[0038] According to an embodiment of the present disclosure, the tumor is selected from at least one of hepatocellular carcinoma, melanoma, pancreatic cancer, colorectal cancer, acute myeloid leukemia, and small cell lung cancer.

[0039] Additional aspects and advantages of the disclosure will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the disclosure.

[0040] The above and / or additional aspects and advantages of the present disclosure will become apparent and be readily understood from the following description of examples taken in conjunction with the drawings. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 shows an SDS-PAGE detection diagram of ADI or mADI according to an embodiment of the present disclosure after induction and expression with IPTG. The analysis samples in the electrophoresis lanes from left to right are as follows: electrophoresis lane 1 is Marker, electrophoresis lane 2 is AWT, electrophoresis lane 3 is A1C, electrophoresis lane 4 is A2C, and electrophoresis lane 5 is A3C. Here, the molecular weights (kDa) of the markers are 94.0, 66.2, 45.0, 33.0, 26.0, 20.0, and 14.4, respectively, from top to bottom. [Figure 2] FIG. 1 shows an SDS-PAGE analysis of ADI or mADI after preparation and purification according to an embodiment of the present disclosure. The analysis samples in the electrophoresis lanes from left to right are as follows: Lane 1 is A1C, Lane 2 is Marker, and Lane 3 is AWT. Here, the molecular weights (kDa) of the markers are 97.4, 66.2, 43.0, 31.0, 22.0, and 14.4, respectively, from top to bottom. [Figure 3]FIG. 1 shows a comparison of SEC-HPLC detection results after preparation and purification of ADI or mADI according to an embodiment of the present disclosure, where the main peak with a retention time of 26.577 min is A1C and the main peak with a retention time of 29.693 min is AWT. [Figure 4] FIG. 1 is a SEC-HPLC detection diagram after preparation and purification of PEG-A1C-5K according to an embodiment of the present disclosure. [Figure 5] 1 shows blood drug concentration-time curves after a single administration of PEG-A1C-5K or PEG-AWT-5K into a rat body according to an embodiment of the present disclosure. [Figure 6] 1 shows blood drug concentration-time curves after multiple administrations of PEG-A1C-5K or PEG-AWT-5K into rats according to an embodiment of the present disclosure. [Figure 7] 1 shows a blood drug concentration-time curve after a single administration of PEG-A1C-5K into a cynomolgus monkey according to an embodiment of the present disclosure. [Figure 8] 1 shows blood drug concentration-time curves after multiple administrations of PEG-A1C-5K into cynomolgus monkeys according to an embodiment of the present disclosure. [Figure 9] 1 shows the in vivo tumor volume-time curves for each administration group in a situation where melanoma was transplanted into mice according to an embodiment of the present disclosure. [Figure 10] 1 shows the internal tumor weight-time curves for each administration group in a situation where melanoma was transplanted into mice according to an embodiment of the present disclosure. [Figure 11] FIG. 10 is a comparison diagram of serum arginine concentrations in vivo after the final administration of each administration group in a situation where melanoma was transplanted into mice according to an embodiment of the present disclosure. [Figure 12] FIG. 10 is a comparison diagram of the serum citrulline concentration in vivo after the final administration of each administration group in a situation where melanoma was transplanted into mice according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0042] Examples of the present disclosure will be described in detail below. The examples described below are illustrative and are used only to interpret the present disclosure, and should not be understood as limitations on the present disclosure.

[0043] It should be noted that the terms "first," "second," etc. are for descriptive purposes only and cannot be considered to denote or imply relative importance or to denote the number of technical features. Thus, a feature qualified as "first" or "second" may expressly or imply the inclusion of one or more of the feature. Furthermore, in the description of this disclosure, unless specifically stated otherwise, "plurality" means two or more.

[0044] As used herein, the terms "arginine deiminase mutant," "arginine deiminase analog," "arginine deiminase derivative," "covalent dimeric arginine deiminase mutant," etc. may be used interchangeably in certain cases. All of these terms refer to the achievement of the objectives of this embodiment, including stabilization of protein structure, maintenance of enzymatic activity, and reduction of immunogenicity, by modifying the protein structural sequence of arginine deiminase, such as substituting, deleting, or adding some amino acids, while retaining the catalytic activity of arginine deiminase to specifically convert arginine into citrulline and ammonia.

[0045] As used in this disclosure, the terms "modifying agent," "modifying group," and "polymer" can be used interchangeably and refer to polymers, such as polyethylene glycol, used to modify protein and peptide pharmaceuticals.

[0046] As used in this disclosure, the terms "conjugate," "conjugate," and "modified product" can be used interchangeably and refer to the modified product obtained after arginine deiminase or a variant thereof is modified with a modifying group.

[0047] As used in this disclosure, the terms "PEG," "polyethylene glycol," etc. refer to polyethylene glycol (PEG, HO-(CHCHO)-CHCHOH) as a linear polymer with hydroxyl groups at both ends, and it exists in branched, straight-chain, and multi-armed forms. A typical polyethylene glycol has one hydroxyl group at each end, and when one end is blocked with a methyl group, methoxypolyethylene glycol (mPEG) is obtained. This derivative is most frequently used in the polyethylene glycol modification technology for proteins, where polyethylene glycol is activated to have functional groups.

[0048] The terms "covalently linked dimeric arginine deiminase mutant conjugate," "polyethylene glycolated arginine deiminase mutant," "PEGylated arginine deiminase mutant," "polyethylene glycolated arginine deiminase," and the like can be used interchangeably in certain contexts and all refer to a conjugate formed by modifying arginine deiminase or a mutant thereof with a modifying group such as activated polyethylene glycol.

[0049] The terms "average degree of modification," "average number of modifications," "average number of bonds," "average number of modification groups," etc. may be used interchangeably in certain cases, and all refer to the number of PEGs attached to one arginine deiminase subunit.

[0050] In this application, the molecular weight represented by "molecular weight of polyethylene glycol" or "relative molecular weight of polyethylene glycol" has the general meaning in the art. For example, when the total relative molecular weight of PEG after activation with an active group is within the range of 5 KDa ± 10%, both are expressed as 5 KDa.

[0051] For ease of explanation, in this specification, unmutated non-covalent dimeric arginine deiminase will be referred to as ADI or AWT, covalent dimeric arginine deiminase mutants will be collectively referred to as mADI, and specific mutant structural types of mADI will be referred to as A1C, A2C, A3C, etc.

[0052] One aspect of the present disclosure provides arginine deiminase mutants, which form covalent disulfide bonds between arginine deiminase subunits by mutating amino acid sites on arginine deiminase, either forming covalent disulfide bonds between the mutated sites or between the mutated sites and other cysteines in the arginine deiminase subunits, ultimately forming covalent dimers.

[0053] According to specific embodiments of the present disclosure, the mutants promote the formation of covalent dimers between subunits of homologous non-covalent dimeric arginine deiminase by site-specific mutation of amino acid sites on arginine deiminase.

[0054] According to a specific embodiment of the present disclosure, the mutant is derived from human mycoplasma or a homologous sequence thereof, and the mutation site is a cysteine ​​mutation at least one amino acid selected from positions 46 (A before mutation), 145 (E before mutation), and 337 (E before mutation) of the sequence shown in SEQ ID NO:1. Subsequently, a covalent disulfide bond is formed between the mutated sites (e.g., C46-C46, C145-C145, C337-C337, etc.) or between the mutated site and another cysteine ​​in the subunit (e.g., C46-C396, C145-C396, C337-C396, etc.), ultimately forming a stable covalent dimer. The sequence shown in SEQ ID NO:1 represents a sequence derived from human mycoplasma. Those skilled in the art will understand that the homologous sequence differs from the sequence set forth in SEQ ID NO: 1. According to specific embodiments of the present disclosure, the sequence derived from human mycoplasma is derived from a sequence having at least 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the sequence of SEQ ID NO: 1. According to specific embodiments, the mutation site of the provided variant is a mutation to cysteine ​​of at least one amino acid selected from the corresponding positions 46, 145, and 337 of the sequence set forth in SEQ ID NO: 1. The sequence shown in SEQ ID NO:1 is a sequence derived from human mycoplasma, and those skilled in the art will understand that its homologous sequences differ from the sequence shown in SEQ ID NO:1, and therefore, those skilled in the art cannot exhaustively list corresponding sequences. However, the provided variants may be any variants that contain the corresponding mutations at amino acid positions 46, 145, and 337 of the sequence shown in SEQ ID NO:1 or its homologous sequences, or at positions corresponding to these amino acids.The term "corresponding position" as used herein refers to the position in a homologous sequence where the amino acid at position 37, 146, 338, or other positions exhibits the same function, as long as the homologous sequence exhibits the same or similar effect. For specific details, see the contents listed in Tables 1 and 2 below.

[0055] According to a specific embodiment of the present disclosure, the mutant is derived from another mycoplasma, and the mutation site is a mutation of at least one amino acid at positions 46, 145, and 337 corresponding to the sequence shown in SEQ ID NO: 1 to cysteine.

[0056] It should be understood that arginine deiminases derived from other organisms or pathways such as structural modification may also have mutations at the corresponding sites of the corresponding arginine deiminases derived from human mycoplasma. For example, see the homology comparison of arginine deiminases derived from different organisms in Table 1 below. Arginine deiminases derived from seal brain mycoplasma, arginine mycoplasma, arthritis mycoplasma, etc., have amino acid sequences that are at least 80% identical to those of human mycoplasma, share similar three-dimensional structures, and mutations or differences in surface amino acids do not affect the enzymatic activity characteristic of reducing arginine. Those skilled in the art can obtain covalently bonded dimers using technical methods well known in the art. For example, one method for achieving this includes mutating at least one amino acid at the structurally corresponding positions 46, 145, and 337 of the sequence represented by SEQ ID NO: 1 to cysteine, which corresponds to the mutation site. When arginine deiminase is derived from different sources, the mutation position may vary, but all of these variations fall within the scope of protection of the present disclosure. According to a specific embodiment of the present disclosure, for specific mutation sites, please refer to Table 2, "Comparison of mutation sites of arginine deiminases derived from different sources."

[0057] [Table 1] [Table 2]

[0058] According to a specific embodiment of the present disclosure, the mutant is derived from human mycoplasma, and the amino acid mutation site of the provided mutant is a mutation of the amino acid at position 46 in the sequence shown in SEQ ID NO: 1 to cysteine, and the amino acid sequence of the formed arginine deiminase subunit is shown in SEQ ID NO: 2. According to a specific embodiment of the present disclosure, the mutant is derived from human mycoplasma, and the amino acid mutation site of the provided mutant is a mutation of the amino acid at position 145 in the sequence shown in SEQ ID NO: 1 to cysteine, and the amino acid sequence of the formed arginine deiminase subunit is shown in SEQ ID NO: 3. According to a specific embodiment of the present disclosure, the mutant is derived from human mycoplasma, and the amino acid mutation site of the provided mutant is a mutation of the amino acid at position 337 in the sequence shown in SEQ ID NO: 1 to cysteine, and the amino acid sequence of the formed arginine deiminase subunit is shown in SEQ ID NO: 4.

[0059] According to a specific embodiment of the present disclosure, the mutant is derived from human Mycoplasma, and the amino acid mutation site of the mutant is a mutation of the amino acid at position 46 in the sequence shown in SEQ ID NO:1 to cysteine. According to a specific embodiment of the present disclosure, the mutant is derived from human Mycoplasma, and the amino acid mutation site of the mutant is a mutation of the amino acid at position 145 in the sequence shown in SEQ ID NO:1 to cysteine. According to a specific embodiment of the present disclosure, the mutant is derived from human Mycoplasma, and the amino acid mutation site of the mutant is a mutation of the amino acid at position 337 in the sequence shown in SEQ ID NO:1 to cysteine. The provided mutants may have any two or any three of the above amino acid mutation sites. The present disclosure further provides a method for producing a covalent dimeric arginine deiminase mutant, which enhances the structural stability of the arginine deiminase dimeric mutant and reduces the exposure of antigen epitopes at the interaction interface.

[0060] [Table 3]

[0061] The present disclosure further provides a covalent dimer, wherein the covalent dimer comprises an arginine deiminase mutant described above.

[0062] In another aspect of the present disclosure, a method for preparing a covalently dimeric arginine deiminase mutant is provided.

[0063] In other preferred examples, the covalent dimeric arginine deiminase variants described in the present disclosure, whether from different species or from the same species with at least 80% homology, can be obtained through many routes, including, but not limited to, chemical synthesis, genetic engineering recombinant expression, etc.

[0064] In another preferred example, the vector is prepared by a method of constructing a recombinant expression strain using E. coli or yeast as a host, and more preferably, recombinant expression is carried out using E. coli as a host.

[0065] The covalent dimeric arginine deiminase mutants described in the present invention can be obtained in large quantities by recombinant expression in Escherichia coli, and the expressed arginine deiminase mutants can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, highly purified covalent dimeric arginine deiminase mutants can be obtained using methods well known to those skilled in the art. Examples of these methods include, but are not limited to, centrifugation, cell disruption, salting out, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and combinations of various other techniques.

[0066] The present disclosure further provides a covalent conjugate comprising the arginine deiminase mutant or the covalent dimer and a modifying group chemically bonded thereto, the modifying group being capable of reducing immunogenicity and extending half-life in vivo.

[0067] According to a specific embodiment of the present disclosure, the modifying group forms an amide bond with the ε-amino group of a lysine residue or the α-amino group of the N-terminal amino acid residue in the arginine deiminase mutant.

[0068] According to a specific embodiment of the present disclosure, the number of modifying groups is 9 to 20, and the modifying groups are at least one of polyethylene glycol, dextran, and polypeptide repeat sequences having an active group. According to a preferred embodiment of the present disclosure, the modifying group is polyethylene glycol.

[0069] According to a specific embodiment of the present disclosure, the molecular weight of the modifying group is 2KDa-20KDa, and according to a preferred embodiment of the present disclosure, the modifying group is 5KDa.

[0070] According to specific embodiments of the present disclosure, the activating group is selected from at least one of succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, succinimidyl succinate.

[0071] According to a specific embodiment of the present disclosure, the modifying group forms an amide bond with the ε-amino group of a lysine residue or the α-amino group of the N-terminal amino acid residue in the arginine deiminase mutant, the number of modifying groups is 9 to 20, and the modifying group is polyethylene glycol having an active group. According to a specific embodiment of the present disclosure, the polyethylene glycol (PEG) is linear or branched, preferably linear. For example, one end of the polyethylene glycol has a blocking group, and the blocking group referred to here includes, but is not limited to, a monomethoxy group, an ethoxy group, glucose, or galactose, and includes at least one type. For example, a monomethoxy group is preferred.

[0072] According to a specific embodiment of the present disclosure, the molecular weight of said polyethylene glycol is 2KDa-20KDa, preferably 5KDa.

[0073] According to a specific embodiment of the present disclosure, the activating group used on the polyethylene glycol is selected from at least one of succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate, and may be, for example, succinimidyl propionate.

[0074] According to specific embodiments of the present disclosure, the covalent complex has at least one of the following characteristics: (a) The amino acid sequence of the covalently dimeric arginine deiminase mutant subunit is shown in SEQ ID NO:2. (b) The modifying group of the complex is at least one, preferably 9 to 20, modified groups randomly bonded by forming an amide bond with the ε-amino group of a lysine residue or the α-amino group of the N-terminal amino acid residue in the covalently bonded dimeric arginine deiminase mutant. (c) The modifying group is polyethylene glycol having an active group. (d) The polyethylene glycol is linear. (e) The polyethylene glycol has a monomethoxy blocking group at one end. (f) The molecular weight of the polyethylene glycol is 5 KDa. (g) the activating group is succinimidyl propionate. (h) The complex is a polyethylene glycolated arginine deiminase mutant, and the obtained polyethylene glycolated arginine deiminase mutant has the characteristics of retaining the enzymatic activity of the covalently bonded dimeric arginine deiminase mutant by 50% or more, being structurally stable, having low immunogenicity, and being able to extend the half-life in the body.

[0075] When the arginine deiminase mutant is modified with polyethylene glycol, it can be prepared by a method commonly used in the art. When purifying and obtaining each sample, a method known in the art can be used. This includes, but is not limited to, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, ultrafiltration, or a combination thereof. Molecular sieve chromatography and ultrafiltration are more preferred.

[0076] Another object of the present disclosure is to provide a use of an arginine deiminase mutant or a conjugate thereof, which has the characteristics of retaining the enzymatic activity of the covalently bonded dimeric arginine deiminase mutant at 50% or more, being structurally stable, having low immunogenicity, and being able to extend the half-life in the body, and can deplete arginine alone or in combination, and can be used for the treatment of arginine-sensitive tumors.

[0077] The present disclosure further provides pharmaceutical compositions comprising the above-described arginine deiminase mutant, covalent dimer, or covalent complex and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intrathecal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, antibody, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The provided pharmaceutical compositions can be administered by many methods known in the art. The antibody can be formulated with a suitable carrier, including, for example, sustained-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

[0078] The present disclosure further provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described arginine deiminase mutant, covalent dimer, covalent complex, or pharmaceutical composition. The mutant, covalent dimer, or covalent complex can be administered alone in the form of a salt or other pharmaceutical composition to deplete arginine and be used to treat arginine-sensitive tumors.

[0079] According to a specific embodiment of the present disclosure, the conjugate is used to treat arginine-sensitive tumors, including, but not limited to, ASS expression-deficient tumors, P53 wild-type tumors, or a combination thereof, including hepatocellular carcinoma, melanoma, pancreatic cancer, colorectal cancer, acute myeloid leukemia, small cell lung cancer, etc.

[0080] The frequency and dosage of administration of the conjugate or other pharmaceutical composition of the present disclosure can be determined based on several relevant factors, including the type of disease being treated, the route of administration, the age, sex, and weight of the patient, the severity of the disease, and the type of pharmaceutical agent as the active ingredient.

[0081] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease and / or adverse effects caused by the disease are partially or completely cured. As used herein, "treatment" includes the treatment of a disease in a mammal, particularly a human. Specifically, "treatment" includes (a) preventing a disease in an individual who is susceptible to the disease but has not yet developed the disease; (b) inhibiting a disease, e.g., preventing the progression of a disease; or (c) alleviating a disease, e.g., reducing symptoms associated with a disease. As used herein, "treatment" encompasses all administration of a pharmaceutical agent or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or prevent a disease in the individual, including, but not limited to, administration of a covalent dimeric arginine deiminase mutant complex described herein to an individual in need thereof.

[0082] "Therapeutically effective amount" or "effective amount" refers to the amount of a variant, covalent dimer, or pharmaceutical composition needed to elicit a desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of a variant, covalent dimer, or pharmaceutical composition needed to treat and / or prevent a disease.

[0083] The therapeutically effective amount provided herein will vary depending on the relative activity (e.g., cell proliferation suppression activity) of the mutants or covalent dimers and combinations thereof, and will also vary depending on the subject and disease state to be treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., but a therapeutically effective amount can be easily determined by one skilled in the art. The dosage used for administration can be within the following ranges. For example, about 0.1 mg / kg to about 50 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 1 mg / kg to about 50 mg / kg, about 2 mg / kg to about 50 mg / kg, about 3 mg / kg to about 50 mg / kg, about 5 mg / kg to about 50 mg / kg, about 8 mg / kg to about 50 mg / kg, about 10 mg / kg to about 50 mg / kg, about 15 mg / kg to about 50 mg / kg, about 20 mg / kg to about 50 mg / kg, about 25 mg / kg to about 50 mg / kg, about 30 mg / kg to about 50 mg / kg, or about 40 mg / kg to about 50 mg / kg of the variant or covalently bonded dimer is administered.

[0084] As used herein, "subject" refers to any animal, including rodents (e.g., mice or rats), primates (e.g., cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens)). Preferably, the subject is a primate, more preferably a human. As used herein, "prophylaxis" refers to a method aimed at preventing or delaying the onset of a disease.

[0085] As used herein, examples of cancer include, but are not limited to, carcinoma, lymphoma, germ cell tumor, sarcoma, and leukemia. More specific examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioma (such as glioblastoma and neurofibromatosis), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. In specific embodiments, the cancer treated or diagnosed using the methods disclosed herein is selected from melanoma, breast cancer, ovarian cancer, kidney cancer, gastrointestinal / colon cancer, lung cancer, and prostate cancer. According to preferred embodiments of the present disclosure, the cancer includes, but is not limited to, melanoma, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, or liver cancer.

[0086] Unless specific techniques or conditions are specified in the examples, they are performed in accordance with the techniques or conditions described in the literature or in accordance with the product instructions. If the manufacturer of the reagents or equipment used is not specified, they are all commercially available products.

[0087] Example 1 Construction of expression plasmids for arginine deiminase and its mutants and transformation The researchers in this disclosure designed the mADI mutants shown in Table 4 based on the unmutated ADI (amino acid sequence: SEQ ID NO: 1, gene sequence: SEQ ID NO: 5). Based on the codon usage data of Escherichia coli (E. coli), they designed and synthesized different mutant genes taking into account factors such as codon preference and GC content.

[0088] [Table 4]

[0089] The pET-30a plasmid was used as the expression vector for ADI and mADI. Double restriction enzyme digestion confirmed that both the ADI and mADI expression vector sequences were consistent with the theoretical sequence. The expression host strain BL21(DE3) was transformed using the CaCl2 method to obtain a recombinant expression strain. The recombinant expression strain was screened using the kanamycin-containing plating method and then screened for high-expression clones using IPTG-induced expression. The strains were then stored at -80°C in the original seed library. The expressed gene sequences of ADI and mADI are as follows:

[0090] [Table 5] JPEG2026506061000007.jpg137144 JPEG2026506061000008.jpg137144 JPEG2026506061000009.jpg131144

[0091] According to the research, as shown in Figure 1, the constructed strains were cultured in an Erlenmeyer flask with shaking under IPTG induction, and both the unmutated ADI (AWT) and mutant mADI strains were successfully constructed. Expression bands for the target protein, ADI or mADI subunit, were confirmed at a molecular weight of approximately 46 kDa, and the expression levels were almost consistent.

[0092] Example 2 Preparation and purification of arginine deiminase and its mutants Inclusion bodies of ADI or mADI were obtained by fermentation. Specifically, the original strain of ADI or mADI was taken and fermentation expression was carried out in a fermenter in a two-stage scale-up. The conditions were controlled as follows: first, OD was reached at 30-34°C and pH 7.0-7.4. 600The cells were cultured until the value reached 30 or higher, then the temperature was raised to 36-38°C, IPTG was added to 0.5 mmol / L, and induction was continued for 3 hours or more to allow the accumulation of ADI or mADI. The cells were collected by centrifugation and then stored at -15°C or below.

[0093] The inclusion bodies of ADI or mADI produced by the fermentation described above were denatured and renatured to obtain crude ADI or mADI. Specifically, the resulting bacterial cells were suspended in a disruption buffer containing 20 mmol / LK2HPO4 and 5 mmol / L E-Di-TA. The cells were disrupted using a high-pressure homogenizer at 500–800 bar, and the ADI or mADI inclusion bodies were collected by centrifugation. The inclusion body precipitates were washed with a disruption buffer containing 1–3% Triton X-100 and a 20 mmol / LK2HPO4 buffer, respectively. The washed inclusion bodies were dissolved in a denaturation buffer containing 8 mol / L urea, 20 mmol / LK2HPO4, and 5 mM DTT. The mixture was stirred at room temperature for at least 1 hour to dissolve the inclusion bodies, followed by centrifugation and collection of the supernatant. The dilution regeneration method was used, in which the sample was slowly diluted with a regeneration buffer of 20 mM KHPO (pH 7.0-7.5) at a ratio of 1:30 to 1:50 (V / V) and regenerated by stirring at 10-15°C for more than 36 hours to obtain active ADI or mADI.

[0094] To obtain highly pure ADI or mADI, further purification was performed using multi-step chromatography. Briefly, crude purification was performed using anion exchange chromatography, followed by advanced purification using hydrophobic chromatography, anion exchange chromatography, and other methods, followed by permeation or elution at different salt concentrations. Finally, buffer exchange and concentration were performed using ultrafiltration to obtain purified ADI or mADI samples of the required concentration.

[0095] The results of the preparation and purification studies showed representative spectra (Figure 2). Analysis by non-reducing SDS-PAGE and SEC-HPLC indicated that the purity of the purified ADI or mADI products was 95% or higher. The representative mADI structure A1C exhibited a single band at approximately 92 kDa, indicating that the target protein A1C aggregates as a covalent dimer. In contrast, the unmutated ADI structure AWT did not form a covalent dimer and instead became a monomer under denaturing conditions, exhibiting a single band at approximately 46 kDa. Further SEC-HPLC studies showed that, as shown in Figure 3, reduction of mADI structure A1C with DTT resulted in its conversion to a monomeric subunit, resulting in an extended SEC-HPLC retention time consistent with that of the unmutated ADI structure AWT. These results further demonstrated that A1C exists in the form of a covalent dimer and is more stable in different buffers than the noncovalent dimer of AWT.

[0096] Example 3 Measurement of enzyme activity of samples In Example 3, an improved method was used to measure enzyme activity, which included the following steps: An appropriate amount of ADI or mADI enzyme was added to 10 mol / L L-arginine (L-Arg) and 0.1 mol / L sodium phosphate buffer (pH 6.0), mixed uniformly at 37°C, and reacted for 5 minutes. The reaction was stopped by adding a mixed acid iron solution, and after 10 minutes in a metal bath at 100°C, the absorbance was measured at 530 nm. A standard curve was created using different citric acid standards, and the content of the reaction substrate, citric acid, was calculated from this standard curve. Definition of enzyme activity unit (U): The amount of enzyme required to produce 1 micromole of L-citric acid per minute at 37°C is defined as 1 unit (U). The results of the ADI and mADI enzyme measurements are shown in Table 6.

[0097] [Table 6]

[0098] Research results on ADI or mADI enzymes showed that ADI undergoes mutation to form mADI covalent dimers. Except for the A3C structure, in which the enzymatic activity of mADI is slightly lower than that of ADI, the enzymatic activities of the other A1C and A2C mADI structures were almost identical to those of ADI (AWT).

[0099] Example 4 Preparation of a complex of arginine deiminase and its mutant In Example 4, conjugates of arginine deiminase and its mutants were prepared. Monomethoxypolyethylene glycol (M-SPA), with molecular weights of 2 kDa, 5 kDa, 10 kDa, 20 kDa, etc. and an activating group of succinimidyl propionate, was selected as the modifying agent. The resulting conjugate mixture was further purified by ultrafiltration and ion exchange chromatography to obtain pure PEG-modified conjugates of ADI or mADI.

[0100] The purified ADI or mADI protein was diluted with 100 mol / L phosphate (pH 7.5–9.5) modification buffer to adjust the ADI or mADI protein concentration to 5–10 mg / mL. The ADI or mADI protein and polyethylene glycol M-SPA-5K were weighed at different molar ratios (1:20–1:160) and reacted for at least 1 hour under room temperature with stirring to obtain modified complex mixtures with different degrees of modification.

[0101] The resulting mixture of modified conjugates with different degrees of modification was first subjected to ultrafiltration using a 30 kD or 100 kD hollow fiber column to remove unbound PEG and other modification by-products. Next, unmodified ADI or mADI and endotoxin were removed using ion exchange chromatography. Finally, the buffer was exchanged for 20 mmol / L phosphate buffer (pH 7.0-8.0) using a 30 kD hollow fiber column. The resulting PEG-modified conjugates (PEG-ADI-5K, PEG-mADI-5K, PEG-mADI-10K, PEG-mADI-20K, etc.) were obtained by aliquoting (see Figure 4). Purity testing by SEC-HPLC demonstrated that the purity of the purified PEG-A1C-5K reached >99%.

[0102] Example 5 Quality evaluation of complexes of arginine deiminase and its mutants According to the method provided in Example 3, the enzyme activity and content of the samples before and after modification were measured.

[0103] The average degree of modification was determined according to the principles described by Kantian et al. in "Online characterization of polyethylene glycol-modified proteins." Briefly, the refractive index (RI) and ultraviolet (UV) absorbance of proteins both show a linear relationship with protein concentration, while the RI absorbance of PEG also shows a linear relationship with its concentration. Furthermore, since the PEG and protein moieties do not interfere with each other in the RI and / or UV absorbance of PEG-modified proteins, the average degree of modification of PEG-modified proteins was measured using a differential refractometer and UV detector in SEC-HPLC.

[0104] The results of the assay for the average modification degree and enzymatic activity are shown in Table 7. For PEG-A1C-5K or PEG-AWT-5K samples with different degrees of modification obtained by modification at different feed ratios, the specific activity of the PEG-mADI-5K or PEG-ADI-5K samples tended to decrease with increasing average modification degree compared to the activity of unmodified A1C and AWT. However, within the same modification degree range, the retention of enzymatic activity of PEG-A1C-5K was significantly higher than that of PEG-AWT-5K. This is likely because, when the average modification degree of PEG-AWT-5K exceeds 10 (e.g., 11.5), the PEG-AWT-5K structure becomes unstable and depolymerization begins, resulting in a significant decrease in enzymatic activity. In contrast, even when the average modification degree of PEG-A1C-5K exceeds 10, the A1C structure does not undergo depolymerization, resulting in a high retention of enzymatic activity. The decrease in enzyme activity may be due to an increase in the degree of modification that affects the binding between the enzyme and the substrate, or may be due to modification of some of the enzyme's active sites, resulting in inactivation.

[0105] The above quality assessment further demonstrated that the structure of the covalent dimeric arginine deiminase mutant is more stable than the structure of the non-covalent dimeric arginine deiminase, and the enzymatic activity is also more stable.

[0106] [Table 7]

[0107] The binding strength of the modified samples with A1C rabbit polyclonal antibody was assayed using a competitive ELISA. Briefly, A1C rabbit polyclonal antibody (10 μg / ml, 100 μl / well) was coated onto wells, washed three times with PBS, and blocked overnight with 2% BSA. After washing three times with PBS, biotin-A1C was diluted to 100 ng / ml in PBS, and the samples A1C, PEG-A1C-2K, PEG-A1C-5K, PEG-A1C-10K, and PEG-A1C-20K were each diluted to 100 ng / ml. These were mixed with biotin-A1C in equal volumes, and 50 μl of each mixture was added to each well and shaken at 25°C for 30 minutes. PBS was used as a control, followed by five washes with PBS. Avidin-HRP was diluted 1:2500 and added at 100 μl per well. The plate was shaken at 150 rpm at 25°C for 30 minutes. 50 μl per well of TMB Chromogen Solution A was added, followed by 50 μl per well of TMB Chromogen Solution B. The plate was then incubated at room temperature for 10 minutes in the dark. 50 μl per well of stop solution was added to terminate the reaction. The OD was measured at 450 nm within 30 minutes using an enzyme-labeled plate reader.

[0108] The results are shown in Table 8. When A1C was PEG-modified, all PEG-modified products of different sizes clearly reduced immunogenicity (the higher the OD value, the closer it was to the OD value of PBS, indicating lower immunogenicity). Among them, A1C modified with 5K (PEG-A1C-5K) was the most effective in reducing immunogenicity.

[0109] [Table 8]

[0110] Example 6 In vitro growth inhibition experiments of different tumor cells Cell lines, including mouse melanoma B16F10 and human hepatoma SK-Hep-1, were harvested in logarithmic growth phase, diluted with complete medium, and cultured in 96-well plates. After serial dilutions of the PEG-A1C-5K sample, 10 μl of equal volume of the sample solution was added to each well, and the culture was continued for an additional 3 days. The inhibitory effect of different dose concentrations on the cell proliferation was measured using the CCK-8 assay, and the IC50 was calculated.

[0111] [Table 9]

[0112] The results are shown in Table 9. Here, Imax (maximum inhibition rate) is a parameter that represents the inhibitory effect on cell proliferation, with a higher value indicating a better effect. As a result, PEG-A1C-5K, PEG-A2C-5K, PEG-A3C-5K, and PEG-AWT-5K all showed clear inhibitory effects on the selected cells.

[0113] Example 7: Pharmacokinetic evaluation of a single dose of arginine deiminase and its mutant complex in rats The PK of PEG-AWT-5K and PEG-A1C-5K was evaluated in rats. PEG-AWT-5K and PEG-A1C-5K were injected into the tail vein of six rats in each group at a dose of 1 mg / kg body weight, and blood samples were taken on days 1, 3, 7, 10, and 14 after administration to measure PK. The results are shown in Figure 5. The PEG-A1C-5K group maintained its drug efficacy for more than 10 days, while the PEG-AWT-5K group maintained its efficacy for only 7 days. This result suggests that the duration of drug concentration maintenance in the blood of PEG-A1C-5K is clearly superior to that of PEG-AWT-5K.

[0114] Example 8: Multiple-dose pharmacokinetic and immunogenicity evaluation of arginine deiminase and its mutant conjugates in rats Twelve male SD rats were randomly divided into two groups and received multiple intravenous injections of PEG-AWT-5K and PEG-A1C-5K at a dose of 1.0 mg / kg once a week for 4 weeks. Blood samples were taken on the 3rd and 7th days after each injection to measure PK and anti-protein antibodies. As shown in Figure 6, after the third administration, drug accumulation in the blood was observed in the PEG-A1C-5K group, but not in the PEG-AWT-5K group. This suggests that PEG-A1C-5K has a longer and more stable retention time in the blood than EG-AWT-5K. Furthermore, antibody testing revealed that no anti-protein antibodies were produced in the PEG-A1C-5K group, whereas approximately 30% of the individuals in the PEG-AWT-5K group produced anti-protein antibodies. These results indicate that PEG modification provides a superior shielding effect on the structural antigenic epitopes of the covalently linked dimeric arginine deiminase A1C formed by mutation compared to the unmutated AWT structure.

[0115] Example 9: Pharmacokinetics of a single dose of arginine deiminase and its mutant complex in monkeys The PK of PEG-A1C-5K was evaluated in cynomolgus monkeys. Two cynomolgus monkeys were divided into each group, and PEG-A1C-5K was administered intravenously to each monkey in each group at doses of 1 mg / kg and 3 mg / kg, respectively. Plasma samples were collected before administration, and 5 minutes (±1 minute), 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 168 hours, 240 hours, 336 hours, 408 hours, and 504 hours after administration, and the PK of blood drug concentrations was measured. The results are shown in Figure 7. PEG-A1C-5K maintained its efficacy for more than 21 days, with a half-life of 131 to 192 hours.

[0116] Example 10: Multiple-dose pharmacokinetic and immunogenicity evaluation of a conjugate of arginine deiminase and its mutants in monkeys The PK and immunogenicity of PEG-A1C-5K were evaluated in cynomolgus monkeys. Two cynomolgus monkeys were divided into each group, and PEG-A1C-5K was administered intravenously to each monkey in each group at doses of 1 mg / kg and 3 mg / kg, respectively. Blood samples were taken on the 3rd and 7th days after administration, and PK and ADA detection analyses were performed. As shown in Figure 8, the blood drug concentration of PEG-A1C-5K in rhesus monkeys increased with increasing dose. Furthermore, antibody testing confirmed only low titer (1:10) anti-protein antibodies in PEG-A1C-5K.

[0117] Example 11 In vivo inhibitory effect of arginine deiminase mutant complex on tumor cells Female C57 mice were subcutaneously inoculated with mouse melanoma B16F10 cells and then randomly divided into five groups. The administration method is shown in Table 10, and tumor volume was measured two to three times weekly. After the experiment, blood samples were collected and the animals were euthanized. The tumor masses were excised and removed, and their weights were measured. The arginine and citrulline contents in the serum were also measured.

[0118] [Table 10]

[0119] The results of tumor volume and tumor weight are shown in Figures 9 and 10. Compared with the model control group, PEG-AWT-5K, PEG-A1C-5K, PEG-A2C-5K, and PEG-A3C-5K were all able to inhibit tumor growth, and the tumor growth inhibitory effects of PEG-A1C-5K, PEG-A2C-5K, and PEG-A3C-5K were significantly superior to those of PEG-AWT-5K.

[0120] The results of serum arginine and citrulline content are shown in Figures 11 and 12. Compared with the model control group, PEG-AWT-5K, PEG-A1C-5K, PEG-A2C-5K, and PEG-A3C-5K were all able to reduce serum arginine and maintain serum citrulline levels. Among them, PEG-A1C-5K, PEG-A2C-5K, and PEG-A3C-5K were significantly better than PEG-AWT-5K in maintaining low levels of arginine and high levels of citrulline. This may be related to the appearance of anti-protein antibodies in PEG-AWT-5K after the third administration, which reduced its efficacy, consistent with its tumor growth inhibition.

[0121] In the description herein, a statement referring to terms such as "one example," "some examples," "embodiments," "specific embodiments," or "some embodiments" means that a particular feature, structure, material, or characteristic described with reference to that example or example is included in at least one example or example of the present disclosure. In this specification, general references to the above terms do not necessarily refer to the same example or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more examples or examples. Furthermore, those skilled in the art can combine and combine the various examples or examples described herein and the features of the various examples or examples without mutual contradiction.

[0122] Although the embodiments of the present disclosure have been shown and described, the above embodiments are illustrative and should not be construed as limiting the present disclosure. It will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present disclosure.

[0123] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to and the benefit of patent application number 202310115527.8, filed with the State Intellectual Property Office of China on February 15, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. An arginine deiminase mutant, wherein the mutant promotes the formation of covalent disulfide bonds between subunits of arginine deiminase by mutation of amino acid sites on arginine deiminase, thereby promoting the formation of covalent dimers between subunits of homologous noncovalent dimer arginine deiminase.

2. The arginine deiminase mutant according to claim 1, wherein, based on a sequence derived from human mycoplasma or a homologous sequence thereof, the mutation site of the mutant is selected from those in which at least one amino acid among the amino acids at positions 46, 145, and 337 shown in SEQ ID NO: 1 is substituted with cysteine.

3. The arginine deiminase mutant according to claim 1, wherein the mutant is derived from human mycoplasma, and the amino acid mutation site is a mutation in which the amino acid at position 46 in the sequence shown in SEQ ID NO: 1 is changed to cysteine, and the amino acid sequence of the arginine deiminase subunit of the mutant is shown in SEQ ID NO:

2.

4. The arginine deiminase mutant according to claim 1, wherein the mutant is derived from human mycoplasma, and its amino acid mutation site is a mutation in which the amino acid at position 145 in the sequence shown in SEQ ID NO: 1 is changed to cysteine, and the amino acid sequence of the arginine deiminase subunit of the mutant is shown in SEQ ID NO:

3.

5. The arginine deiminase mutant according to claim 1, wherein the mutant is derived from human mycoplasma, and its amino acid mutation site is a mutation in which the amino acid at position 337 in the sequence shown in SEQ ID NO: 1 is changed to cysteine, and the amino acid sequence of the arginine deiminase subunit of the mutant is shown in SEQ ID NO:

4.

6. A covalent complex comprising the arginine deiminase mutant described in claim 1 and a modifying group chemically bound thereto.

7. The covalent complex according to claim 6, wherein the modifying group forms an amide bond with the ε-amino group of the lysine residue or the α-amino group of the N-terminal amino acid residue in the arginine deiminase mutant.

8. The covalent complex according to claim 6, wherein the modifying group is selected from at least one of polyethylene glycol, dextran, and polypeptide repeat sequences having an active group, and is preferably polyethylene glycol.

9. The covalent complex according to claim 6, wherein the molecular weight of the modifying group is 2 kDa to 20 kDa, preferably 5 kDa.

10. The covalent complex according to claim 6, wherein the ratio of the arginine deiminase mutant subunit to the modifying group is 1:9 to 1:

20.

11. The covalent complex according to claim 6, wherein the modifying group has an active group, and the active group is selected from at least one of succinimidyl carbonate, succinimidylpropionate, succinimidyl acetate, and succinimidyl succinate, preferably the active group is succinimidylpropionate.

12. A pharmaceutical composition comprising the covalent complex described in claim 6 and a pharmaceutically acceptable carrier.

13. The arginine deiminase mutant or Use of the covalent complex according to any one of claims 6 to 11 or the pharmaceutical composition according to claim 12 in the preparation of a pharmacopoeia for the prevention or treatment of tumors.

14. The use according to claim 13, wherein the tumor is an arginine-sensitive tumor.

15. The use according to claim 13, wherein the tumor is an ASS expression-deficient tumor, a P53 wild-type tumor, or a combination thereof.

16. The use according to claim 13, wherein the tumor is selected from at least one of hepatocellular carcinoma, melanoma, pancreatic cancer, colorectal cancer, acute myeloid leukemia, and small cell lung cancer.

Images