Directed chemical coupling asparaginase mutant and its preparation method and application

Abstract

The present invention relates to the fields of biotechnology and pharmaceuticals, and specifically to directed chemically conjugated asparaginase mutants, their preparation methods, and uses. The present invention provides an asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof, wherein at least one amino acid on the surface of asparaginase is mutated to lysine. The newly formed lysine is then subjected to directed chemical conjugation to form a novel asparaginase mutant conjugate; the novel asparaginase mutant conjugate has lower immunogenicity and a longer half-life in vivo. The present invention also provides pharmaceutical compositions and products comprising the mutant chemical conjugate, as well as their use in the treatment of acute lymphoblastic leukemia, Hodgkin's lymphoma, chronic leukemia, lymphosarcoma cells, and hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and pharmaceuticals, and in particular to a directed chemical coupling asparaginase mutant and a preparation method and application thereof. Background Art

[0002] Asparaginase is a protein that catalyzes the hydrolysis of L-asparagine into aspartic acid and ammonia. It is found in a variety of organisms, such as bacteria, actinomycetes, fungi, yeast, algae, and plants (Verma, Critical Reviews in Biotechnology 27.1 (2007): 45-62). Asparaginases derived from Escherichia coli and Erwinia have been successfully used to treat childhood acute lymphoblastic leukemia (ALL) for many years.

[0003] In recent years, asparaginase has also been used in research to treat Hodgkin's disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma, pancreatic cancer, and melanoma (Archana, 3 Biotech 8.6 (2018): 278). These cells are believed to be unable to synthesize the amino acid asparagine, which is essential for growth, and must rely on the host for supply. Asparaginase hydrolyzes asparagine, depriving tumor cells of asparagine and thus inhibiting their growth. Normal human cells contain asparagine synthetase and can synthesize asparagine on their own, making them less affected.

[0004] In 1978, Escherichia coli-derived asparaginase (E. coli ASNase) was approved by the U.S. Food and Drug Administration (FDA) for the treatment of childhood acute lymphoblastic leukemia (ALL). For over 40 years, asparaginase and combination chemotherapy regimens containing it have achieved good results in the treatment of ALL. However, due to its exogenous origin, it is highly immunogenic, with a 45%-75% positive rate of enzyme antibody positivity in patients during clinical use. Clinically, adverse reactions such as progressive immune reactions and systemic anaphylaxis are common. Furthermore, the half-life of the naturally occurring E. coli-derived asparaginase molecule in humans is only 1.24±0.17 days, requiring frequent medication use. These limitations have limited the clinical use of asparaginase (Asselin, Drug resistance in leukemia and lymphoma III. Springer, Boston, MA, 1999. 621-629).

[0005] To address these issues, researchers have focused their efforts on modifying asparaginase in the following areas: 1. Screening for new sources of asparaginase. In recent years, asparaginases from a variety of microorganisms have been extensively studied, including Erwinia chrysanthemi, Erwinia chrysanthemi, Bacillus mesentericus, Marine actinomycetes, Aspergillus niger, and Saccharomyces cervisiae. In 2011, Erwinia chrysanthemi-derived asparaginase (Erwinaze) was launched in the United States. Because it does not cross-react with antibodies produced by E. coli-derived asparaginase in patients, it was approved by the FDA as a second-line drug for the treatment of ALL patients with hypersensitivity to E. coli ASNase (Keating, BioDrugs 27.4 (2013): 413-418). Second, nanoencapsulation techniques, such as encapsulating asparaginase in liposomes or red blood cells, are also being explored. French biopharmaceutical company Erytech Pharma is using GRASPA (encapsulating E. coli ASNase in red blood cells) to extend the drug's in vivo half-life for the treatment of acute lymphoblastic leukemia and pancreatic cancer. The drug is currently in Phase III clinical trials (Thomas, NCT0151851 (2015): 2492-2492; Hammel, Annals of Oncology, 2017, 28. suppl_5). ③ Structural modification through genetic engineering. Genetic engineering of asparaginase primarily involves enzyme activity, glutamine hydrolysis, immunogenicity, stability, and resistance to proteolysis. To date, no related products have entered clinical trials. Marc N. Offman et al. (Offman, Blood 117.5 (2011): 1614-1621) found that the N24A and R195S mutations increased E. coli-ASP's resistance to proteolytic cleavage and simultaneously enhanced its activity. Mehta RK et al. (Mehta, Journal of Biological Chemistry 289.6 (2014): 3555-3570) significantly reduced the immunogenicity and glutamine hydrolysis of E. coli-ASP by introducing the K288S and Y176F mutations in the asparaginase sequence. The combination of genetic engineering and other asparaginase modification technologies may further advance the development of asparaginase drugs.Chemical modification: Compounds used for drug modification include polyethylene glycol, peptides and their derivatives, fatty acids, and carbohydrates. PEGylation is often used to extend the clinical half-life of drugs and improve their immunogenicity. Two PEGylated asparaginase drugs (Oncaspar and Asparlas) are already marketed overseas. Patent US10174302B1 covalently couples asparaginase with a peptide composed of repeating amino acids (proline and alanine) and its derivatives to improve its immunogenicity and in vivo half-life.

[0006] Polyethylene glycol (PEG) is a linear or branched polyether formed by the polymerization of ethylene oxide. Due to its excellent hydrophilicity, biocompatibility, and biological inertness, it is widely used to modify protein drugs. PEG modification can effectively reduce the immunogenicity of protein drugs and prolong their half-life in vivo. Clinical studies of PEGylated drugs have shown that clinical side effects of various drugs are related to the immune response induced by PEG in the human body. In clinical studies of Palynziq, 93% of patients experienced hypersensitivity adverse events (HAEs) during the early stages of treatment. At this time, patients had high levels of anti-protein antibodies and anti-PEG antibodies in their bodies. The onset of allergic reactions may be related to the formation of circulating immune complexes in the body by these antibodies (Gupta, EBioMedicine 37 (2018): 366-373). In clinical studies of pegloticase, 32% of patients developed anti-PEG antibodies, of which 78% developed both anti-PEG IgG and IgM, 20% developed only IgM antibodies, and 2% developed only IgG antibodies. These antibodies have been shown to be associated with drug clearance in the body (Ganson, Arthritis research & therapy, 2005, 8(1): R12). In 2015, Mima et al. used PEGylated ovalbumin as a model for studying PEGylated protein drugs and demonstrated that anti-PEG IgM is an important factor in the clearance of PEGylated drugs in the body (Mima, Molecular pharmaceutics, 2015, 12(7): 2429-2435). Armstrong et al. reported that in pediatric patients with acute lymphoblastic leukemia treated with pegylated asparaginase (Oncaspar), 32% developed anti-PEG-specific antibodies by serological testing, and 46% developed anti-PEG-specific antibodies by flow cytometry. The anti-PEG antibody type was mainly IgM, which was associated with the rapid clearance of the subsequently injected drug (Armstrong, Cancer, 2007, 110(1):103-111). In another study, 3 / 4 of the patients developed allergic reactions after receiving pegylated asparaginase, and all of these patients developed anti-PEG IgG antibodies (Rau, Pediatric blood & cancer, 2018, 65(3):e26873).

[0007] The in vivo immune response to PEGylated drugs is related to the immunogenicity of the carrier protein, the size of the PEG, the presence of PEG branches, and the degree of PEGylation. Using tetanus toxoid (TT), bovine serum albumin (BSA), and chicken ovalbumin (OVA) as model proteins, Xue Wan et al. studied the factors influencing the immune response to PEGylated proteins by coupling them with different types of polyethylene glycol. The results showed that the degree of PEGylation can help attenuate the immune response to PEG and proteins (Wan X, Process Biochemistry, 2017, 52:183-191).

[0008] For many years, polyethylene glycol modification technology has been successfully applied in the development of Escherichia coli-derived asparaginase. The earliest PEGylated asparaginase drug (Oncaspar) was approved for marketing by the FDA in 1994 and has been approved as a first-line treatment for ALL in children and adults since 2006 (Dinndorf, Oncologist 12.8 (2007): 991). Oncaspar is a drug that uses polyethylene glycol succinimidyl succinate (SS-PEG) to randomly modify the lysine and N-terminal amino groups in asparaginase. The half-life of the drug in humans is 5.73±3.24 days. At the current clinical dosage, it can be administered once a week or once every two weeks. However, due to the unstable ester bond in the drug structure, the polyethylene glycol is easily detached in the blood and has poor in vivo stability. After PEG detachment, the protein and the succinic acid termini remaining on the protein are prone to cause immune reactions and systemic allergic reactions, which limits the clinical effect (Asselin, Springer, Boston, MA, 1999: 621-629; Heo, Drugs, 2019, 79(7): 767-777; Würthwein, European journal of drug metabolism and pharmacokinetics, 2017, 42(6): 955-963). Calaspargase pegol, which was launched at the end of 2019, uses polyethylene glycol succinimidyl carbonate (SC-PEG) instead of SS-PEG to modify asparaginase, which improves the stability of the drug to a certain extent, and the half-life can reach 13.4 ± 1.4 days. Clinically, a dosing frequency of once every three weeks is basically achieved (Angiolillo, Journal of Clinical Oncology Official Journal of the American Society of Clinical Oncology 32.34 (2014): 3874-82). Calaspargase pegol and Oncaspar are polyethylene glycol saturated modified drugs, that is, an asparaginase protein molecule is covalently coupled to 7-9 polyethylene glycols, shielding the immunogenic region on the surface of asparaginase as much as possible, extending the half-life of the drug. However, clinical studies have found that these two listed drugs can still produce a certain degree of anti-drug antibodies in the human body, indicating that the coupled PEG may not completely cover the immune epitope on the surface of asparaginase.In 2016, Schore, Reuven J et al. reported that among 166 newly diagnosed acute lymphoblastic leukemia patients receiving calaspargase pegol and Oncaspar treatment, 3.6% (Calaspargase pegol group) and 10.0% (Oncaspar group) of the patients developed antibodies against asparaginase, and 13% (Calaspargase pegol group) and 19.2% (Oncaspar group) of the patients developed antibodies against PEG. The production of these antibodies accelerated the clearance of drugs in the body (Schore, Children's Oncology Group (COG) study AALL07P4 (2016)).

[0009] Some researchers have used bioinformatics software and bioanalysis methods to identify possible immunodominant regions of asparaginase and reduce immunogenicity by amino acid mutations in these regions. However, since immunodominant regions often involve a large number of amino acids and protein structural conformations, single or multiple amino acid mutations often cannot significantly improve the immunogenicity of the protein (Belén, Biologicals 59(2019):47-55; US Pat. No: US20120148559A1).

[0010] Therefore, there is an urgent need to develop a compound that can reduce immunogenicity in vivo and prolong half-life. Summary of the Invention

[0011] The present invention aims to at least partially address one of the technical problems in the related art. To this end, one object of the present invention is to provide an asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof, wherein at least one amino acid on the surface of asparaginase is mutated to lysine. The newly formed lysine is then subjected to directed chemical conjugation to form a novel asparaginase mutant conjugate; this novel asparaginase mutant conjugate has lower immunogenicity and a longer half-life in vivo. The present invention also provides pharmaceutical compositions and products comprising this mutant chemical conjugate, as well as their use in the treatment of acute lymphoblastic leukemia, Hodgkin's lymphoma, chronic leukemia, lymphosarcoma cells, and hepatocellular carcinoma.

[0012] To this end, the present invention provides, in one aspect, an asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof. According to an embodiment of the present invention, the asparaginase mutant conjugate comprises an asparaginase mutant and a group chemically coupled thereto that is capable of reducing in vivo immunogenicity and extending in vivo half-life.

[0013] The asparaginase mutant is a mutant in which the amino acid at at least one site in the unmutated asparaginase is mutated to lysine, and the group capable of reducing in vivo immunogenicity and prolonging in vivo half-life is chemically coupled to the asparaginase mutant via the lysine in the asparaginase mutant.

[0014] The inventors used bioinformatics tools to selectively mutate at least one amino acid accessible on the protein surface to lysine in an immunodominant region away from the active center of asparaginase, while ensuring that the modified asparaginase retained at least 85% of the pre-modification enzyme activity. Furthermore, they used targeted saturation modification technology to introduce at least one polyethylene glycol modification site, increasing the number of polyethylene glycol chemical conjugates on the asparaginase surface and improving the coverage of polyethylene glycol on the enzyme surface, thereby achieving a PEGylated asparaginase mutant with lower immunogenicity and a longer in vivo half-life. While reducing in vivo immunogenicity and extending half-life, the asparaginase mutant conjugate still retains the activity of catalyzing the hydrolysis of asparagine, with the enzyme activity retained at least 50% of the pre-modification level. The asparaginase mutant conjugate can sustainably reduce asparagine in the body, maintaining serum asparagine levels below 2 μmol / L for up to 21 days.

[0015] According to an embodiment of the present invention, the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof further has one of the following additional technical features:

[0016] According to an embodiment of the present invention, the non-mutated asparaginase is derived from Erwinia or Escherichia coli.

[0017] According to an embodiment of the present invention, the amino acid sequence of the unmutated asparaginase has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0018] According to an embodiment of the present invention, the unmutated asparaginase has the activity of hydrolyzing asparagine and can be derived from Escherichia coli or Erwinia, but is not limited to Escherichia coli and Erwinia, and can be extracted from natural strains or recombinantly expressed. In a specific embodiment of the present invention, the asparaginase before modification has at least about 80% sequence identity with a protein comprising the sequence of SEQ ID NO: 1, more particularly, has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% sequence identity with a protein comprising SEQ ID NO: 1.

[0019] Fragments of the protein of SEQ ID NO: 1 are also included in the definition of proteins used in the conjugates of the present invention. The term "fragment of the protein of SEQ ID NO: 1" refers to a polypeptide sequence that may include fewer amino acids than SEQ ID NO: 1.

[0020] The sequence of SEQ ID NO: 1 is as follows:

[0021] LPNITILATGGTIAGGGDSATKSNYTAGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDDVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVDAGYDGIVSAGVGNGNLYKTVFDTLATAAKNGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY

[0022] The "multiple amino acids accessible to the surface of asparaginase" refers to the solvent accessible amino acids in the asparaginase tetramer model evaluated by bioinformatics tools. The asparaginase tetramer model can be derived from a model published in an existing database (such as Protein Data Bank) or a protein model constructed by homology modeling. The solubility accessibility refers to the amino acid solvent accessible area calculated by one or more bioinformatics tools to be greater than or equal to

[0023] According to an embodiment of the present invention, the amino acid sequence of the asparaginase mutant has at least 80% sequence identity with the non-mutated asparaginase, and the asparaginase mutant retains at least 85% of the enzyme activity compared with the non-mutated asparaginase.

[0024] According to an embodiment of the present invention, the sites where amino acid mutations occur in the asparaginase mutant include at least one of N37K, D64K, N143K, D233K, T252K, and Q317K.

[0025] According to an embodiment of the present invention, the sites where amino acid mutations occur in the asparaginase mutant include D64K, N143K, D233K, and Q317K, and the amino acid sequence of the asparaginase mutant is shown in SEQ ID NO: 2.

[0026] SEQ ID NO: 2 is as follows:

[0027] LPNITILATGGTIAGGGDSATKSNYTAGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDKVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASAKRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVKAGYDGIVSAGVGNGNLYKTVFDTLATAAKNGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPKQIQQIFNQY

[0028] According to an embodiment of the present invention, the sites where amino acid mutations occur in the asparaginase mutant include D64K, D233K, and Q317K, and the amino acid sequence of the asparaginase mutant is shown in SEQ ID NO: 3.

[0029] SEQ ID NO:3 is as follows:

[0030] LPNITILATGGTIAGGGDSATKSNYTAGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDKVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKT NTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVKAGYDGIVSAGVGNGNLYKTVFDTLATAAKNGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPKQIQQIFNQY

[0031] According to an embodiment of the present invention, the group capable of reducing in vivo immunogenicity and prolonging in vivo half-life is directionally coupled by forming an amide bond with the amino group of lysine in the asparaginase mutant, and the group is methoxypolyethylene glycol with an activated group.

[0032] According to an embodiment of the present invention, the activating group is selected from succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate.

[0033] According to an embodiment of the present invention, the molecular weight of the polyethylene glycol is 2KDa-20KDa, preferably 5KDa.

[0034] According to an embodiment of the present invention, the directional coupling rate of the newly formed lysine in the asparaginase mutant to the group is not less than 50%.

[0035] According to an embodiment of the present invention, the number of the coupled groups in the asparaginase mutant conjugate is A+75%N, wherein A and N are integers, 30≤A≤40; and N is the number of lysine residues increased in the asparaginase mutant compared to the unmutated asparaginase.

[0036] The directed coupling of the groups to lysine in the asparaginase mutant may be heterogeneous, so the asparaginase mutant conjugate is essentially a mixture, and the number of the coupled groups in the asparaginase mutant conjugate may be an integer or not.

[0037] According to some specific embodiments of the present invention, the unmutated asparaginase is an asparaginase derived from Escherichia coli, which has a sequence of SEQ ID NO: 1; the mutation sites of the asparaginase mutant include but are not limited to any combination of N37K, D64K, N143K, D233K, T252K, and Q317K; more specifically, it contains D64K, N143K, D233K, and Q317K mutation points, and the amino acid sequence is such as SEQ ID NO: 2; more specifically, it contains D64K, D233K, and Q317K mutation points, and the amino acid sequence is such as SEQ ID NO: 3. Here, N37K refers to the mutation of asparagine at position 37 in SEQ ID NO: 1 to lysine, D64K refers to the mutation of aspartic acid at position 64 in SEQ ID NO: 1 to lysine, N143K refers to the mutation of asparagine at position 143 in SEQ ID NO: 1 to lysine, D233K refers to the mutation of aspartic acid at position 233 in SEQ ID NO: 1 to lysine, T252K refers to the mutation of threonine at position 252 in SEQ ID NO: 1 to lysine, and Q317K refers to the mutation of glutamine at position 317 in SEQ ID NO: 1 to lysine.

[0038] Through sequence alignment (such as Blast sequence alignment), the mutation of the amino acid at the same position in the asparaginase sequence to lysine is also within the scope of protection of this patent.

[0039] A polypeptide can be modified by replacing, inserting, deleting and / or adding one or more amino acids while retaining its enzymatic activity. For example, it is common to replace an amino acid at a given position by a chemically equivalent amino acid without affecting the functional properties of the protein.

[0040] The mutant conjugate is formed by forming an amide bond between a compound and the amino group of lysine in the asparaginase mutant, wherein the compound includes but is not limited to polyethylene glycol.

[0041] The polyethylene glycol molecule may have a molecular weight of 2 KDa to 20 KDa, preferably 5 KDa, depending on the degree of polymerization.

[0042] Polyethylene glycol chemically reacts with the amino group of lysine in the asparaginase mutant through its activating group to form an amide bond. The activating group of polyethylene glycol includes but is not limited to succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate. In a preferred embodiment, the activating group of polyethylene glycol is succinimidyl carbonate and succinimidyl propionate.

[0043] A second aspect of the present invention provides a method for preparing the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to the first aspect. According to an embodiment of the present invention, the method comprises mixing the asparaginase mutant with polyethylene glycol in a buffer solution to cause a coupling reaction, thereby obtaining the asparaginase mutant conjugate.

[0044] The method of the present invention produces an asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof that, while reducing in vivo immunogenicity and extending half-life, retains activity in catalyzing the hydrolysis of asparagine, with the enzyme activity retained at least 50% of its pre-modification level. The asparaginase mutant conjugate can sustainably reduce asparagine levels in the body, maintaining serum asparagine levels below 2 μmol / L for up to 21 days or more.

[0045] According to an embodiment of the present invention, the preparation method further has one of the following additional technical features:

[0046] According to an embodiment of the present invention, when the coupling reaction occurs, the pH of the buffer solution is 7.5-10.5.

[0047] According to an embodiment of the present invention, the coupling reaction takes 1-2 hours.

[0048] According to an embodiment of the present invention, the buffer solution has an ionic strength of 10 to 200 mmol / L.

[0049] According to an embodiment of the present invention, the buffer solution is selected from at least one of phosphate, carbonate, and borate.

[0050] According to an embodiment of the present invention, the weight ratio of the asparaginase mutant to polyethylene glycol is not less than 1:5.

[0051] According to an embodiment of the present invention, the asparaginase mutant is a mutant in which the amino acid at at least one site in the unmutated asparaginase is mutated to lysine.

[0052] According to an embodiment of the present invention, the non-mutated asparaginase is derived from Erwinia or Escherichia coli.

[0053] According to an embodiment of the present invention, the amino acid sequence of the unmutated asparaginase has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0054] According to an embodiment of the present invention, the amino acid sequence of the asparaginase mutant has at least 80% sequence identity with the non-mutated asparaginase, and the asparaginase mutant retains at least 85% of the enzyme activity compared with the non-mutated asparaginase.

[0055] According to an embodiment of the present invention, the sites where amino acid mutations occur in the asparaginase mutant include at least one of N37K, D64K, N143K, D233K, T252K, and Q317K.

[0056] According to an embodiment of the present invention, the sites where amino acid mutations occur in the asparaginase mutant include D64K, N143K, D233K, and Q317K, and the amino acid sequence of the asparaginase mutant is shown in SEQ ID NO: 2.

[0057] According to an embodiment of the present invention, the sites where amino acid mutations occur in the asparaginase mutant include D64K, D233K, and Q317K, and the amino acid sequence of the asparaginase mutant is shown in SEQ ID NO: 3.

[0058] According to an embodiment of the present invention, the molecular weight of the polyethylene glycol is 2KDa-20KDa.

[0059] According to an embodiment of the present invention, the polyethylene glycol is selected from at least one of succinimidyl carbonate polyethylene glycol (SC-PEG), succinimidyl propionate polyethylene glycol (SPA-PEG), succinimidyl acetate polyethylene glycol (SCM-PEG), and succinimidyl succinate polyethylene glycol (SS-PEG).

[0060] The third aspect of the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof described in the first aspect, or the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof prepared by the preparation method described in the second aspect.

[0061] The fourth aspect of the present invention provides the use of the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof described in the first aspect, or the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof prepared by the preparation method described in the second aspect, in the preparation of tumor-killing drugs.

[0062] According to an embodiment of the present invention, the tumor-killing drug includes a drug that inhibits and kills tumors by consuming asparagine in serum.

[0063] According to an embodiment of the present invention, the tumor is selected from acute lymphoblastic leukemia, melanoma cells, Hodgkin's lymphoma, chronic leukemia, lymphosarcoma cells, and hepatocellular carcinoma.

[0064] The asparaginase mutant conjugate can sustainably reduce asparagine in the body, maintaining serum asparagine levels below 2 μmol / L for up to 21 days or more. The asparaginase mutant conjugate can be used alone or in combination in drugs that inhibit and kill tumors by depleting serum asparagine. These tumors include, but are not limited to, acute lymphoblastic leukemia, Hodgkin's lymphoma, chronic leukemia, lymphosarcoma cells, and hepatocellular carcinoma.

[0065] The asparaginase mutant conjugates of the present invention are used as drugs for treating tumors. The combined drugs can be selected from anti-tumor drugs, drugs for treating and / or preventing complications caused by or associated with tumors, examples of which include: vincristine, cyclophosphamide, cytarabine, daunorubicin, etoposide, steroids (prednisone or dexamethasone), thioguanine and mercaptopurine, etc.

[0066] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0068] Figure 1 The in vivo inhibitory effects of A0, M0, and M4 on tumor cells were shown;

[0069] Figure 2 Drug concentrations in rat serum after single intravenous administration of M0 and M4 are shown;

[0070] Figure 3 Drug concentrations in rat serum after multiple intravenous administration of M0 and M4 are shown;

[0071] Figure 4 The effects of anti-asparaginase antibodies on the pharmacokinetics of pegaspargase (Hengrui Pharmaceuticals) and M4 in rats are shown;

[0072] Figure 5 Shown is the effect of anti-PEG antibodies on the pharmacokinetics of pegaspargase (Hengrui Pharmaceuticals) and M4 in rats. DETAILED DESCRIPTION

[0073] Definition of terms:

[0074] The asparaginase mutant of the present invention is obtained by mutating at least one amino acid accessible on the surface of asparaginase into lysine through genetic engineering.

[0075] In the present invention, the terms "mutation" and "modification" can be used interchangeably.

[0076] The terms "compound", "modifier" and "group" used in the present invention are used interchangeably to refer to polymers used for modification of protein and polypeptide drugs, such as polyethylene glycol.

[0077] As we all know, ordinary polyethylene glycol has a hydroxyl group at each end. If one end is blocked with a methyl group, it becomes methoxy polyethylene glycol (mPEG). Activated polyethylene glycol refers to polyethylene glycol derivatives with functional groups (or activated groups), which are currently mainly used for protein and peptide drug modification.

[0078] The polyethylene glycol used to modify or chemically couple the asparaginase mutants in the present invention is a methoxy polyethylene glycol with an activating group. Activating groups on polyethylene glycol include, but are not limited to, succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate. In specific embodiments, the polyethylene glycol is 5K-SC-PEG and 5K-SPA-PEG, wherein 5K-SC-PEG refers to methoxy polyethylene glycol-succinimidyl carbonate with a molecular weight of 5 kDa, and 5K-SPA-PEG refers to methoxy polyethylene glycol-succinimidyl propionate with a molecular weight of 5 kDa.

[0079] The terms "modification", "coupling", "chemical coupling" and "covalent coupling" used in the present invention are used interchangeably, and refer to the covalent binding of a compound to asparaginase and its mutants by chemical reaction under certain conditions.

[0080] The terms "asparaginase mutant conjugate" and "asparaginase mutant chemical conjugate" are used interchangeably in this application and refer to the modified product obtained by polyethylene glycol-modifying the multi-subunit asparaginase protein. These modified products of asparaginase modified with polyethylene glycol may be referred to herein as "SC-PEG-ASP, SPA-PEG-ASP," and collectively referred to as PEG-ASP or asparaginase PEGylated conjugates. "ASP" is the abbreviation for asparaginase.

[0081] In the present invention, the asparaginase and its mutants are tetrameric proteins, and the number of their covalent coupling groups is the total number of coupling groups of the tetrameric protein.

[0082] The average degree of modification of the asparaginase mutant conjugate in the present invention refers to the number of asparaginase mutant conjugate compounds. Those skilled in the art can determine the number of covalent coupling groups of asparaginase and its mutants by conventional technical means. For example, in Example 3, the average degree of modification of PEGylated asparaginase is determined by utilizing the different UV and RI absorption characteristics of protein and polyethylene glycol.

[0083] In the present invention, "coupling rate is not less than 50%" means that the number of newly formed lysines after mutation that are directed coupled by the compound is not less than 50% of the total number of newly formed lysines. It will be understood by those skilled in the art that they can determine whether a specific amino acid site is modified by a compound molecule by conventional technical means, for example, non-PEGylated and PEGylated asparaginase are enzymatically cleaved by one or more enzymes, and the cleaved fragments are separated and determined by liquid chromatography-mass spectrometry (LC-MS) to generate chromatograms, i.e., peptide maps, of non-PEGylated and PEGylated asparaginase. The peptide maps of asparaginase before and after PEG modification are compared to determine the relative proportion of reduction or disappearance of the peptide peak at the specific amino acid site in the PEGylated asparaginase. In the present invention, if the reduction or disappearance ratio of the cleaved peptide is greater than 80%, it is determined that the specific amino acid site on the peptide is modified by the compound.

[0084] According to some specific embodiments of the present invention, the method for preparing the asparaginase mutant conjugate comprises:

[0085] In a phosphate or carbonate buffer with a buffer ion strength of 10 to 200 mmol / L, a modified pH range of 7.5 to 10.5, and an asparaginase mutant concentration of 2 to 20 mg / ml, a sufficient amount of 5K-SC-PEG and / or 5K-SPA-PEG is added, and the reaction is stirred at room temperature for 1 to 2 hours. The free PEG in the reaction solution is removed by ultrafiltration through an ultrafiltration column to obtain an asparaginase mutant conjugate.

[0086] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0087] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0088] Example 1: Recombinant expression and preparation of asparaginase and its mutants

[0089] (1) The nucleic acid sequences SEQ ID NO: 4, 5, and 6 were used to construct an expression vector plasmid (SEQ ID NO: 4 encodes the amino acid shown in SEQ ID NO: 1, SEQ ID NO: 5 encodes the amino acid shown in SEQ ID NO: 2, and SEQ ID NO: 6 encodes the amino acid shown in SEQ ID NO: 3). The correctly constructed expression plasmid was transformed into an Escherichia coli expression host bacterium, and a recombinant expression strain was obtained by screening. The recombinant strain was inoculated into YT culture medium, induced with IPTG, and then fermented. The fermentation broth was centrifuged and the cells were collected and stored at -20°C for later use.

[0090] (2) After the fermentation cells were lysed, asparaginase was isolated and concentrated using a series of purification chromatography columns and methods. Briefly, asparaginase was adsorbed onto an ion exchange resin column and eluted with varying salt concentrations. The eluted sample was collected and further purified by passing it through a phenyl pyrrolidone column, eluted with varying salt concentrations, and the asparaginase-containing sample was collected. Finally, the sample was refined and purified using size exclusion chromatography. The purity of the asparaginase preparation was greater than 98% as assessed by SDS-PAGE, and the integrity of the recombinant enzyme amino acid sequence was verified by LC-MS.

[0091] SEQ ID NO:4 is as follows:

[0092]

[0093] The sequence of SEQ ID NO:5 is as follows:

[0094]

[0095] The sequence of SEQ ID NO:6 is as follows:

[0096]

[0097] Example 2: Preparation of PEGylated conjugates of asparaginase and its mutants

[0098] In carbonate buffer with a buffer ionic strength of 100 mmol / L and a modified pH of 9.0, a protein concentration of 20 mg / ml was added with a predetermined amount of 5K-SC-PEG or 5K-SPA-PEG (protein to 5K-SC-PEG or 5K-SPA-PEG mass ratio of 1:10). The reaction was stirred at room temperature for 2 hours. Free PEG was removed from the reaction solution by size exclusion chromatography, and the solution was sterilely treated and aliquoted.

[0099] Table 1 Comparison table of asparaginase PEGylated conjugates

[0100]

[0101] Example 3: Average modification degree of asparaginase and its mutant PEGylated conjugates

[0102] Kunitani et al. (Journal of Chromatography A, 1991, 588(1-2): 125-137) found that the RI and UV absorption values ​​of proteins were linearly related to protein concentration; the RI absorption value of PEG was also linearly related to its concentration, and after PEG modification, the PEG portion and the protein portion of the protein did not interfere with each other's respective absorption values ​​in RI and / or UV. Using the above mechanism, the average modification degree of PEG-modified proteins was determined by SEC-HPLC differential refractive index and UV coupling. The respective contents of the protein portion and the PEG portion in the PEG-protein were calculated using standard curves of known protein and PEG contents, and the average modification degree was then obtained by the molar ratio of protein to PEG molecules.

[0103] Table 2 Average modification degree of asparaginase mutant conjugates

[0104] sample M0 M1 M2 M3 M4 M5 Average modification 34.0 34.4 50.0 48.4 48.8 49.6

[0105] Example 4: Identification of modification sites of M0, M2 and M4

[0106] PEGylation of asparaginase involves covalent bonding of PEG to the amino group of the lysine in asparaginase. Lys-C cannot recognize PEG-modified lysines. Therefore, the modification site can be inferred by comparing the reduction in peptides before and after asparaginase modification. Samples before and after modification are digested with Lys-C and analyzed by HPLC-MS to identify the modification site.

[0107] Table 3-1 Molecular weight of enzyme-cleaved peptides before and after M0 modification

[0108]

[0109] Table 3-2 Molecular weight of M2 cleaved peptides before and after modification

[0110]

[0111] Table 3-3 Molecular weight of peptide fragments before and after M4 modification

[0112]

[0113]

[0114] Example 5: In vitro enzyme activity assays of A0, A1, A2, M0, M2, and M4

[0115] Asparaginase hydrolyzes L-asparagine to release ammonia, and the enzyme activities of A0, A1, A2, M0, M2, and M4 described in the previous examples were determined by colorimetric analysis of ammonia with Nessler's reagent. Briefly, 50 μL of enzyme solution was diluted to a certain ratio with 100 mM PB buffer (pH 8.0), mixed with a 100 mM PB, 20 mM asparagine solution, and incubated at 37°C for 10 min. The reaction was terminated by the addition of trichloroacetic acid, and 100 μL of Nessler's reagent was added for colorimetric development. The absorbance of the reaction solution was measured at a wavelength of 450 nm. The activity was calculated using a calibration curve obtained using ammonium sulfate as a reference. One unit (U) of enzyme activity is defined as the amount of enzyme required to generate 1 micromole of ammonia in 1 minute at the optimal reaction temperature of 37°C and pH 8.0. Table 4 shows the in vitro enzyme activities of A0, A1, A2, M0, M2, and M4.

[0116] Table 4

[0117] sample A0 A1 A2 M0 M2 M4 Enzyme activity 190.1 U / mg 179.0 U / mg 189.3 U / mg 110.4 U / mg 115.7 U / mg 106.2 U / mg

[0118] The results in Table 4 show that the asparaginase mutant conjugate still maintains the activity of catalyzing the hydrolysis of asparagine.

[0119] Example 6: In vitro inhibitory effects of M0, M2 and M4 on different tumor cells

[0120] HL-60 (human acute promyelocytic leukemia cells), THP-1 (human mononuclear macrophages), Raji (human lymphoma cells), and L1210 (mouse lymphocytic leukemia cells) were selected, and the toxicity of M0, M2, and M4 on tumor cells was determined using the CCK-8 method. 90ul of cell suspension was added to a 96-well plate and cultured at 37°C, 5% CO2, and saturated humidity for 12 hours. Different concentrations of drugs were added and placed in an incubator for 10 hours. The plates were then taken out and 10ul of CCK-8 was added. The plates were cultured under the same conditions for 2 hours, and the absorbance at a wavelength of 450nm was measured to investigate the inhibition rate of different drug concentrations and calculate the IC 50 .

[0121] Table 5 In vitro inhibitory effects of A0, M0, M2 and M4 on different tumor cells

[0122]

[0123] The results in Table 5 show that the asparaginase mutant conjugate can inhibit the growth of HL-60, THP-1, Raji, and L1210 tumor cells in vitro.

[0124] Example 7: Inhibitory effects of A0, M0 and M4 on tumor cells in vivo

[0125] L1210 cells (mouse lymphocytic leukemia cells) in the logarithmic growth phase were collected, counted, and cell suspension was prepared. The cells were subcutaneously injected into the axilla of the right upper limb of DBA / 2 mice at a rate of 2×10 6 After 24 hours, the mice were randomly divided into four groups: PBS, A0, M0, and M4. Each group had 10 mice. The mice were injected intravenously with a dose of 50 U / kg once a week for 50 days. The results are shown in the attached figure. Figure 1 .

[0126] Result: By Figure 1 It can be seen that mice in the control group began to die on the 30th day, and all mice died on the 39th day; mice in the A0 group began to die on the 33rd day, and the survival rate of mice on the 50th day was 1 / 5; mice in the M0 group began to die on the 34th day, and the survival rate of mice on the 50th day was 7 / 10; mice in the M4 group began to die on the 41st day, and the survival rate of mice on the 50th day was 4 / 5.

[0127] Example 8: Single-dose pharmacokinetic study of A0, M0 and M4 in rats

[0128] To evaluate the pharmacokinetics of A0, M0, and M4 in rats, 24 male rats were randomly divided into four groups (n=6 in each group): PBS group (control group), A0 group, M0 group, and M4 group. Each group received a single intravenous injection of 200 U / kg. Blood samples were collected 1 hour before administration and 3, 7, 11, 17, and 21 days after the first administration. Serum asparaginase levels were determined by the enzyme activity method (α-KG method) (Fernandez, International journal of clinical and experimental medicine, 2013, 6(7):478).

[0129] Result: By Figure 2 It can be seen that after a single intravenous administration, the asparaginase levels in the serum of the two groups of rats showed a downward trend, and the asparaginase level in the serum of the rats in the M4 group was always higher than that in the M0 group.

[0130] Example 9: Multiple pharmacokinetic and immunogenicity studies of A0, M0 and M4 in rats

[0131] The pharmacokinetics and immunogenicity of A0, M0, and M4 were evaluated in rats. 18 male rats were randomly divided into four groups, each with 6 rats: PBS group (control group), A0 group, M0 group, and M4 group. The rats were administered intravenously at a dose of 70 U / kg once a week for four times. Blood was collected 3 and 7 days after each administration. The serum asparaginase content was measured by enzyme activity (α-KG method) (High-throughput asparaginase activity assay in serum of children with leukemia). Anti-asparaginase protein antibodies and anti-PEG antibodies in serum were measured by ELISA.

[0132] result: Figure 3 Results showed that during the four intravenous administrations, serum asparaginase levels in the M4 group increased with each dose, and after the fourth (final) dose, serum asparaginase levels increased significantly compared to the first dose. In the M0 group, serum asparaginase levels did not differ significantly after each dose. Throughout the administration period, serum asparaginase levels in the M4 group remained higher than those in the M0 group. LC-MS analysis revealed no asparagine was detected in the serum of either group.

[0133] At a serum dilution of 1:10, one animal (1 / 6) in the M0 group developed anti-PEG antibodies 7 days after the second dose, 6 / 6 3 days after the third dose, 5 / 6 7 days after the third dose, 5 / 6 3 days after the fourth dose, and 5 / 6 7 days after the fourth dose. In the M4 group, one animal (1 / 6) developed anti-PEG antibodies 3 days after the first dose, 1 / 6 7 days after the third dose, 1 / 6 3 days after the fourth dose, and 2 / 6 7 days after the fourth dose. This suggests that M4 is superior to M0 in immunogenicity, likely due to the increased degree of asparaginase PEGylation, which weakens the protein's immunogenicity (Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins). Furthermore, since PEG modification reduces the surface charge of proteins, and since cells (such as macrophages and B cells) carry a negative surface charge, M4's affinity for immune cells decreases, weakening the immune response in vivo. At a serum dilution factor of 1:50, the anti-PEG antibody test in the sera of the two groups of rats was negative, indicating that the anti-PEG antibody titers in the two groups of rats were low.

[0134] The serum antibody titers of the rats in group A0 reached 1:100 after two weeks, and the rats in groups M0 and M4 did not produce antibodies against asparaginase protein after multiple intravenous administration.

[0135] Example 10: Effect of anti-asparaginase antibodies on the pharmacokinetics of pegaspargase (Hengrui Pharmaceuticals) and M4 in rats

[0136] Twelve male SD rats were subcutaneously administered asparaginase (Qianhong Pharmaceutical) once daily at 100 U / kg for three days. After a one-week rest, the anti-ASP antibody titer in the rat serum was determined by ELISA. When the titer reached 1:100, the asparaginase activity in the serum disappeared. The rats were then randomly divided into two groups (pegaspargase group and M4 group), each with six rats (n=6). The rats were injected intravenously at a dose of 200 U / kg. Blood samples were collected one, three, five, and seven days after administration. The serum asparaginase content was determined by the enzyme activity method (α-KG method) (Fernandez, International journal of clinical and experimental medicine, 2013, 6(7):478).

[0137] result: Figure 4The results showed that the asparaginase levels in the serum of the two groups of rats showed a downward trend after intravenous injection of pegaspargase (Hengrui) and M4, and the asparaginase level in the serum of the rats in the M4 group was always higher than that in the pegaspargase group. One day after administration, the asparaginase level in the serum of the rats in the M4 group was 2501.7±435.7 mU / ml, and the asparaginase level in the serum of the rats in the pegaspargase group was 1473.8±139.9 mU / ml.

[0138] Example 11: Binding of pegaspargase (Hengrui Pharmaceuticals) and M4 to anti-asparaginase antibodies in vitro

[0139] Four rats were subcutaneously injected with asparaginase (Qianhong Pharmaceutical) and Freund's adjuvant once a week at 2 mg / kg each time. One month after administration, the rats were killed and their serum was collected. Anti-asparaginase antibodies were prepared by affinity chromatography. The binding of pegaspargase (Hengrui Pharmaceutical) and M4 to anti-asparaginase antibodies was detected by ELISA.

[0140] Table 6

[0141]

[0142] The results in Table 6 show that in the Elisa experiment, after the same amount of pegaspargase and M4 were coated and incubated with anti-asparaginase antibody, the binding force between pegaspargase and mouse anti-asparaginase antibody was stronger than that between pegaspargase and M4.

[0143] Example 12: Effect of anti-PEG antibodies on the pharmacokinetics of pegaspargase (Hengrui Pharmaceuticals) and M4 in rats

[0144] Twelve male SD rats were subcutaneously administered 1.5 mg / kg of PEGylated phenylalanine lyase. Blood was collected on the 7th day after administration to test for PEG antibodies. After confirming that the serum PEG antibodies were positive, the rats were randomly divided into two groups and injected intravenously with pegaspargase (Hengrui Pharmaceuticals) and M4 at a dose of 100 U / kg. Blood was collected on the 1st, 3rd, 5th, and 7th day after administration. After the 7th day, the administration was repeated once, and blood was collected on the 1st, 3rd, 5th, and 7th day after administration. The serum asparaginase content was measured by enzyme activity method (α-KG method) (Fernandez, International journal of clinical and experimental medicine, 2013, 6(7):478).

[0145] Results: PEGylated phenylalanine lyase is a highly immunogenic metabolic enzyme. After subcutaneous injection, rats produce a large amount of anti-PEG antibodies. In this experiment, the rats were randomly divided into two groups and injected intravenously with pegaspargase (Hengrui Pharmaceuticals) and M4 respectively. No asparaginase was detected in the serum of the two groups of rats 1 day, 3 days, 5 days, and 7 days after administration. The asparaginase content in the serum of the two groups of rats was as follows: Figure 5 As shown, it can be seen that the asparaginase content in the serum of rats in the M4 group was significantly higher than that in the pegaspargase group.

[0146] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0147] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention. Sequence Listing <110> Chongqing Paijin Biotechnology Co., Ltd. <120> Directed chemical coupling asparaginase mutant and its preparation method and application <130> PIDC3204372 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 326 <212> PRT <213> Escherichia coli <400> 1 Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly 1 5 10 15 Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Ala Gly Lys Val Gly Val 20 25 30 Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 35 40 45 Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Asp 50 55 60 Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 65 70 75 80 Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 85 90 95 Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 100 105 110 Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 115 120 125 Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg 130 135 140 Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 145 150 155 160 Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 165 170 175 Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 180 185 190 Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 195 200 205 Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 210 215 220 Asp Leu Pro Ala Lys Ala Leu Val Asp Ala Gly Tyr Asp Gly Ile Val 225 230 235 240 Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Thr Val Phe Asp Thr 245 250 255 Leu Ala Thr Ala Ala Lys Asn Gly Thr Ala Val Val Arg Ser Ser Arg 260 265 270 Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 275 280 285 Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 290 295 300 Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys Asp Pro Gln Gln Ile Gln 305 310 315 320 Gln Ile Phe Asn Gln Tyr 325 <210> 2 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Asparaginase with mutations at D64K, N143K, D233K, and Q317K <400> 2 Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly 1 5 10 15 Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Ala Gly Lys Val Gly Val 20 25 30 Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 35 40 45 Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Lys 50 55 60 Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 65 70 75 80 Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 85 90 95 Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 100 105 110 Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 115 120 125 Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Lys Arg 130 135 140 Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 145 150 155 160 Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 165 170 175 Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 180 185 190 Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 195 200 205 Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 210 215 220 Asp Leu Pro Ala Lys Ala Leu Val Lys Ala Gly Tyr Asp Gly Ile Val 225 230 235 240 Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Thr Val Phe Asp Thr 245 250 255 Leu Ala Thr Ala Ala Lys Asn Gly Thr Ala Val Val Arg Ser Ser Arg 260 265 270 Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 275 280 285 Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 290 295 300 Leu Leu Gln Leu Ala Leu Thr Gln Thr Lys Asp Pro Lys Gln Ile Gln 305 310 315 320 Gln Ile Phe Asn Gln Tyr 325 <210> 3 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Asparaginase with mutations at D64K, D233K, and Q317K sites <400> 3 Leu Pro Asn Ile Thr Ile Leu Ala Thr Gly Gly Thr Ile Ala Gly Gly 1 5 10 15 Gly Asp Ser Ala Thr Lys Ser Asn Tyr Thr Ala Gly Lys Val Gly Val 20 25 30 Glu Asn Leu Val Asn Ala Val Pro Gln Leu Lys Asp Ile Ala Asn Val 35 40 45 [[ID=三十八]]Lys Gly Glu Gln Val Val Asn Ile Gly Ser Gln Asp Met Asn Asp Lys 50 55 60 Val Trp Leu Thr Leu Ala Lys Lys Ile Asn Thr Asp Cys Asp Lys Thr 65 70 75 80 Asp Gly Phe Val Ile Thr His Gly Thr Asp Thr Met Glu Glu Thr Ala 85 90 95 Tyr Phe Leu Asp Leu Thr Val Lys Cys Asp Lys Pro Val Val Met Val 100 105 110 Gly Ala Met Arg Pro Ser Thr Ser Met Ser Ala Asp Gly Pro Phe Asn 115 120 125 Leu Tyr Asn Ala Val Val Thr Ala Ala Asp Lys Ala Ser Ala Asn Arg 130 135 140 Gly Val Leu Val Val Met Asn Asp Thr Val Leu Asp Gly Arg Asp Val 145 150 155 160 Thr Lys Thr Asn Thr Thr Asp Val Ala Thr Phe Lys Ser Val Asn Tyr 165 170 175 Gly Pro Leu Gly Tyr Ile His Asn Gly Lys Ile Asp Tyr Gln Arg Thr 180 185 190 Pro Ala Arg Lys His Thr Ser Asp Thr Pro Phe Asp Val Ser Lys Leu 195 200 205 Asn Glu Leu Pro Lys Val Gly Ile Val Tyr Asn Tyr Ala Asn Ala Ser 210 215 220 Asp Leu Pro Ala Lys Ala Leu Val Lys Ala Gly Tyr Asp Gly Ile Val 225 230 235 240 Ser Ala Gly Val Gly Asn Gly Asn Leu Tyr Lys Thr Val Phe Asp Thr 245 250 255 Leu Ala Thr Ala Ala Lys Asn Gly Thr Ala Val Val Arg Ser Ser Arg 260 265 270 Val Pro Thr Gly Ala Thr Thr Gln Asp Ala Glu Val Asp Asp Ala Lys 275 280 285 Tyr Gly Phe Val Ala Ser Gly Thr Leu Asn Pro Gln Lys Ala Arg Val 290,295,300 Leu Leu Gln Leu Ala Leu Thr Thr Gln Lys Asp Pro Lys Gln Ile Gln 305 310 315 320 Gln Ile Phe Asn Gln Tyr 325 <210> 4 <211> 1047 <212> DNA <213> Escherichia coli <400> 4 atggagttct ttaagaaac cgcgctggcg gcgctggtga tggttttcag cggtgcggcg 60 ctggcgctgc cgaacatcac cattctggcg accggtggca ccattgcggg tggcggtgac 120 agcgcgacca agcaacta caccgcgggt aaagtggggcg ttgagaacct ggtgaacgcg 180 gttccgcagc tgaggatat cgcgaacgtg aaggtgaac aggtgttaa cattggcagc 240 caagacatga acgacgatgt ttggctgacc ctggcgaaga aaatcaacac cgactgcgat 300 aaaaccgacg gtttcgtgat tacccacggc accgatacca tggaggaaac cgcgtacttt 360 ctggacctga ccgtgaagtg cgataaaccg gtggttatgg ttggtgcgat gcgtccgagc 420 accagcatga gcgcggatgg tccgttcaac ctgtataacg cggtggttac cgcggcggat 480 aaggcgagcg cgaaccgtgg tgttctggtg gttatgaacg acaccgtgct ggacggccgt 540 gatgttacca agaccaacac caccgatgtg gcgaccttca aaagcgttaa ctacggtccg 600 ctgggctata tccacaacgg caagattgac tatcagcgta ccccggcgcg taaacacacc 660 agcgacaccc cgtttgatgt gagcaagctg aacgagctgc cgaaagtggg tatcgtttac 720 aactatgcga acgcgagcga tctgccggcg aaagcgctgg ttgacgcggg ttacgatggc 780 attgtgagcg cgggcgttgg taacggcaac ctgtataaga ccgtgtttga taccctggcg 840 accgcggcga aaaacggtac cgcggtggtt cgtagcagcc gtgttccgac cggtgcgacc 900 acccaggacg cggaagtgga cgatgcgaag tacggtttcg ttgcgagcgg caccctgaac 960 ccgcaaaaag cgcgtgttct gctgcagctg gcgctgaccc aaaccaagga cccgcagcaa 1020 atccagcaaa tttttaacca atattaa 1047 <210> 5 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding asparaginase with mutations at D64K, N143K, D233K, and Q317K sites <400> 5 atggagttct ttaagaaaac cgcgctggcg gcgctggtga tgggtttcag cggtgcggcg 60 ctggcgctgc cgaacatcac cattctggcg accggtggca ccattgcggg tggcggtgac 120 agcgcgacca agagcaacta caccgcgggt aaagtgggcg ttgagaacct ggtgaacgcg 180 gttccgcagc tgaaggatat cgcgaacgtg aaaggtgaac aggtggttaa cattggcagc 240 caagacatga acgacaaggt ttggctgacc ctggcgaaga aaatcaacac cgactgcgat 300 aaaaccgacg gtttcgtgat tacccacggc accgatacca tggaggaaac cgcgtacttt 360 ctggacctga ccgtgaagtg cgataaaccg gtggttatgg ttggtgcgat gcgtccgagc 420 accagcatga gcgcggatgg tccgttcaac ctgtataacg cggtggttac cgcggcggat 480 aaggcgagcg cgaaacgtgg tgttctggtg gttatgaacg acaccgtgct ggacggccgt 540 gatgttacca agaccaacac caccgatgtg gcgaccttca aaagcgttaa ctacggtccg 600 ctgggctata tccacaacgg caagattgac tatcagcgta ccccggcgcg taaacacacc 660 agcgacaccc cgtttgatgt gagcaagctg aacgagctgc cgaaagtggg tatcgtttac 720 aactatgcga acgcgagcga tctgccggcg aaagcgctgg ttaaggcggg ttacgatggc 780 attgtgagcg cgggcgttgg taacggcaac ctgtataaga ccgtgtttga taccctggcg 840 accgcggcga aaaacggtac cgcggtggtt cgtagcagcc gtgttccgac cggtgcgacc 900 acccaggacg cggaagtgga cgatgcgaag tacggtttcg ttgcgagcgg caccctgaac 960 ccgcaaaaag cgcgtgttct gctgcagctg gcgctgaccc aaaccaagga cccgaagcaa 1020 atccagcaaa tttttaacca atattaa 1047 <210> 6 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Asparaginase nucleic acid sequence encoding mutations at D64K, D233K, and Q317K sites <400> 6 atggagttct ttaagaaaac cgcgctggcg gcgctggtga tgggtttcag cggtgcggcg 60 ctggcgctgc cgaacatcac cattctggcg accggtggca ccattgcggg tggcggtgac 120 agcgcgacca agagcaacta caccgcgggt aaagtgggcg ttgagaacct ggtgaacgcg 180 gttccgcagc tgaaggatat cgcgaacgtg aaaggtgaac aggtggttaa cattggcagc 240 caagacatga acgacaaggt ttggctgacc ctggcgaaga aaatcaacac cgactgcgat 300 aaaaccgacg gtttcgtgat tacccacggc accgatacca tggaggaaac cgcgtacttt 360 ctggacctga ccgtgaagtg cgataaaccg gtggttatgg ttggtgcgat gcgtccgagc 420 accagcatga gcgcggatgg tccgttcaac ctgtataacg cggtggttac cgcggcggat 480 aaggcgagcg cgaaccgtgg tgttctggtg gttatgaacg acaccgtgct ggacggccgt 540 gatgttacca agaccaacac caccgatgtg gcgaccttca aaagcgttaa ctacggtccg 600 ctgggctata tccacaacgg caagattgac tatcagcgta ccccggcgcg taaacacacc 660 agcgacaccc cgtttgatgt gagcaagctg aacgagctgc cgaaagtggg tatcgtttac 720 aactatgcga acgcgagcga tctgccggcg aaagcgctgg ttaaggcggg ttacgatggc 780 attgtgagcg cgggcgttgg taacggcaac ctgtataaga ccgtgtttga taccctggcg 840 accgcggcga aaaacggtac cgcggtggtt cgtagcagcc gtgttccgac cggtgcgacc 900 acccaggacg cggaagtgga cgatgcgaag tacggtttcg ttgcgagcgg caccctgaac 960 ccgcaaaaag cgcgtgttct gctgcagctg gcgctgaccc aaaccaagga cccgaagcaa 1020 atccagcaaa tttttaacca atattaa 1047

Claims

1. An asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The asparaginase mutant conjugate comprises an asparaginase mutant and a group chemically coupled thereto that is capable of reducing in vivo immunogenicity and prolonging in vivo half-life. The asparaginase mutant is a mutant in which the amino acid at at least one site in the unmutated asparaginase is mutated to lysine, and the group capable of reducing in vivo immunogenicity and prolonging in vivo half-life is chemically coupled to the asparaginase mutant via the lysine in the asparaginase mutant; The amino acid mutation sites in the asparaginase mutant are D64K, N143K, D233K and Q317K, and the amino acid sequence of the asparaginase mutant is shown in SEQ ID NO: 2; or The amino acid mutation sites in the asparaginase mutant are D64K, D233K and Q317K, and the amino acid sequence of the asparaginase mutant is shown in SEQ ID NO: 3; The group capable of reducing in vivo immunogenicity and prolonging in vivo half-life is directionally coupled by forming an amide bond with the amino group of lysine in the asparaginase mutant, and the group is methoxy polyethylene glycol with an activated group; The activating group is selected from succinimidyl carbonate, succinimidyl propionate, succinimidyl acetate, and succinimidyl succinate.

2. The asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The non-mutated asparaginase is derived from Erwinia or Escherichia coli.

3. The asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The amino acid sequence of the unmutated asparaginase is shown in SEQ ID NO:

1.

4. The asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The molecular weight of polyethylene glycol is 2KDa-20KDa.

5. The asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The molecular weight of the polyethylene glycol is 5KDa.

6. The method for preparing the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized in that: The preparation method comprises mixing an asparaginase mutant with polyethylene glycol in a buffer solution to produce a coupling reaction, so as to obtain an asparaginase mutant conjugate; The polyethylene glycol is selected from succinimidyl carbonate polyethylene glycol (SC-PEG), succinimidyl propionate polyethylene glycol (SPA-PEG), succinimidyl acetate polyethylene glycol (SCM-PEG), and succinimidyl succinate polyethylene glycol (SS-PEG).

7. The preparation method according to claim 6, characterized in that When the coupling reaction occurs, the pH of the buffer solution is 7.5-10.

5.

8. The preparation method according to claim 6, characterized in that The reaction time is 1-2h.

9. The preparation method according to claim 6, characterized in that The buffer solution has an ionic strength of 10 to 200 mmol / L.

10. The preparation method according to claim 6, characterized in that The buffer solution is selected from at least one of phosphate, carbonate and borate.

11. The preparation method according to claim 6, characterized in that The weight ratio of the asparaginase mutant to polyethylene glycol is not less than 1:

5.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof prepared by the preparation method according to any one of claims 6 to 11.

13. Use of the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, or the asparaginase mutant conjugate or a pharmaceutically acceptable salt thereof prepared by the preparation method according to any one of claims 6 to 11 in the preparation of a tumor-killing drug; The tumor is selected from acute lymphoblastic leukemia, Hodgkin's lymphoma, and chronic leukemia.

14. The use according to claim 13, characterized in that The tumor-killing drug includes a drug that inhibits and kills tumors by consuming asparagine in serum.

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