WO2016106941A1 - 聚乙二醇定点修饰的门冬酰胺酶及其制备方法与应用 - Google Patents

聚乙二醇定点修饰的门冬酰胺酶及其制备方法与应用 Download PDF

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WO2016106941A1
WO2016106941A1 PCT/CN2015/072167 CN2015072167W WO2016106941A1 WO 2016106941 A1 WO2016106941 A1 WO 2016106941A1 CN 2015072167 W CN2015072167 W CN 2015072167W WO 2016106941 A1 WO2016106941 A1 WO 2016106941A1
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asparaginase
polyethylene glycol
modified
peg
asp
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PCT/CN2015/072167
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English (en)
French (fr)
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马永
王俊
吴鼎龙
闻兴元
王和
徐春林
陈一飞
王耀方
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江苏众红生物工程创药研究院有限公司
常州京森生物医药研究所有限公司
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Priority to US15/522,445 priority Critical patent/US10537620B2/en
Publication of WO2016106941A1 publication Critical patent/WO2016106941A1/zh
Priority to US16/746,264 priority patent/US11103565B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)

Definitions

  • the invention relates to a polyethylene glycol modified protein drug, in particular to a polyethylene glycol modified asparaginase, a preparation method thereof and the application thereof in the preparation of a medicament, and the application thereof in clinical treatment.
  • Asparaginase is a protein with L-asparagine aminohydrolase activity (commonly known as L-asparaginase, L-asparaginase, or asparaginase), active form A homotetramer structure consisting of 4 subunits, each subunit consisting of 326 amino acids.
  • L-asparaginase is effective in the treatment of acute lymphoblastic leukemia (ALL) in children or adults.
  • ALL acute lymphoblastic leukemia
  • L-asparaginase-containing drugs have been used in combination with chemotherapy to treat NK/T-cell lymphoma, and have achieved good therapeutic effects.
  • NK/T-cell lymphoma is a special type of non-Hodgkin's lymphoma, which is more common in Asia and Latin America, and its incidence is relatively high in China. According to the site of tumorigenesis, NK/T cell lymphoma can be divided into nasal NK/T cell lymphoma and non-nasal NK/T cell lymphoma.
  • L-asparaginase is also used to treat Hodgkin's disease, acute myeloid leukemia, acute myeloid monocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum sarcoma and melanoma (Kotzia and labrou, J. Biotechnol. 127 (2007) 657-669).
  • L-asparaginase was originally purified from several organisms, including E. coli and Erwinia carotovora. In mammals, slightly higher amounts of L-asparaginase were found only in guinea pigs (Cavioidea superfamily) and certain New World monkeys. Because it is derived from exogenous organisms, it is a foreign protein for humans, has strong immunogenicity, and is clinically common for progressive immune response and systemic allergic reactions, which limits its clinical application. (Zhang Lina, Gong Daohua. Jiangsu Medicine. L-asparaginase in the treatment of acute lymphoblastic leukemia in children. 2005, 31 (5): 392; Wang Ningling, Liu Zhiqi et al.
  • the prior art has improved the drug in various aspects, for example: 1 for L-asparaginase-sensitive patients of Escherichia coli, replacing the Escherichia coli L-asparaginase with the enzyme of Escherichia coli; 2L-asparaginase For example, preparation of its liposome and the like; 3 structural modification by protein engineering, mutation of E. coli L-asparaginase antigen site to reduce immunogenicity; 4 chemical modification according to L-asparaginase structure. Modification with polyethylene glycol (PEG) is one of chemical modification.
  • PEG polyethylene glycol
  • Polyethylene glycol is a linear, uncharged polymer that is free to curl in solution, with non-toxic, weak antigenicity and good biocompatibility. Use it to covalently modify proteins, which can increase the in vivo circulating half-life of proteins. And reducing its antigenicity, increasing the solubility of proteins and altering the biological distribution of proteins in the human body. Since 1977, Abuchowski, Davis (J. Biol. Chem. 1977, 252: 3578-3581.) et al. first reported the use of PEG to modify proteins, PEG modification technology has been widely used in biomedical and biotechnology fields, PEG has It is widely used in the modification of proteins and peptide drugs. At present, protein PEGylation technology has become one of the most effective methods for reducing the immunogenicity of protein biopharmaceuticals and improving their pharmacokinetic/pharmacodynamic properties, and has been approved by the FDA for use in pharmaceuticals, foods and cosmetics.
  • polyethylene glycol modification technology has undergone several decades of development and is now mature. However, it is not possible to find a universal polyethylene glycol modifier and modification method to modify all protein drugs.
  • polyethylene glycol modifiers are the most important factors affecting the physical properties of modification, biological activity in vitro and in vivo, pharmacokinetics, pharmacodynamics and clinical manifestations of modified products. Therefore, the choice of modifier (type of modifier, molecular weight) and control of modification reactions play an important role in polyethylene glycol modification technology.
  • the activity of a pegylated protein drug is reduced compared to an unmodified native protein, and generally the protein activity after modification is only 30% to 40% or even lower of the original protein.
  • Schering-Plough's PEG-Intron uses a PEG with a molecular weight of 5000 to modify interferon, which is modified to have only 8% of the original protein.
  • Yoshihiro et al. modified E. coli asparaginase with a branched PEG (2-0-methoxypolyethylene glycol-4,6-dichloro-s-triazine) having a molecular mass of 750, 1900, and 5000 U, respectively.
  • Bailon et al. modified Interferon- ⁇ -2a with a branched 40 kDa PEG, and the resulting single modification achieved a longer circulating half-life, but retained only 7% of in vitro activity (Bailon P, Bioconjugate Chem., 2001, 12: 195-202. ).
  • Oncaspar (Enzon Inc.), a product of L-asparaginase modified with polyethylene glycol, has been on the market since 1994 and has been approved as a first-line therapy for ALL in children and adults since 2006.
  • Oncaspar is modified with succinimide succinate (SS-PEG)
  • SS-PEG succinimide succinate
  • the product is not uniform, the modification product has a large loss of activity, and contains defects such as an ester bond which is sensitive to hydrolysis of the enzyme or unstable at a slightly alkaline pH (U.S. Patent No. 4,670,417).
  • These properties significantly reduce stability in vitro and in vivo and have a greater side effect.
  • the PEG-modified L-asparaginase currently listed in China is only the “Pendoorase” of Hengrui Company. It is a generic drug of Oncaspar, and the above problems also exist.
  • Both products are products of random modification, so the modified product is not uniform and the composition of the product consists of coupling different numbers of PEG molecules.
  • the active part of asparaginase has a large steric hindrance due to more modification sites, so the activity has a large loss, and only 60% of the activity of the original protein is retained.
  • Related studies have been carried out on the N-terminal fixed-point modification of asparaginase, and the obtained modified product has only about 36% of the original protein. (Chi Shaoping, Wang Bochu, Preliminary study on chemical modification of L-asparaginase by drug biotechnology polyethylene glycol aldehyde).
  • the present invention aims to provide a polyethylene glycol fixed-point modified aspartame which has improved stability, reduced immunogenicity, and high activity and uniform composition.
  • Amidase (ASP) its preparation method and its use in the preparation of a medicament for treating diseases such as childhood or adult acute lymphoblastic leukemia and non-Hodgkin's lymphoma.
  • a first object of the present invention is to provide a polyethylene glycol site-directed modified L-asparaginase which is coupled to the N-terminal amino group of one or two subunits of L-asparaginase,
  • the polyethylene glycol has a molecular weight of 30-40 kDa, polyethylene glycol is branched, and the activating group is an aldehyde group.
  • the polyethylene glycol is coupled to the N-terminal primary amino group of the two subunits of L-asparaginase, the polyethylene glycol having a molecular weight of 30 kDa or 40 kDa, and the polyethylene glycol being branched.
  • the activating group is acetaldehyde, propionaldehyde, butyraldehyde or valeraldehyde.
  • the structural formula of the polyethylene glycol site-modified L-asparaginase is as follows:
  • R is H or a C1-C4 alkyl group; n is an integer value from 100 to 500, p is an integer from 1 to 4; AA is an N-terminal L-amino acid residue, m is an integer from 0 to 5, and s is 1 Or 2.
  • the alkyl group is a methyl group
  • n is an integer value between 320 and 455
  • p is 2
  • m is 0.
  • the polyethylene glycol preferably has a molecular weight of 40 KDa.
  • the PEGylated L-asparaginase purified product is preferably a second modified product.
  • the reducing agent is preferably sodium cyanoborohydride.
  • a third object of the present invention is to provide a use of the above-mentioned polyethylene glycol site-directed modified ASP for the preparation of a medicament for treating diseases such as childhood or adult acute lymphoblastic leukemia and non-Hodgkin's lymphoma. It has been reported in the literature that PEG-modified asparaginase is used in combination with chemotherapy for NK/T cell lymphoma or acute lymphocytic leukemia, and has achieved good results. Therefore, the PEG-modified asparaginase prepared by the site-directed modification technique provided in the present invention can also be used for the combined treatment of NK/T cell lymphoma or acute lymphocytic leukemia.
  • a fourth object of the present invention is to provide a polyethylene glycol site-directed modified ASP or a pharmaceutically acceptable salt or complex thereof.
  • the complex refers to a combination of two or more different substances.
  • a fifth object of the present invention is to provide a pharmaceutical composition comprising the above polyethylene glycol site-modified ASP or a pharmaceutically acceptable salt or complex thereof and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition is a liquid injection or a lyophilized powder injection.
  • the excipients include pharmaceutically acceptable carriers and/or excipients and the like.
  • the site-modified L-asparaginase and pharmaceutical compositions thereof are administered by the muscle, vein or subcutaneous route.
  • the polyethylene glycol fixed-point modified ASP provided by the invention has the advantages of significantly prolonging half-life, uniformity of structural stability, and the like, and the biological activity thereof is improved to a greater extent.
  • FIG. 1 High performance liquid phase analysis of different PEG-ASP conjugates.
  • Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di), Y-PALD-30K-ASP (Mono) and Y-PALD-30K-ASP (Di) for analysis.
  • Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di)
  • Y-PALD-30K-ASP (Mono)
  • the purity of Y-PALD-30K-ASP (Di) can reach 98% or more, slightly higher than the purity of Pembellase 97%.
  • Y-PALD-40K-ASP (Di) Y-PALD-30K-ASP (Di) has a higher molecular weight than the perigase.
  • FIG. 1 SDS-PAGE analysis of different PEG-ASP conjugates.
  • Lanes 1-4 of Figure 2a are high molecular weight Marker, Pembellase, Y-PALD-40K-ASP (Mono), and Y-PALD-40K-ASP (Di), respectively.
  • Lanes 1-3 of Figure 2b are otherwise high molecular weight Marker, Y-PALD-30K-ASP (Di) and Y-PALD-30K-ASP (Mono).
  • ASP is a homotetramer composed of four subunits
  • four subunits will depolymerize.
  • Each subunit will show a different band in SDS-PAGE due to the degree of modification.
  • the enzymatic enzyme showed a diffuse band, indicating that the protein was once poor, compared with Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di).
  • the two bands with modified subunits and unmodified subunits are equally high.
  • the larger molecular weight bands shown in lanes 2 and 3 in lanes 3 and 4 of Figure 2a indicate that they have been modified.
  • the ASP single subunit, a smaller molecular weight band represents an unmodified ASP single subunit.
  • FIG. 3 Identification of modified sites for PEG-ASP conjugates.
  • Y-PALD-40K-ASP (Mono) is one of the four subunits of the asparaginase molecule, and only one subunit has a PEG molecule coupled to the N-terminus.
  • Y-PALD-40K-ASP (Di) is the four subunits of the asparaginase molecule, and two N-terminals of the subunits are each coupled with a PEG molecule.
  • Figure 6 Detection of pharmacokinetic properties of asparaginase and its PEG conjugate.
  • Figure 6a shows the pharmacokinetic results of intravenous injection
  • Figure 6b shows the pharmacokinetic results of intramuscular injection.
  • Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di) have better pharmacokinetic properties compared with asparaginase, of which Y-PALD-40K The increase in the pharmacokinetic properties of -ASP(Di) is more pronounced.
  • Figure 8 Enzyme activity assay for different PEG-ASP conjugated objects.
  • PEG polyethylene glycol
  • PEG modifier polyethylene glycol modifier
  • Polyethylene glycol (PEG, HO-(CH2CH2O)n-CH2CH2OH) is a linear polymer with hydroxyl groups at both ends. Polyethylene glycol is polymerized from ethylene oxide by repeated oxyethylene groups. Composition, branched, linear and multi-armed. PEG is also known as poly(ethylene oxide) (PEO), poly(oxy-ethylene) (POE), or polyoxirane. In general, a molecular weight of less than 20,000 is called PEG, and a larger molecular weight is called PEO. Ordinary polyethylene glycol has a hydroxyl group at each end. If one end is blocked with a methyl group, methoxypolyethylene glycol (mPEG) is obtained, which is the most commonly used in protein pegylation technology.
  • PEG poly(ethylene oxide)
  • POE poly(oxy-ethylene)
  • PEO poly(oxy-ethylene)
  • Ordinary polyethylene glycol has a hydroxyl group at each end. If one end is blocked with a methyl group
  • the polyethylene glycol modifier refers to a polyethylene glycol derivative with a functional group, and refers to an activated polyethylene glycol, which is mainly used for protein and polypeptide drug modification, and is also called modified polyethylene glycol. Modified PEG.
  • Y-PALD-40K a branched polyethylene glycol propionaldehyde with a molecular weight of 40KDa, the structural formula is Wherein R is methyl, n is 455, and p is 2;
  • Y-PALD-30K branched polyethylene glycol propionaldehyde with a molecular weight of 30KDa, the structural formula is Wherein R is a methyl group, n is 320, and p is 2.
  • conjugate refers to a modified product obtained after modification of an ethylene glycol-modified asparaginase
  • Y-PALD-40K-ASP Single modified product purified after modification of asparaginase with PEG propionaldehyde having a molecular weight of 40K, only one subunit is coupled with PEG at the N-terminus
  • Y-PALD-40K-ASP Double modified product purified after modification of asparaginase with PEG propionaldehyde having a molecular weight of 40K, only one subunit is coupled with PEG at the N-terminus
  • Y-PALD-40K-ASP Di
  • the PEG-propionaldehyde-modified asparaginase-purified two modified products with two subunits coupled to the N-terminus of PEG Y-PALD-30K-ASP (Mono) (using PEG propionaldehyde with a molecular weight of 30K)
  • Y-PALD-30K-ASP Di
  • the polyethylene glycol modifier used in the present invention is preferably the following: aldehyde-activated polyethylene glycol, and more specifically, the polyethylene glycol modifier is propionaldehyde-activated polyethylene glycol.
  • the modified asparaginase protein may be of any origin, and the asparaginase may be extracted from Escherichia coli, including but not limited to Escherichia coli. It can also be expressed recombinantly.
  • the asparaginase has at least about 60% sequence identity to the protein comprising the sequence of SEQ ID NO: 1. More particularly with at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the protein comprising SEQ ID NO: , 97%, 98% or 100% sequence identity.
  • the protein is an E. coli-derived asparaginase having the sequence of SEQ ID NO: 1.
  • L-asparaginase (derived from Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd.) was prepared as a 20 mg/mL solution by dissolving with 40 mM pH 5.0 acetic acid-sodium acetate buffer.
  • PEG was Y-PALD-40K (purchased from Y-PALD-40K) Beijing Keykai Technology Co., Ltd., reacted with asparaginase: PEG: sodium cyanoborohydride at a molar ratio of 1:4:200. After reacting at 4 ° C for 12 h, the reaction was terminated by adding 1 M glycine.
  • a single modified product Y-PALD-40K-ASP (Mono) and a second modified product Y-PALD-40K-ASP (Di) were prepared.
  • Elution 0-50% B solution, elution volume of 10 column volumes, time 20 minutes, step-by-step sample collection.
  • HPLC Waters, e2695 HPLC
  • Superdex 20010/300GL available from GE
  • mobile phase was 0.1 M Na 2 SO 4 in PBS (pH 7.4)
  • flow rate was 0.4 mL / min
  • detection wavelength was 280nm
  • injection volume 50 ⁇ L detection time is 60min.
  • the protein concentrate was 8% and the separation gel was 7%.
  • the concentrated gel buffer was 0.5 M Tris-HCl buffer (pH 6.8); the separation gel buffer was 1.5 mol/L Tris-HCl buffer (pH 8.8).
  • a 10 ug protein sample was taken, mixed with the sample buffer in equal volume, boiled at 100 ° C for 5 min, and then loaded, and after electrophoresis, stained with Coomassie blue dye solution. The results of the identification are shown in Figure 2a.
  • Preparation Example 2 PEG is Y-PALD-30K, the specific parameters and yields are shown in Table 1 below, and the method steps and parameters not listed in Table 1 are the same as in Preparation Example 1:
  • lanes 3 and 4 of Figure 2a two uniform bands are shown, and the first band in lanes 3 and 4 corresponds to the molecule.
  • the amount is about 200KDa. Since the PEG molecule binds a large amount of water molecules in the solution, its apparent molecular weight is about 4 times its theoretical molecular weight. Therefore, it can be preliminarily judged that this band is a subunit of an asparaginase coupled with a PEG molecule, and the molecular weight corresponding to the second band is about 34 KDa, which is consistent with the molecular weight of a single subunit of asparaginase.
  • the second band is a single subunit of the asparaginase to which the PEG is not coupled. From the software analysis results, it can be concluded that the ratio of the content of the first band and the second band in the third lane is 1:1, indicating that two subunits of the modified product are coupled with PEG, and The other two subunits are not coupled to the PEG molecule and can be determined to be the second modified product. Similarly, in the fourth lane, we analyzed by software, the ratio of the content of the first band to the second band was 1:3, indicating that only one subunit of the modified product was coupled with PEG. The other three subunits were not coupled to the PEG molecule, and it was confirmed that it was a single modified product.
  • the PEG modification site of the PEG-ASP conjugate was determined by comparing the difference between the original protein and the modified product peptide.
  • the specific experimental procedure was as follows. 100 ⁇ L of a sample having a concentration of 0.5 mg/mL was added to 0.9 ⁇ L of a trypsin solution having a concentration of 1 mg/mL, and reacted at 37 ° C for 5 hours. After the end of the reaction, 10% by weight of TFA was added to terminate the reaction.
  • the enzymatic hydrolyzed product was analyzed using a C18 reverse phase column (purchased from Waters).
  • the mobile phases were: liquid A H 2 O + 0.1% wt TFA, liquid B acetonitrile + 0.1% wt TFA, loading 80 ⁇ L, flow rate 0.5. mL/min, running time is 120min. Gradient elution conditions: 0-100 min 5% wt-60% wtB.
  • the comparison results of the peptide map are shown in Fig. 3.
  • the N-terminal peptide of the PEG-ASP conjugate is compared with the original protein, Y-PALD-40K-ASP (Mono) and Y-PALD-40K-
  • the peak area of the ASP(Di) N-terminal peptide has been reduced by 25% and 50%, respectively, indicating that PEG is indeed coupled to the N-terminal amino acid of ASP, consistent with the expected results.
  • Y-PALD-40K-ASP(Mono) is a single modified product
  • only one PEG is coupled to one subunit of the asparaginase, and the other three subunits are not coupled to PEG, so After trypsin digestion, compared with the original protein, only the peak of the N-terminal peptide of one subunit will shift, and the other three subunits will not be coupled with PEG, so the peak of the N-terminal peptide will not occur. Variety. Therefore, the peak area of the final N-terminal peptide was only reduced by 25%.
  • Y-PALD-40K-ASP (Di) each asparaginase molecule has two subunits coupled to PEG, so the peak area of the final N-terminal peptide is reduced by 50% compared to the original protein.
  • Example 2 Combined with the experimental results in Example 1, it was further verified that the two modified products purified by us were single-modified and two-modified products modified at the N-terminus of the protein. The same results were obtained by analyzing the enzymatic peptide map with a PEG modified product with a molecular weight of 30 KDa, indicating that the two modified products obtained by modification with PEG having a molecular weight of 30 KDa are single and two modified products modified at the N-terminus of the protein. .
  • the secondary and tertiary structures of the modified and unmodified proteins can be characterized by a circular dichroism spectrometer.
  • the protein concentration range is from 0.1 to 0.2 mg/mL.
  • the sample was added to a circular dichroic cuvette with a diameter of 1 mm to detect a circular dichroism spectrum in the far ultraviolet region (190 nm to 250 nm) and the near ultraviolet region (253 nm to 480 nm) with a scanning bandwidth of 1 nm and a scanning speed of 500 nm/min. Each test was taken as the background with the corresponding buffer, and the average was measured three times. As can be seen from Fig.
  • the near-ultraviolet circular dichroism of the PEG-ASP conjugate showed almost no peak shift in the spectrum of the conjugate compared to the original protein, although the peaks showed some changes, indicating that the PEG modification did not affect the ASP.
  • Three-level structure Overall, the high-order structure of ASP was substantially unchanged by the conjugate prepared by site-directed modification. Since the conjugate is not modified by PEG modification, its activity is less than that of the original protein.
  • the endogenous fluorescence detection of the modified protein and the unmodified protein has an excitation wavelength of 280 nm and an emission wavelength range of 300 to 400 nm.
  • the scanning speed was 1200 nm/min.
  • the slit widths of the excitation and emission were both 5 nm, and the test was performed at room temperature using a 0.1 cm sample cell.
  • the concentration of the protein to be tested ranges from 0.1 to 0.2 mg/ml.
  • the activity of the modified product obtained by modifying the asparaginase with branched PEG having a molecular weight of 30 KDa and 40 KDa did not differ much. However, from the perspective of pharmacokinetics, the modified product with a larger molecular weight generally has better pharmacokinetic performance. Therefore, in this example, the two-modified and single-modified products obtained by modifying the branched PEG having a molecular weight of 40 KDa were selected for pharmacokinetic studies, and compared with the unmodified original protein and the commercially available product, Pembellase.
  • the samples were labeled with 125 I using the IODOGEN labeling method.
  • the labeled sample was purified and verified by SHPLC.
  • the labeled series of samples were assayed for protein concentration using the BCA Protein Assay Kit, and then mixed with a respective amount of unlabeled sample, diluted with 1 ⁇ solvent (10 ⁇ solvent diluted to 1 ⁇ with water for injection). 1.175 mg/mL of the injection, and take the prepared drug (about 5 ⁇ L) to measure the radioactivity.
  • Specific activity radioactivity / protein concentration. Rats were sampled at a certain time after administration.
  • the blood concentration at the last blood collection time of each intravenous administration group was not less than 1/20 of the blood concentration at the 2 min time point, then the blood was taken again once a day. Until its blood concentration is less than 1/20 of the 2min time point. If the blood concentration at the last blood collection time of each muscle administration group is not less than 1/10 of the peak blood concentration, then continue to take blood once a day until the blood concentration is lower than the peak blood concentration. 1/10. After the blood was taken, the blood sample was immediately placed in an EP tube supplemented with heparin sodium (1000 IU/mL, 10 ⁇ L), and inverted 5 to 10 times, and the plasma was separated by centrifugation at 4000 rpm for 5 minutes.
  • heparin sodium 1000 IU/mL, 10 ⁇ L
  • TCA trichloroacetic acid
  • NCA Non-compartmental model method
  • Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di) are significantly longer than those of asparaginase, whether it is intravenous or intramuscular, and the half-life is significantly prolonged.
  • the area under the curve (AUC) has been significantly improved.
  • the clearance rate of Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di) in the blood is also much lower than that of asparaginase.
  • Y-PALD-40K-ASP (Mono) and Y-PALD-40K-ASP (Di) have a certain degree of stability in vivo compared with asparaginase, and Y-PALD-
  • the 40K-ASP (Di) enhancement range is more obvious, so its half-life is significantly improved, the metabolic rate in the blood is significantly reduced, and the drug efficacy time is effectively prolonged, as shown in Fig. 6.
  • the half-life of Y-PALD-40K-ASP (Di) is higher than that of Pembellase, whether it is intramuscular or intravenous, compared with the published Pembellase.
  • L-asparaginase can hydrolyze the amide group of asparagine, and according to this principle, the activity of asparaginase can be measured.
  • the specific measurement method is carried out by referring to the method described on page 31 of the second edition of the Pharmacopoeia 2005.
  • the reagents required for the measurement were purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the asparaginase-modified product prepared in Example 1 of the present invention has a significantly higher activity than the commercially available products of Pembellase and Oncaspar.
  • Commercially available aspartase and Oncaspar are products obtained by random modification using PEG having a molecular weight of 5000 Da.
  • the activity of the modified asparaginase retains only 62.9% and 53.5% of the activity of the original protein; and the present invention uses a molecular weight of 30 kDa.
  • ⁇ 40kDa PEG propionaldehyde is N-terminally modified to the asparaginase, and the modified asparaginase activity retains about 90% of the original protein.
  • the same type of PEG is used to modify the protein.
  • the modified product prepared in the present invention unexpectedly does not completely follow such a rule, and not only the activity of the modified substance is higher than that of the enzyme, but also the modification of the PEG modifier having a molecular weight of 30 KDa and 40 KDa in the present invention.
  • the activity of the modified product substantially retains about 90% of the activity of the original protein, wherein the modified product obtained by modification with 40KDa of PEG is slightly more active than the modified product obtained by modification with 30KDa of PEG.
  • each of the asparaginases in the two modified products is coupled with a PEG molecule, so the two modified The molecular weight is significantly higher than the single modified product.
  • the activity of the two modified products should be significantly lower than that of the single modified product.
  • asparaginase which is a multi-subunit protein consisting of four subunits with identical amino acid sequences at the active center.
  • asparaginase which is a multi-subunit protein consisting of four subunits with identical amino acid sequences at the active center.
  • the PEG molecules are coupled to the N-terminus of two non-adjacent subunits, respectively, so the two PEG molecules are relatively independent, and there is no large steric hindrance between them, thus making the single The activity of the modified product and the second modified product did not differ much.
  • N-terminal fixed-point modification of asparaginase using a site-directed modification technique there are also examples of N-terminal fixed-point modification of asparaginase using a site-directed modification technique.
  • the linear PEGs were used in these examples, and the modified products prepared were single-modified products of the N-terminal site, and the activity was only about 40% of the original protein, which was far lower than the modified product prepared in the present invention. Since all of them belong to the N-terminal fixed-point modification, and both are linear and small molecular weight PEG, the modified modified product should theoretically be superior to the present invention in terms of activity, but the result is unexpectedly the activity of the modified product prepared in the present invention. Higher.
  • THP-1 derived from human monocyte leukemia cell line
  • Raji derived from human The three lymphocytes (Lymphoma cell line)
  • L1210 leukemia cells derived from mice
  • the killing effect of the asparaginase modified by PEG on the above three kinds of tumor cells is generally higher than that of the unmodified asparaginase, and their antitumor activities are significantly higher than that of Pedangase and Oncaspar.
  • a variety of cell lines have shown good anti-tumor effects. Since the specific modified product of the asparaginase prepared in the present invention has higher specific activity than that of Pedangase and Oncaspar, the inhibitory effect on tumor cells is better.

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Abstract

本申请公开了一种聚乙二醇化的门冬酰胺酶及其应用。本申请的聚乙二醇在L-门冬酰胺酶的1或2个亚基的N端氨基上偶联，所述聚乙二醇的分力量为30-40KDa。聚乙二醇优先采用分支型、活化基团为醛基的聚乙二醇。所述聚乙二醇化的门冬酰胺酶适用于抗肿瘤药物的制备。

Description

聚乙二醇定点修饰的门冬酰胺酶及其制备方法与应用 技术领域
本发明涉及聚乙二醇修饰的蛋白类药物，特别是聚乙二醇修饰的门冬酰胺酶及其制备方法和其在药物制备中的应用，以及其在临床治疗中的应用。
背景技术
门冬酰胺酶(Asparaginase，ASP)是具有L-天冬酰胺氨基水解酶活性的蛋白(通常被称为L-天冬酰胺酶，L-门冬酰胺酶，或天门冬酰胺酶)，活性形式为4个亚基组成的同源四聚体结构，每一亚基由326个氨基酸组成。L-门冬酰胺酶可以有效的治疗儿童或成人中的急性淋巴细胞白血病(ALL)。最近几年，含有L-门冬酰胺酶的药物已经用于联合化疗方案来治疗NK/T细胞淋巴瘤，并取得了较好的治疗效果。NK/T细胞淋巴瘤是种特殊类型的非霍奇金淋巴瘤，多见于亚洲和拉丁美洲，我国发病率相对较高。根据肿瘤发生部位，NK/T细胞淋巴瘤可分为鼻型NK/T细胞淋巴瘤和非鼻型NK/T细胞淋巴瘤。另外L-门冬酰胺酶还被用于治疗何杰金氏病，急性骨髓白血病，急性骨髓单核细胞白血病，慢性淋巴细胞白血病，淋巴肉瘤，网状细胞肉瘤和黑素肉瘤(Kotzia and labrou,J.Biotechnol.127(2007)657-669)。
L-门冬酰胺酶最初从若干生物体中纯化，包括大肠杆菌(E.coli)和软腐欧文氏菌(Erwinia carotovora)。在哺乳动物中，仅在豚鼠(Cavioidea超科)和某些阔鼻猴(New World monkey)中发现略高于痕量的L-门冬酰胺酶。由于它来源于外源生物，对人而言是一种外源蛋白，有较强的免疫原性，临床上常见进行性免疫反应和全身性过敏反应，而限制了其临床应用。(张丽娜，宫道华.江苏医药.左旋门冬酰胺酶治疗小儿急性淋巴细胞性白血病的毒副反应.2005，31(5)：392；王宁玲，刘芝璋等.左旋门冬酰胺酶治疗儿童白血病的毒副作用及防治.中国小儿血液，2005，10(3)：133)。另一方面，由于天然结构的L—asparaginase分子在人体内半衰期短，用药间隔短。这些特点使患者对该药的依从性下降。
针对这些弊端，现有技术从多方面对该药物进行了改进，例如：①对于大肠杆菌L—asparaginase敏感患者，以欧文氏菌来源的酶代替大肠杆菌L—asparaginase；②L—asparaginase的剂型改造，如制备其脂质体等形式；③通过蛋白质工程进行结构改造，对大肠杆菌L—asparaginase抗原部位进行突变以减弱免疫原性；④根据L—asparaginase结构进行的化学修饰。而以聚乙二醇(PEG)修饰则是化学修饰的一种。
聚乙二醇(PEG)是一种线性、在溶液中可自由卷曲的不带电荷的聚合物，具有无毒、微弱的抗原性和良好的生物相容性。用它来共价修饰蛋白质，可以增加蛋白质的体内循环半衰期 和减小其抗原性，增加蛋白质的溶解性并会改变蛋白质在人体内的生物学分布。自1977年Abuchowski，Davis(J.Biol.Chem.1977,252:3578-3581.)等人首次报道用PEG修饰蛋白质以来，PEG修饰技术在生物医学和生物技术领域得到了广泛的应用，PEG已经被广泛地应用于蛋白质、多肽类药物的修饰研究。目前蛋白质PEG化技术已经成为降低蛋白质生物药物的免疫原性，以及改善其药代动力学/药效学性质最有效的方法之一，且已通过FDA认证可用于药物、食品和化妆品。
聚乙二醇修饰技术经历了几十年的发展，目前虽然已经比较成熟。但并不能找出一种通用的聚乙二醇修饰剂以及修饰方法来对所有的蛋白药物进行修饰。蛋白本身的结构以及所用PEG的分子量、形状以及修饰的位点等对聚乙二醇化的蛋白质的生物活性以及药效有很大的影响。对于特定药物的修饰，聚乙二醇修饰剂是影响修饰产物理化性质、体内外生物活性、药代动力学、药效学以及修饰产物临床表现最重要的因素。因此，修饰剂的选择(修饰剂种类、分子量大小)以及修饰反应的控制在聚乙二醇修饰技术中占有重要地位。蛋白质结构的解析并不能准确预测天然蛋白质的药代动力学行为，而在蛋白偶联上PEG以后由于引入了许多新的变量如分子量、修饰剂种类等，对于PEG偶联物的药代动力学行为预测变得更加不可能。为此，针对不同的蛋白质药物，需要通过选择不同种类以及不同分子量的修饰剂，并通过理化性质检测、动物实验评价等确定最佳方案。
例如，在大多数情况下，与未修饰的原蛋白相比，聚乙二醇化的蛋白药物的活性会降低，一般修饰后的蛋白活性仅有原蛋白的30％-40％,甚至更低。例如Schering-Plough公司的PEG-Intron,是用分子量为5000的PEG修饰干扰素，修饰后其活性只有原蛋白的8％。又如Yoshihiro等分别用分子质量为750，1900，5000U的分支状活化PEG(2-0-methoxypolyethylene glycol-4，6-dichloro-s-triazine)对大肠杆菌门冬酰胺酶进行修饰。其中5000u-PEG修饰了门冬酰胺酶所有92个游离氨基(88个δ-氨基与4个α-氨基)中的73个，保留原酶活性的7％，修饰后产物完全失去与门冬酰胺酶抗性血清结合能力。此外，一般随着PEG分子量的增大，修饰后的蛋白质活性降低更明显。例如用分子量为20KDa、30KDa、40KDa的PEG修饰促红细胞生成素(EPO)后，活性随着PEG分子量的增大而显著减小(Yin-jue Wang,journal of controlled release,2010(145):306-313)。Bailon等采用分支型40kDa PEG修饰Interferon-α-2a，所得单修饰物获得较长的循环半衰期，但是仅仅保留了7％的体外活性(Bailon P,Bioconjugate Chem.,2001,12:195-202.)。
目前针对L-门冬酰胺酶的聚乙二醇修饰研究也有很多。国外利用聚乙二醇修饰的L-门冬酰胺酶的产品Oncaspar(Enzon Inc.)早在1994年就已上市，并在2006年起被批准作为儿童和成人的ALL的一线疗法。Oncaspar使用琥珀酰亚胺琥珀酸酯(SS-PEG)进行修饰，具有修 饰产物不均一，修饰产物活性损失较大，且含有对酶的水解敏感的、或者在微碱性PH值时不稳定的酯键(美国专利号4670417)等缺陷。这些性质显著降低了体外和体内的稳定性并且副作用也较大。而国内目前上市的PEG修饰的L-门冬酰胺酶只有恒瑞公司的“培门冬酶”，它是Oncaspar的仿制药，也存在着上述问题。
这两种产品都是随机修饰的产物，因此修饰产物不均一，其产品的组成是由偶联了不同数目的PEG分子而组成的。另外随机修饰后，由于修饰位点较多，对门冬酰胺酶的活性部分具有较大的位阻作用，因此活性有较大损失，只保留了原蛋白60％的活性。已有文献进行了门冬酰胺酶N端定点修饰的相关研究，得到的修饰产物的活性只有原蛋白的36％左右。(迟少萍，王伯初，药物生物技术聚乙二醇醛对L-门冬酰胺酶化学修饰的初步研究)。而国内另一个对门冬酰胺酶进行N端定点修饰的专利(申请号200810112025.5)中，使用的也是直链PEG，分子量为20KDa，最终纯化得到的修饰产物中，PEG除了偶联在门冬酰胺酶其中一个亚基上的N端外，另一个是和亚基的氨基酸侧链上的氨基偶联。修饰产物最终只保留了原蛋白40％左右的生物活性，活性损失较大。上述两个例子都是用的直链PEG对门冬酰胺酶进行定点修饰。
发明内容
解决的技术问题：为了克服现有技术中的上述技术问题，本发明旨在提供稳定性提高、免疫原性降低的同时，也具有高活性，组分均一的聚乙二醇定点修饰的门冬酰胺酶(ASP)，及其制备方法和在制备治疗儿童或成人急性淋巴细胞白血病以及非霍奇金淋巴瘤等疾病的药物中的应用。
更进一步的说：
本发明的第一目的是提供聚乙二醇定点修饰的L-门冬酰胺酶，所述聚乙二醇在L-门冬酰胺酶的1或2个亚基的N端氨基上偶联，所述聚乙二醇分子量为30-40kDa，聚乙二醇为分支型，活化基团为醛基。
优选的，所述聚乙二醇在L-门冬酰胺酶的2个亚基的N端伯氨基上偶联，所述聚乙二醇分子量为30kDa或40kDa，聚乙二醇为分支型，活化基团为乙醛、丙醛、丁醛或戊醛。
所述聚乙二醇定点修饰的L-门冬酰胺酶的结构通式如下所示：
Figure PCTCN2015072167-appb-000001
其中R为H 或C1-C4的烷基；n为100到500的整数值，p为1-4的整数；AA为N端的L-氨基酸残基，m为0-5的整数,s为1或者2。
优选地，所述烷基为甲基，n为320-455之间的整数值，p为2，m为0。
本发明的第二目的是提供一种上述的聚乙二醇定点修饰的ASP的制备方法，包括如下步骤：步骤1，用40mM pH4-6的乙酸-乙酸钠缓冲液配制15-20mg/mL的L-门冬酰胺酶溶液；步骤2，按摩尔比L-门冬酰胺酶:聚乙二醇:还原剂＝1:(4-5):(100-200)在4℃的条件下反应12-24h；步骤3，反应结束后以离子交换色谱法纯化处理，最终得到单修饰及二修饰的聚乙二醇化的L-门冬酰胺酶。
所述聚乙二醇优选分子量40KDa。
所述聚乙二醇化的L-门冬酰胺酶纯化产物优选二修饰产物。
所述还原剂优选为氰基硼氢化钠。
本发明的第三目的是提供一种上述聚乙二醇定点修饰的ASP在制备治疗儿童或成人急性淋巴细胞白血病以及非霍奇金淋巴瘤等疾病的药物中的应用。目前已有文献报道用PEG修饰的门冬酰胺酶用来联合化疗治疗NK/T细胞淋巴瘤或急性淋巴细胞白血病，并取得较好疗效。因此本发明中提供的定点修饰技术制备的PEG修饰的门冬酰胺酶也可用于联合治疗NK/T细胞淋巴瘤或急性淋巴细胞白血病。
本发明的第四目的是提供一种聚乙二醇定点修饰的ASP或其药学上可接受的盐或复合物。所述复合物是指两种或两种以上不同物质所形成的结合体。
本发明的第五目的是提供一种药物组合物，所述药物组合物含有上述聚乙二醇定点修饰的ASP或其药学上可接受的盐或复合物和药学上可接受的辅料。
所述药物组合物为液态针剂或冻干粉针剂。
所述的辅料包括药学上可接受的载体和/或赋形剂等。
所述定点修饰的L-门冬酰胺酶及其药物组合物通过肌肉、静脉或皮下途径给药。
有益效果：本发明提供的聚乙二醇定点修饰的ASP，在具有半衰期明显延长、结构稳定均一等优点的基础上，更大程度的提高了其生物活性。
附图说明
图1：不同PEG-ASP偶联物高效液相分析。
使用SEC法分别对培门冬酶、Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)、Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)进行分析。从结果中可以看出，经过纯化后，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)，Y-PALD-30K-ASP(Mono) 及Y-PALD-30K-ASP(Di)纯度均能达到98％以上，略高于培门冬酶的纯度97％。且与培门冬酶相比，Y-PALD-40K-ASP(Di)、Y-PALD-30K-ASP(Di)具有更高的分子量。
图2：不同PEG-ASP偶联物SDS-PAGE分析。
使用还原性SDS-PAGE对培门冬酶、Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)、Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)进行分析。图2a泳道1-4分别为高分子量Marker、培门冬酶、Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)。图2b泳道1-3别为高分子量Marker、Y-PALD-30K-ASP(Di)及Y-PALD-30K-ASP(Mono)。由于ASP为四亚基组成的同源四聚体，在还原环境中，四个亚基会发生解聚。由于修饰度不同各亚基在SDS-PAGE中将显示出不同的条带。如图所示，培门冬酶呈现出弥散条带，说明其蛋白均一度较差，与之相比，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)显示出被修饰亚基与未被修饰亚基两条清晰的谱带，均一度较高，图2a中泳道3、4图2b中泳道2、3中显示的分子量较大的条带表示经修饰后的ASP单亚基，分子量较小的条带表示未经修饰的ASP单亚基。
图3：PEG-ASP偶联物的修饰位点鉴定。
从PEG-ASP偶联物和原蛋白的酶解肽图可以看出，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)的PEG是修饰在蛋白的N端的。其中Y-PALD-40K-ASP(Mono)是门冬酰胺酶分子的4个亚基中，只有一个亚基的N端偶联上了一个PEG分子。其中Y-PALD-40K-ASP(Di)是门冬酰胺酶分子的4个亚基中，有两个亚基的N端各偶联上了一个PEG分子。
图4：不同PEG-ASP偶联物圆二色光谱分析
使用圆二色光谱分别对门冬酰胺酶、Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)进行结构鉴定。从结果看，远紫外扫描结果显示，与门冬酰胺酶相比，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)圆二色特征谱图未发生明显变化，近紫外结果显示出相同结果。由此认为，经过Y-PALD-40K-PEG修饰后，蛋白主体结构未发生变化。
图5：不同PEG-ASP偶联物荧光光谱分析
使用荧光分光光度计对门冬酰胺酶、Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)进行内源荧光光谱扫描，结果表明PEG修饰后没有改变门冬酰胺酶的三级结构。
图6：门冬酰胺酶及其PEG偶联物药代动力学性质检测。
图6a表示静脉注射的药代动力学结果，图6b表示肌肉注射的药代动力学结果。
使用125I同位素标记示踪法对PEG修饰后的门冬酰胺酶的体内血药浓度进行了研究。从结果看，与门冬酰胺酶相比，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)均具有 更好的药代动力学性质，其中Y-PALD-40K-ASP(Di)药代动力学性质提升更为显著。
图7：不同PEG-ASP偶联物热处理后的活性测定
图8：不同PEG-ASP偶联物体外酶活测定。
使用《中国药典》所附方法分别对门冬酰胺酶、Oncaspar、培门冬酶、Y-PALD-40K-ASP(Mono)、Y-PALD-40K-ASP(Di)、Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)的体外酶活进行检测。从结果可以看出，门冬酰胺酶相比，经过修饰后，各修饰产物均有不同程度活性损失。其中Oncaspar和培门冬酶损失较大，本发明的修饰产物的活性损失相对较小。
具体实施方式
定义：
本发明使用的缩写含义如下：
PEG，聚乙二醇；PEG修饰剂，聚乙二醇修饰剂。
聚乙二醇(PEG，HO-(CH2CH2O)n-CH2CH2OH)是一种两端带羟基的线型聚合物，聚乙二醇是经环氧乙烷聚合而成的，由重复的氧乙烯基组成，有分支型，直链型和多臂型。PEG也被称为poly(ethyleneoxide)(PEO)，poly(oxy-ethylene)(POE)，或者polyoxirane。一般情况下，分子量低于20,000的被称为PEG，分子量更大的被称为PEO。普通的聚乙二醇两端各有一个羟基，若一端以甲基封闭则得到甲氧基聚乙二醇(mPEG)，这种衍生物是蛋白质聚乙二醇化技术中最常用到。
聚乙二醇修饰剂，则是指带有官能团的聚乙二醇衍生物，是指经过活化的聚乙二醇，目前主要用于蛋白质以及多肽药物修饰，又叫修饰性聚乙二醇，修饰性PEG。
Y-PALD-40K，分子量为40KDa的分支聚乙二醇丙醛，结构通式为
Figure PCTCN2015072167-appb-000002
其中R为甲基，n为455，p为2；
Y-PALD-30K，分子量为30KDa的分支聚乙二醇丙醛，结构通式为
Figure PCTCN2015072167-appb-000003
其中R为甲基，n为320，p为2。
ASP，门冬酰胺酶。
本申请中所用，术语“偶联物”，是指聚乙二醇修饰门冬酰胺酶后得到的修饰产物；
几种聚乙二醇修饰门冬酰胺酶的修饰产物可在本申请中称为“Y-PALD-40K-ASP(Mono) (用分子量为40K的PEG丙醛修饰门冬酰胺酶后纯化得到的单修饰产物，只有一个亚基的N端偶联了PEG)及Y-PALD-40K-ASP(Di)(用分子量为40K的PEG丙醛修饰门冬酰胺酶后纯化得到的二修饰产物，有两个亚基的N端偶联了PEG)，Y-PALD-30K-ASP(Mono)(用分子量为30K的PEG丙醛修饰门冬酰胺酶后纯化得到的单修饰产物，只有一个亚基的N端偶联了PEG)及Y-PALD-30K-ASP(Di)(用分子量为30K的PEG丙醛修饰门冬酰胺酶后纯化得到的二修饰产物，有两个亚基的N端偶联了PEG)”统称为PEG-ASP或PEG修饰ASP的偶联物。Oncaspar，是原研药的产品名称。
本发明使用的聚乙二醇修饰剂优选下面几种：醛基活化的的聚乙二醇，更具体地，聚乙二醇修饰剂为丙醛活化的聚乙二醇。
在本发明中，所修饰的门冬酰胺酶蛋白，可以是任何来源的，门冬酰胺酶可从大肠杆菌中提取，包括但不限于大肠杆菌。也可以是重组表达的。在本发明的偶联物的特定实施方式中，门冬酰胺酶与包含SEQ ID NO:1的序列的蛋白具有至少约60％的序列一致性。更特别地与包含SEQ ID NO:1的蛋白具有至少约65％，70％，75％，80％，85％，90％，91％，92％，93％，94％，95％，96％，97％，98％或100％的序列一致性。
在特定的实施方式中，所述蛋白为大肠杆菌来源的门冬酰胺酶，其具有SEQ ID NO:1的序列。
实施例1：ASP的PEG偶联物的制备及分析
制备例一，本发明聚乙二醇定点修饰的ASP通过如下方法制备、纯化、鉴定：
1.PEG偶联物样品制备
用40mM pH5.0的乙酸-乙酸钠缓冲液溶解将L-门冬酰胺酶(来源于常州千红生化制药股份有限公司)配制成为20mg/mL的溶液，PEG采用Y-PALD-40K(购自北京键凯科技有限公司)，按门冬酰胺酶:PEG:氰基硼氢化钠为1:4:200的摩尔比进行反应，在4℃下反应12h后，加入1M甘氨酸终止反应。制备得到单修饰产物Y-PALD-40K-ASP(Mono)及二修饰产物Y-PALD-40K-ASP(Di)。
2.PEG偶联物样品纯化
色谱法条件：Q离子交换柱(购自GE公司，HiTrap Q HP 5mL)，A液：20mM的Tris-HCl(pH9.0)，B液：含1M NaCl的20mM的Tris-HCl(pH9.0)，流速2.5mL/min，检测波长为280nm。
上样：上述修饰反应产物用0.5M的NaOH溶液调节至pH 9.0，结合至Q离子交换柱。
平衡：A液冲洗5个柱体积。
洗脱：0-50％B液，洗脱体积为10个柱体积，时间20分钟，分步进行收样。
2.PEG偶联物样品检测
2.1色谱方法检测：
色谱法条件：HPLC(Waters，e2695HPLC)，Superdex 20010/300GL(购自GE公司)，流动相为含0.1M Na2SO4的PBS(pH7.4)，流速为0.4mL/min，检测波长为280nm，进样量50μL，检测时间为60min。
分析结果见图1a所示。如图可以看出，制备的两种产物Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)的纯度均高于98％。同时，Y-PALD-40K-ASP(Di)修饰产物的分子量也高于培门冬酶(购自江苏恒瑞医药股份有限公司)的分子量。
2.2电泳方法鉴定：
蛋白浓缩胶为8％，分离胶为7％。浓缩胶缓冲液为0.5M Tris-HCl缓冲液(pH 6.8)；分离胶缓冲液为1.5mol/L Tris-HCl缓冲液(pH8.8)。取10ug蛋白样品，与样品缓冲液等体积混合，在100℃下煮沸5min后上样运行，电泳结束后用使用考马斯亮蓝染液进行染色。鉴定结果如图2a所示。
制备例二PEG为Y-PALD-30K，具体参数与得率如下表1所示，表1未列出的方法步骤和参数与制备例一相同：
表1
实验条件 对应实验参数
pH值 5.0
修饰剂 Y-PALD-30K
摩尔比(蛋白:PEG:还原剂) 1:5:100
反应温度 4℃
反应时间(小时) 24
蛋白浓度(mg/mL) 15
同时也对制备例二所得修饰产物进行色谱法与电泳法进行鉴定，所得结果分别如图1b、2b所示。
制备例二所制备的单修饰及二修饰产物的活性及纯度与制备例一中的所得的单修饰及二修饰产物无显著性差异，Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)的纯度均高于98％。
由图2a、2b可以看出，修饰产物Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)，Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)的电泳条带非常均一。比较国内市场上的同类产品——培门冬酶(购自江苏恒瑞医药股份有限公司)，均一性得到很大提高。
图2a中第3、4泳道都显示出两条均一的条带，第3、4泳道中的第一个条带所对应的分子 量大概是200KDa左右，由于PEG分子在溶液中会结合大量水分子，使得其表观分子量是其理论分子量的4倍左右。因此可以初步判断这个条带是偶联了一个PEG分子的门冬酰胺酶的亚基，而第二个条带所对应的分子量是34KDa左右，和门冬酰胺酶的单个亚基的分子量一致，因此可以初步判断第二个条带是没有偶联上PEG的门冬酰胺酶的单个亚基。通过软件分析结果可以得出，第3泳道的第一个条带和第二个条带的含量的比例为1：1，说明该修饰产物中，有两个亚基偶联上了PEG，而另两个亚基没有偶联上PEG分子，可以确定是二修饰产物。同样，在第4泳道上我们通过软件分析得出，第一个条带和第二个条带的含量的比例为1：3，说明该修饰产物中，只有一个亚基偶联上了PEG，而另三个亚基没有偶联上PEG分子，可以确定是单修饰产物。结果和我们的预期相一致。而第2泳道已上市的产品培门冬酶确显示出弥散的条带，这是由于培门冬酶采用的是随机修饰方式，在最终的修饰产物中，每个门冬酰胺酶上的亚基所偶联的PEG个数并不相同，因此在电泳中显示出来的是弥散的条带。从产品的均一性来看，本发明中制备的定点修饰产物均一性较好，具有较大的优势。和图2a比较，在图2b中也得到了类似的结果，有所区别的是，由于所使用的PEG是分子量为30KDa的，因此图2b中第2，3泳道的第一个条带的大小对应的分子量是150KDa左右，略小于用分子量为40KDa的PEG修饰得到的修饰产物。
实施例2：PEG-ASP偶联物修饰位点的鉴定
为了确定PEG-ASP偶联物的PEG修饰位点，我们对PEG-ASP偶联物进行了胰蛋白酶的酶解实验，并和原蛋白的酶解图谱做比较。通过比较原蛋白和修饰产物肽段的差异，可以确定PEG的修饰位点。具体实验步骤如下，取浓度为0.5mg/mL样品100μL加入0.9μL浓度为1mg/mL的胰蛋白酶溶液，37℃反应5h。反应结束后加入10％wt的TFA对反应进行终止。酶解产物使用C18反相色谱柱(购自Waters公司)进行分析，流动相分别为：A液H2O+0.1％wtTFA，B液乙腈+0.1％wtTFA，上样量为80μL，流速为0.5mL/min，运行时间为120min。梯度洗脱条件：0-100min 5％wt-60％wtB。
肽图的比较结果如图3所示，由图可以看出，PEG-ASP偶联物的N末端肽段和原蛋白比较，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)N末端肽段峰面积已经分别降低了25％和50％，说明PEG确实是偶联到了ASP的N末端氨基酸上，与预期结果一致。其中Y-PALD-40K-ASP(Mono)是单修饰产物，只有一个PEG偶联在门冬酰胺酶的一个亚基上，而另外3个亚基并没有偶联上PEG，因此在对其进行胰蛋白酶酶解后，和原蛋白比较，只有一个亚基的N端肽段的峰会发生位移，而另外三个亚基由于并没有偶联上PEG，因此N端肽段的出峰不会发生变化。因此最终N端肽段的峰面积只降低了25％。而对于二修饰产物 Y-PALD-40K-ASP(Di)，每个门冬酰胺酶分子有两个亚基偶联上了PEG，因此和原蛋白比较，最终N端肽段的峰面积降低了50％。
结合实施例1中的实验结果，进一步验证了我们纯化得到的两个修饰产物是修饰在蛋白N端的单修饰和二修饰产物。用分子量为30KDa的PEG修饰产物进行酶解肽图的分析也得到了相同的结果，说明我们用分子量为30KDa的PEG修饰后得到的两个修饰产物是修饰在蛋白N端的单修饰和二修饰产物。
实施例3：PEG-ASP偶联物和原蛋白的圆二色谱图分析
用圆二色光谱仪可以表征修饰蛋白以及未修饰蛋白的二级结构及三级结构。蛋白浓度范围是0.1～0.2mg/mL。样品加入1mm光径的圆二色比色杯，检测其远紫外区(190nm-250nm)和近紫外区(253nm-480nm)的圆二色光谱，扫描带宽为1nm，扫描速度为500nm/min。每次检测以相应缓冲液为背景，测三次取平均值。由图4可以看出，PEG-ASP偶联物的远紫外区圆二色谱图和原蛋白相比，偶联物的光谱几乎没有发生峰的位移，峰值没有明显变化，说明用分枝状PEG修饰后的二级结构没有差别，这个结果符合PEG的特性，由于PEG在溶液中是一种柔性的两亲性大分子，因此偶联到蛋白表面后，不会对其结构有显著影响。说明PEG修饰没有影响ASP的二级结构。同样的，PEG-ASP偶联物的近紫外区圆二色谱图和原蛋白相比，偶联物的光谱也几乎没有发生峰的位移，虽然峰值有一定变化，这说明PEG修饰没有影响ASP的三级结构。总体看来，通过定点修饰制备的偶联物，ASP的高级结构基本没有发生改变。由于偶联物经过PEG修饰后，其结构未变化，因此其活性和原蛋白比较，损失较少。
实施例4：PEG-ASP偶联物和原蛋白的荧光谱图分析
修饰蛋白以及未修饰蛋白的内源荧光检测的激发波长为280nm，发射波长范围为300～400nm。扫描速度为1200nm/min。激发和发射的缝隙宽度(slit widths)均为5nm，使用0.1cm的样品池，在室温下进行检测。待测蛋白质的浓度范围是0.1～0.2mg/ml。
通过内源荧光(Intrinsic Fluorescence)来检测PEG修饰对门冬酰胺酶三级结构的影响。如图5所示，当激发波长为280nm时，门冬酰胺酶及其修饰产物的发射荧光峰值在315nm处。单修饰、二修饰和门冬酰胺酶，其荧光发射光谱基本一致。这表明PEG对门冬酰胺酶的修饰不影响其三级结构。PEG修饰蛋白的发射光谱强度比未修饰蛋白略有降低，这可能跟PEG链对蛋白光谱发射产生的屏蔽作用有关。
实施例5：PEG-ASP偶联物的药代动力学研究
用分子量为30KDa和40KDa的分支PEG修饰门冬酰胺酶得到的修饰产物的活性差别不大。但从药代动力学的角度考虑，一般分子量越大的修饰产物，其药代动力学的表现越好。因此本实施例中选择用分子量为40KDa的分支PEG修饰得到的二修饰和单修饰产物进行药代动力学研究，并和未修饰的原蛋白以及已上市的的产品培门冬酶进行比较。
我们分别考察了静脉注射和肌肉注射给药的药代动力学。对样品采用IODOGEN标记方法进行125I标记。并对标记后的供试品进行纯化，经SHPLC验证纯度。标记后的系列样品采用BCA蛋白测定试剂盒进行蛋白浓度测定，然后分别与各自一定量的未标记的样品混合，用1×的溶媒(10×的溶媒用注射用水稀释到1×)将样品稀释1.175mg/mL的注射液，并取配制后的药物(5μL左右)，测定放射性。比活性＝放射性/蛋白浓度。大鼠给药后按照一定的时间点进行取样，如果各静脉给药组最后一个取血时间点血药浓度未低于2min时间点血药浓度的1/20，那么接下来继续取血每天一次，直至其血药浓度低于2min时间点的1/20。如果各肌肉给药组最后一个取血时间点血药浓度未低于达峰血药浓度的1/10，那么接下来继续取血每天一次，直至其血药浓度低于达峰血药浓度的1/10。取血后将血样立即置于加有肝素钠(1000IU/mL，10μL)抗凝的EP管中，颠倒5～10次，4000rpm离心5分钟分离出血浆。取50μL血浆，加入等体积的20％三氯乙酸(TCA)，涡旋混匀后，测定总放射性。之后4500rpm，常温离心10min，弃上清，测定沉淀部分放射性。
代谢动力学参数计算：采用WinNonlin6.2进行非房室模型方法(NCA)拟合计算AUC等主要代谢动力学参数；
2)浓度计算：
Figure PCTCN2015072167-appb-000004
3)数据处理：采用Excel 2007对均值、标准差等数据进行统计描述。
药代动力学各参数的计算结果见表2、表3，图6a、6b所示。
表2PEG-ASP偶联物和门冬酰胺酶的药代动力学参数比较(静脉注射)
Figure PCTCN2015072167-appb-000005
表3PEG-ASP偶联物和门冬酰胺酶的药代动力学参数比较(肌肉注射)
Figure PCTCN2015072167-appb-000006
Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)的药代参数与门冬酰胺酶的参数相比，无论是静脉注射还是肌肉注射，其半衰期明显延长，并且药时曲线下面积(AUC)有了显著提高。其次，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)在血液中的清除速率也远低于门冬酰胺酶。由此可见，与门冬酰胺酶相比，Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)在体内的稳定性均有一定程度的增强，而Y-PALD-40K-ASP(Di)增强幅度更为明显，因此其半衰期显著提高，血液中代谢速度明显降低，有效的延长了药物的药效时间，如图6所示。另外从软件计算的药代参数可以看出，和已上市的培门冬酶比较，不管是肌肉注射还是静脉注射，Y-PALD-40K-ASP(Di)的半衰期都高于培门冬酶。
实施例6：PEG-ASP偶联物的稳定性研究
将培门冬酶、Oncaspar(购自Sigma Tau制药公司)以及Y-PALD-40K-ASP(Di)分别用Tris-HCl(9.0)的缓冲液稀释到1mg/ml。放入55℃水浴锅中，分别于0h、1h、2h、3h、4h、5h之后各取出100ul，用于酶活性检测。活性测定结果如图7所示。
由图可以看出，经过水浴处理1小时，培门冬酶的活性基本完全损失，活性基本为零，Oncaspar样品的酶活略好于培门冬酶，在处理1小时后保留了30％左右的活性，而Y-PALD-40K-ASP(Di)样品在处理1小时后保留了80％左右的活性，在处理5小时后依然保持了60％左右的生物活性，稳定性显著优于培门冬酶和Oncaspar。分析培门冬酶和Oncaspar活性显著降低的原因，应该和其PEG容易脱落有关，由于PEG分子的不断脱落，使得PEG对门冬酰胺酶的保护作用显著降低，造成活性损失较大。而Y-PALD-40K-ASP(Di)样品在处理过程中没有发生PEG脱落，PEG分子可以很好的增加门冬酰胺酶的热稳定性，因此活性损失相对较慢。
实施例7：PEG-ASP偶联物的体外活性检测
L-门冬酰胺酶可以水解门冬酰胺的酰胺基，根据这一原理，可以对门冬酰胺酶的活性进行测定。具体测定方法参照2005版药典第二部第31页所述方法进行。测活所需的试剂都购自国药集团化学试剂有限公司。检测的样品分别是Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)，Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)以及未修饰 的原蛋白和市场上的同类产品培门冬酶(购自江苏恒瑞医药股份有限公司)，Oncaspar(购自Sigma Tau制药公司)。它们的相对活性比较结果如图8所示。
由图8活性测定的结果可以看出，本发明的PEG修饰门冬酰胺酶后的活性和市场上的同类产品培门冬酶，Oncaspar都有一定程度的降低，但Y-PALD-40K-ASP(Mono)及Y-PALD-40K-ASP(Di)，Y-PALD-30K-ASP(Mono)及Y-PALD-30K-ASP(Di)分别保留了原蛋白90.25％、90.5％、89.5％、85.8％的活性，而培门冬酶和Oncaspar的活性仅保留了原蛋白62.9％、53.5％的活性。
通过酶活测定结果可以看出，本发明实施例1中制备的门冬酰胺酶定点修饰产物，其活性显著高于市场上已有的产品培门冬酶和Oncaspar。市售的培门冬酶和Oncaspar是采用分子量5000Da的PEG通过随机修饰得到的产品，修饰后的门冬酰胺酶的活性只保留了原蛋白62.9％和53.5％的活性；而本发明采用分子量30kDa～40kDa的PEG丙醛对门冬酰胺酶进行N端定点修饰，修饰后的门冬酰胺酶的活性保留了原蛋白90％左右的生物活性。另外，按照PEG修饰的一般规律，使用同类型的PEG对蛋白进行修饰，PEG的分子量越大，修饰后蛋白的活性损失越大。但在本发明中制备的修饰产物却很意外的没有完全遵循这样的规律，不仅修饰物活性高于培门冬酶的活性，而且本发明中使用分子量为30KDa和40KDa的PEG修饰剂修饰后得到的修饰产物的活性基本都保留了原蛋白90％左右的活性，其中用40KDa的PEG修饰后得到的修饰产物的活性略大于用30KDa的PEG修饰后得到的修饰产物。另外，对于使用同一分子量的PEG修饰剂修饰后得到的修饰产物来说，和单修饰产物比较，二修饰产物中每个门冬酰胺酶的分子上多偶联了一个PEG分子，因此二修饰的分子量显著高于单修饰产物。从实施例1中的凝胶过滤分析的图谱上也可以看出这点。因此按照PEG修饰的一般规律，二修饰产物的活性应该显著低于单修饰产物。但本发明中制备的单修饰产物和二修饰产物的活性基本没有差别。我们进一步分析，这可能和门冬酰胺酶的结构特点有密切的关系，门冬酰胺酶是由4个亚基组成的多亚基蛋白，这4个亚基的氨基酸序列完全一致，活性中心处在由4个亚基包裹的空间区域中。我们制备的二修饰产物，PEG分子是分别偶联在2个不相邻亚基的N端，因此这两个PEG分子相对独立，相互之间不会有较大的空间位阻，因此使得单修饰产物和二修饰产物的活性差别不大。
另外，由于已有的文献或专利报道中，也有使用定点修饰技术对门冬酰胺酶进行N端定点修饰的例子。这些例子中使用的都是直链PEG，并且制备出的修饰产物是N端定点的单修饰产物，活性都只有原蛋白40％左右，远低于本发明中制备的修饰产物。由于都属于N端定点修饰，且都是直链、小分子量的PEG，因此修饰后的修饰产物其活性理论上应该更优于本发明，但结果出人意料的是本发明中制备出的修饰产物活性较高。因此可以看出，在用PEG 对蛋白进行修饰，开发长效蛋白药物的过程中，虽然有一些普遍的规律可以遵循，但最为重要的是要结合所修饰的蛋白药物的结构特点，进行大量的优化筛选，这样才能筛选制备出最佳的修饰产物。这是一个具体情况具体分析的过程。
实施例8：PEG-ASP偶联物对肿瘤细胞的抑制率评价
为了评价PEG-ASP偶联物对肿瘤细胞的抑制率，并和培门冬酶以及Oncaspar进行比较，我们选择了THP-1(来源于人的单核细胞白血病细胞系)、Raji(来源于人的淋巴瘤细胞系)、L1210(来源于小鼠的白血病细胞)这3种细胞进行评价。通过MTT法检测细胞的抑制率，考察了不同给药浓度的抑制率，并最终计算出IC50值，计算结果如下表4所示。
表4不同PEG-ASP偶联物及培门冬酶对肿瘤细胞的IC50值
Figure PCTCN2015072167-appb-000007
根据实验结果，PEG定点修饰后的门冬酰胺酶对上述3种肿瘤细胞的杀伤性普遍高于未修饰的门冬酰胺酶，并且它们的抗肿瘤活性明显高于培门冬酶和Oncaspar，对多种细胞株均表现出良好的抗肿瘤作用。由于本发明中制备的门冬酰胺酶的定点修饰产物，其比活都高于培门冬酶和Oncaspar，因此对肿瘤细胞的抑制作用更好。

Claims (11)

  1. 聚乙二醇定点修饰的L-门冬酰胺酶，其特征在于，聚乙二醇在L-门冬酰胺酶的1或2个亚基的N端氨基上偶联，所述聚乙二醇分子量为30-40KDa，聚乙二醇为分支型。
  2. 聚乙二醇定点修饰的L-门冬酰胺酶，其特征在于，聚乙二醇在L-门冬酰胺酶的1或2个亚基的N端氨基上偶联，所述聚乙二醇分子量为30-40KDa，聚乙二醇为分支型，活化基团为醛基。
  3. 根据权利要求1所述的聚乙二醇定点修饰的L-门冬酰胺酶，其特征在于，聚乙二醇在L-门冬酰胺酶的2个亚基的N端伯氨基上偶联，所述聚乙二醇分子量为30KDa或40KDa，聚乙二醇活化基团为乙醛、丙醛、丁醛或戊醛。
  4. 根据权利要求1所述的聚乙二醇定点修饰的L-门冬酰胺酶，其特征在于所述聚乙二醇化的L-门冬酰胺酶的结构通式如下所示：
    Figure PCTCN2015072167-appb-100001
    其中R为H或C1-C4的烷基；n为320-455的整数值，P为1-4的整数；AA为N端的L-氨基酸残基，m为0-5的整数，S为1或2。
  5. 根据权利要求4所述的聚乙二醇定点修饰的L-门冬酰胺酶，其特征在于所述烷基为甲基，n为320-455之间的整数值，P为2，m为0。
  6. 聚乙二醇定点修饰的L-门冬酰胺酶的制备方法，其特征在于包括如下步骤：步骤1，用40mM pH5.0的乙酸-乙酸钠缓冲液配制15-20mg/mL的L-门冬酰胺酶溶液；步骤2，按摩尔比L-门冬酰胺酶:PEG:还原剂＝1:(4-5):(100-200)在4℃的条件下反应12-24h；步骤3，反应结束后以离子交换色谱法纯化处理，最终得到聚乙二醇单修饰或者二修饰的L-门冬酰胺酶；其中，所述还原剂为氰基硼氢化钠。
  7. 权利要求1-5任一项所述聚乙二醇定点修饰的L-门冬酰胺酶药学上可接受的盐或复合物。
  8. 一种药物组合物，其特征在于所述药物组合物含有权利要求1-5任一项聚乙二醇定点修饰的门冬酰胺酶、或其药学上可接受的盐或其复合物和药学上可接受的辅料。
  9. 根据权利要求8所述的药物组合物，其特征在于所述的药物组合物的剂型为冻干粉针制剂。
  10. 权利要求1-5任一项聚乙二醇定点修饰的门冬酰胺酶或权利要求8或9所述的药物组合物在制备治疗肿瘤的药物中的应用。
  11. 权利要求1-5任一项聚乙二醇定点修饰的门冬酰胺酶或权利要求8或9所述的药物组合物在制备治疗急性淋巴细胞白血病、NK/T细胞淋巴瘤、何杰金氏病、急性骨髓白血病、急性骨髓单核细胞白血病、慢性淋巴细胞白血病、淋巴肉瘤、网状细胞肉瘤或黑素肉瘤 的药物中的应用。
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