WO2013097657A1 - 人精氨酸酶和定点聚乙二醇化人精氨酸酶及其应用 - Google Patents

人精氨酸酶和定点聚乙二醇化人精氨酸酶及其应用 Download PDF

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WO2013097657A1
WO2013097657A1 PCT/CN2012/087240 CN2012087240W WO2013097657A1 WO 2013097657 A1 WO2013097657 A1 WO 2013097657A1 CN 2012087240 W CN2012087240 W CN 2012087240W WO 2013097657 A1 WO2013097657 A1 WO 2013097657A1
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arginase
pegylated
cysteine
mutated
amino acid
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PCT/CN2012/087240
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English (en)
French (fr)
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郑宁民
陈丽
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拜奥生物科技（上海）有限公司
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Priority to KR1020147020855A priority Critical patent/KR102076348B1/ko
Priority to JP2014549335A priority patent/JP2015503333A/ja
Priority to US14/368,508 priority patent/US20150010522A1/en
Priority to EP12861903.8A priority patent/EP2799539B1/en
Priority to BR112014015803-7A priority patent/BR112014015803B1/pt
Publication of WO2013097657A1 publication Critical patent/WO2013097657A1/zh

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    • 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)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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)
    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03001—Arginase (3.5.3.1)

Definitions

  • the present invention relates to an arginase, a process for its preparation and its use in the treatment of diseases associated with arginase. Specifically, the present invention provides a point mutation arginase, a preparation method thereof, and use thereof in the treatment of a disease associated with arginase. The present invention also provides a site-specific PEGylated arginase, a preparation method and application thereof. Background technique
  • Arginine is an important amino acid of mammals, including humans, involved in various physiological processes, including cell proliferation and growth.
  • arginine can serve as a direct precursor to the synthesis of a potential signaling molecule, nitrogen oxides (NO).
  • NO acts as a neurotransmitter, a smooth muscle relaxant and a vasodilator.
  • the biosynthesis of NO involves Ca ++ and NADPH-dependent reactions catalyzed by nitric oxide synthase.
  • Another role of arginine is as a precursor to polyamines, spermidine or spermine, involved in different physiological processes.
  • Arginase is an enzyme that catalyzes the hydrolysis of L-arginine to produce ornithine and urea, and is generally contained in animals that produce urea (mammals, squid, two-handed, sea turtles). Liver, kidney, testis, as a urea cycle One link works.
  • Arginase is an enzyme that catalyzes the final step of urea formation in the mammalian urea cycle pathway, converting arginine to ornithine and urea.
  • the human arginase family includes arginase I and arginase II.
  • Arginase I is mainly expressed in liver cells
  • arginase II is mainly expressed in kidney and red blood cells.
  • Arginase can be obtained by two methods, one is isolated from the organism in which it is produced, and the other is obtained by genetic engineering techniques.
  • the superiority of recombinant production of arginase by genetic engineering technology exists. For example, experiments have shown that E. coli can produce large amounts of arginase.
  • recombinant production of arginase by genetic engineering techniques sometimes has technical problems, such as low enzyme activity, poor stability in vivo, and short half-life, which cannot be applied to actual clinical practice.
  • US Pat. No. 7,951,366 B2 discloses a pharmaceutical composition and method for treating human malignancies using arginine deprivation.
  • Recombinant human arginase I with his-tag was used to covalently conjugate a group with an amino group in the N-terminus of the arginase or amino acid in the surface amino acid with a molecular weight of 5,000 (MW 5,000). Coupled with polyethylene glycol modification, the stability of the modified human arginase is increased, and the half-life in human serum is 3 days.
  • US20100247508A1 modified human arginase with his-tag, replacing cysteines 168 and 303 with serine, and using polyethylene glycol with a molecular weight of 20 kDa for human arginase Make modifications.
  • the arginase has an additional polypeptide fragment such as His-tag.
  • Most drug regulatory agencies in most countries do not recommend the use of these peptide fragments.
  • the State Food and Drug Administration of China has proposed in the "Guidelines for Quality Control Technology for Human Recombinant DNA Products" to simplify the production process.
  • the polypeptide fragment, such as His-tag should be removed as much as possible in the final product.
  • WO 2011/008495 A2 discloses the addition of a cysteine residue at the position of the third amino acid at the N-terminus, based on the retention of the three cysteines carried by the human arginase itself.
  • the human arginase to which a cysteine residue is added is then modified with an oxirane polyethylene glycol maleimide having a molecular weight of 20 kDa.
  • the three cysteines carried by the human arginase itself still exist, and the formed PEGylated human arginase product is heterogeneous and has a low yield, which is difficult to separate and purify.
  • the invention provides an isolated and substantially purified arginase.
  • Arginase is an enzyme that catalyzes the final step of urea formation in the mammalian urea cycle pathway, converting arginine to ornithine and urea.
  • the arginase family includes arginase I and arginase II. Sequencing and activity studies of arginase I from various sources have revealed that the sequences of arginase I from different sources may differ, but also have many conserved regions and active sites.
  • wild type human arginase I has the amino acid sequence of SEQ ID NO. 1, with three vesicles therein. The amino acid is located at positions 45, 168 and 303 of the amino acid sequence of SEQ ID NO. 1, respectively, and the gene has the nucleic acid sequence of SEQ ID NO.
  • the term “isolated” refers to a non-natural form.
  • substantially purified means that there may be other ingredients used to produce and/or modify the protein, but the other ingredients are substantially absent or less proportionate throughout the product.
  • the invention provides a mutant arginase, characterized in that the arginase is human arginase I, which has
  • amino acid sequence obtained by substitution, deletion, insertion, addition or inversion of one or several amino acids is also introduced in the above amino acid sequence, and has arginase activity.
  • the cysteine is mutated to a non-polar amino acid in the arginase of the invention.
  • Amino acids can be classified into polar amino acids and non-polar amino acids depending on the nature of the side chain groups.
  • the side chain group of an amino acid is uncharged or has a very weak polarity, such as: glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, color Amino acid, valine.
  • the cysteine mutation in the arginase of the invention is glycine, alanine, valine, leucine, isoleucine, methionine, styrene Amino acid, tryptophan or valine.
  • the cysteine is mutated to alanine in the arginase of the present invention.
  • a cysteine belonging to a non-ionized polar amino acid in a human arginase I sequence is mutated to an amino acid belonging to a non-polar amino acid property (for example, alanine), and the enzyme The activity is significantly increased.
  • a non-polar amino acid property for example, alanine
  • the arginase of the invention is at position 45 of the amino acid sequence of SEQ ID NO.
  • the refined ammonia of the invention is mutated to a cysteine at position 303 of the amino acid sequence of SEQ ID NO. 1, which is preferably mutated to glycine, alanine, valine, leucine, isoleucine, More preferably, methionine, phenylalanine, tryptophan or valine is mutated to alanine.
  • the arginase of the invention is mutated in any two of the cysteines at positions 45, 168 and 303 of the arginase.
  • the arginase of the invention is mutated at positions 45 and 303 of the amino acid sequence of SEQ ID NO. 1, preferably mutated to glycine, Alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine, more preferably, mutated to alanine.
  • the arginase of the invention is mutated to a cysteine at positions 168 and 303 of the amino acid sequence of SEQ ID NO.
  • the arginase of the invention is mutated at positions 45 and 168 of the amino acid sequence of SEQ ID NO. 1, the cysteine preferably being mutated to glycine, Alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine, more preferably, mutated to alanine.
  • the arginase of the invention is mutated at positions 45, 168 and 303 of the amino acid sequence of SEQ ID NO. 1, said cysteine
  • the amino acid is mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan or valine, and more preferably, mutated to alanine.
  • the arginase of the present invention has a stronger activity than wild-type arginase.
  • the specific activity of the arginase of the present invention is generally at least about 500 U/mg, preferably at least about 700 U/mg, and most preferably at least about 800 U/mg.
  • the arginase of the present invention generally has a stronger activity than the wild type arginase.
  • the arginase of the invention has the polynucleotide of the base sequence of SEQ ID NO. 2, and wherein at least one of the codes corresponds to amino acid sequence 45, 168 of SEQ ID NO.
  • the base sequence of the cysteine at position 303 was replaced.
  • one, two or three base sequences encoding cysteine are substituted.
  • Amino acids are encoded by polynucleotides. Messengers in a polynucleotide The three bases on the R A chain that determine an amino acid are called a "codon", also known as a triplet code.
  • the substitution of the base is replaced by TGT to encode glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, guanidine
  • the base sequence of the acid preferably, is replaced by a base sequence encoding alanine.
  • the base sequence encoding alanine is 0 « ⁇ 00,0[,0[0, preferably For GCT.
  • the invention provides a method of making the above arginase comprising expressing an arginase gene or a mutated arginase gene in a host in which the protein can be expressed.
  • the method of preparing an arginase of the present invention generally comprises the following main steps.
  • the gene of interest or the nucleotide fragment of interest is obtained by PCR or synthetic methods.
  • a DNA fragment carrying the gene of interest is ligated to a vector capable of independent replication and having a selectable marker, such as a plasmid, a bacteriophage, and a virus, to form a recombinant DNA molecule.
  • the DNA fragments are ligated to the vector mainly by homopolymeric ends, sticky end connections, flush end connections, and artificial linker molecules.
  • Recombinant DNA must enter the host cell for amplification and expression.
  • recombinant DNA molecules can be introduced into host cells by means of transfection, transformation, transduction, etc., and allowed to multiply.
  • Suitable genetically engineered hosts are well known in the art and may be E. coli, yeast, insect cells, and the like.
  • the invention also provides an isolated and substantially purified PEGylation arginase, characterized in that the pegylation is site specific.
  • the polyethylene glycol molecule is covalently bound to a specific amino acid residue on the arginase molecule to achieve site-specific pegylation.
  • each arginase molecule binds to at least one polyethylene glycol molecule to form a PEGylated arginase.
  • the arginase enzyme of the PEGylated arginase of the invention is a mutant arginase as described above.
  • the invention provides a PEGylated arginase, wherein the arginase is human arginase I, which has
  • amino acid sequence obtained by substitution, deletion, insertion, addition or inversion of one or several amino acids is also introduced in the above amino acid sequence, and has arginase activity.
  • the cysteine point mutation in the PEGylated arginase of the invention is a non-polar amino acid.
  • the structural differences in the 20 protein amino acids depend on the difference in side chain groups.
  • the side chain group of an amino acid is uncharged or has a very weak polarity, such as: glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, color Amino acid, valine.
  • the sequence of the pegylated human arginase I does not have a tag sequence for purifying the protein, for example, a His-tag sequence is added at its C-terminus or N-terminus.
  • His-Tag fusion protein is currently the most A common expression, which has the advantage of being convenient to purify and does not substantially affect the activity of the protein, whether the expressed protein is soluble or the inclusion body can be purified by immobilized metal ion affinity chromatography.
  • the State Food and Drug Administration of China has proposed in the "Guidelines for Quality Control Techniques for Human Recombinant DNA Products" to introduce additional peptide fragments such as His-tags for the purpose of simplifying the production process. It should be removed as much as possible in the final product.
  • pegylation of arginase can be achieved by a chemical modification method which can be carried out by a polyethylene glycol derivative (also referred to as polyethylation) with a coupling agent. a diol modifier) that covalently binds a polyethylene glycol molecule to a group on the arginase.
  • the chemical modification may be specific, i.e., by using a coupling agent that binds to a particular group, the PEG can only bind to a particular amino acid on the arginase.
  • Site-specific PEGylation modification should follow the following principles: (1) Try to avoid modification of the active site of the protein to prevent the activity of the protein; (2) Simplify the whole process as much as possible, which is controllable.
  • a common polyethylene glycol molecule has a hydroxyl group at each end, and if one end is blocked with a thiol group, a decyloxy polyethylene glycol (mPEG) is obtained.
  • mPEG decyloxy polyethylene glycol
  • the most studied polyethylene glycol modifiers in the polyethylene glycol modification of polypeptides and proteins are derivatives of mPEG.
  • One of the common methods of modifying polyethylene glycol is to combine a polyethylene glycol molecule/polyethylene glycol modifier with lysine on the surface of the protein or ⁇ - ⁇ 2 or ⁇ - ⁇ 2 at the N-terminus of the protein, such as mPEG-succinyl.
  • Modifiers such as mPEG-maleimide, mPEG-o-pyridine-disulfide, mPEG-vinyl sulfone, mPEG-iodoacetamide, etc., to modify the sulfhydryl group of a protein or polypeptide.
  • the sparse base is relatively small, and it is easier to form a homogenous product.
  • the human arginase I monomer has three cysteine residues located at positions 45, 168 and 303 of the amino acid sequence, respectively.
  • the PEGylated arginase is site-specifically PEGylated at one or both of the cysteine sites at positions 45, 168 and 303.
  • the site-directed pegylation is achieved by genetic engineering site-directed mutagenesis of the arginine cysteine followed by cysteine-specific PEGylation.
  • the cysteine-specific PEGylation modification is by using a decyloxy polyethylene glycol maleimide coupling agent to occlude PEG with arginase The S group of the acid is covalently bound.
  • one or two cysteine mutations that do not require PEGylation in the arginase of the invention are preferably mutated to glycine, alanine, valine, leucine Acid, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably mutated to alanine.
  • the choice of PEG molecular weight takes into account both biological activity and pharmacokinetic factors. Previous studies have found that the time of action of the modified protein drug in the body is related to the amount and molecular weight of the coupled PEG.
  • the PEG used in the present invention may have a molecular weight in the range of 5K to 40K Daltons, may be linear or branched, and may be a PEG derivative as described in the art.
  • the PEG molecules covalently bound to the arginase molecule in the present invention are not limited to a specific type. In one aspect of the invention, the PEG molecule PEGylated to arginase has an average molecular weight of 20K, 30 or 40K.
  • the PEGylated protein enters the body by intravenous injection, and the average molecular weight of the polyethylene glycol affects its half-life in serum.
  • glomeruli can filter protein molecules with molecular weights less than 70 kDa; for PEG, glomeruli can filter PEG molecules with molecular weights less than 30 kDa.
  • the molecular weight of PEG is generally unpredictable for the half-life and activity of the protein to be bound, and is closely related to the molecular weight of the protein, the mode of action, the active site, the PEG binding site, and the like.
  • the PEGylated arginase is site-specifically PEGylated at positions 45 and 168 of the amino acid sequence defined by SEQ ID NO.
  • the site-directed pegylation is achieved by mutating a cysteine at position 303 of the arginase, wherein the PEG has an average molecular weight of 20K or 30K.
  • the cysteine is preferably mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably to propyl Amino acid.
  • the PEGylated arginase is site-specifically PEGylated at position 168 of the amino acid sequence defined by SEQ ID NO. 1, wherein the site-specific PEGylation This was achieved by mutating the 45th and 303th cysteine of the arginase, wherein the PEG has an average molecular weight of 40K.
  • the cysteine is preferably mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably to propyl Amino acid.
  • the PEGylated arginase is site-specifically PEGylated at position 45 of the amino acid sequence defined by SEQ ID NO. 1, wherein the site-specific PEGylation is This was achieved by mutating the 168th and 303th cysteines of arginase, wherein the PEG has an average molecular weight of 40K.
  • the cysteine is preferably mutated to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, valine, more preferably to propyl Amino acid.
  • the PEGylated arginase enzymes provided herein generally have a purity in excess of more than 70%, preferably in excess of 80%, and most preferably in 90%.
  • the "purity of PEGylated arginase" as used in the present invention means PEGylated arginase (ie, covalently bonded) in a chemically modified production of PEGylation of arginase
  • the arginase of PEG accounts for the ratio of total arginase (arginase covalently bound to PEG, arginase not covalently bound to PEG and polyPEGylated arginase).
  • the product after PEGylation also contains arginase which is not covalently bound to PEG and polyPEGylated arginase
  • purification of the product is required.
  • Commonly used purification means are well known in the art, including cation exchange columns and the like.
  • the PEGylated arginase provided by the present invention has a half-life in blood or serum of at least 0.5 days, preferably at least 2.5 days, and most preferably at least 3.5 days.
  • the half-life of arginase can be measured by methods well known and commonly used in the art.
  • the measurement of the half-life of arginase in blood or serum is related to the animal model used.
  • the half-life data obtained in animal models with faster metabolic rates (e.g., rats) is generally shorter than data obtained in animal models with faster metabolic rates (e.g., humans).
  • the invention also provides a method of preparing a pegylation arginase.
  • the pegylation is site specific.
  • the polyethylene glycol molecule of the PEGylated arginase of the present invention binds to a specific amino acid residue on the arginase molecule to achieve site-specific pegylation.
  • each arginase molecule binds to at least one polyethylene glycol molecule to form a PEGylated arginine.
  • site-directed pegylation of arginase can be achieved by a chemical modification method in which PEG is covalently covalent with a group on arginase by using a coupling agent. Combine.
  • the chemical modification may be specific, i.e., by using a coupling agent that binds to a particular group, the PEG can only bind to a particular amino acid on the arginase.
  • the present invention also provides the use of any of the above-described arginase or pegylated arginase of the present invention or a pharmaceutical composition thereof for the treatment of a disease associated with arginase /use.
  • the diseases associated with arginase are known in the art to include, for example, conditions/diseases/disorders associated with arginine in mammals. Such conditions/diseases/disorders include: hyperarginemia. Due to the lack of arginase activity, the arginine in the patient cannot be lysed into urea and added to the ornithine metabolic cycle.
  • the arginine content in the blood can be 7-10 times higher than normal, and the arginine in the cerebrospinal fluid and urine. It also increased, and the urinary creatinine output increased.
  • such conditions/diseases/disorders also include arginine-dependent cell proliferation or tumors such as liver cancer, melanoma, breast cancer, small cell lung cancer, prostate cancer, lymphoma and leukemia. .
  • the cells can begin to return to the normal physiological cycle; for proliferating cells or tumors It is said that the lack of arginine causes it to enter the S phase through the 'R' point of the G1 phase of the cell cycle, and soon to apoptosis. Such cells or tumors that are proliferating due to lack of arginine are irreversible. Therefore, scientists began to consider the treatment of cell proliferation or tumor by controlling the level of arginine in the body.
  • the present invention also provides a pharmaceutical composition, the active ingredient of which is the above-described arginase or pegylated arginase of the present invention.
  • the pharmaceutical composition may be formulated in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome or the like, wherein the resulting formulation contains one or more human arginase or PEGylated of the present invention.
  • Arginase is the active ingredient and is mixed with an organic or inorganic carrier or excipient suitable for enteral or parenteral application.
  • adjuvants, stabilizers, thickeners, colorants and perfumes can be used.
  • the active ingredient of one or more isolated and substantially purified human arginase or pegylated arginase enzymes is also included in a sufficient amount of the pharmaceutical formulation to produce the desired desired process, condition or disease. effect.
  • the above pharmaceutical compositions may be formulated into a form suitable for oral administration, for example, tablets, tablets, cough drops, hydrated or oleaginous suspensions, dispersed powders or granules, emulsions, hard or soft capsules, or syrups.
  • Oral formulations can be encapsulated according to techniques known in the art to slow the breakdown and absorption in the gastrointestinal tract, thus providing a sustained action over a longer period of time.
  • the formulation may also be in the form of a sterile injectable solution or suspension.
  • the suspension may be formulated according to known methods using dispersing or wetting agents and suspending agents.
  • the pharmaceutical composition of the present invention may be further prepared in the form of a solid, a liquid, a suspension, a micelle, or a liposome.
  • the formulation of the pharmaceutical composition is in a form suitable for oral or injection.
  • Figure 1 shows the nucleic acid sequence of wild type human arginase I.
  • Figure 2 shows the results of a reductive SDS-polyacrylamide gel electrophoresis assay for human arginase expression.
  • Figure 3 shows a liquid chromatogram of human arginase expression.
  • Figure 4 shows the results of gel electrophoresis detection of wild-type arginase I after reduction and non-reduction treatment.
  • Figure 5 shows the results of gel electrophoresis after purification and non-reduction treatment of purified arginase I mutants (rhArgI-A303, rhArgl-Al 68/303, and rhArgI-A45/303).
  • Figure 6 shows the results of gel electrophoresis after purification and non-reduction treatment of purified arginase I mutants (rhArgI-A45/468, and rhArgI-A45/l 68/303).
  • 6a is rhArgI-A45/303
  • rhArgI-A45/l 68/303 is detected by gel electrophoresis after non-reduction treatment fruit.
  • 6b is rhArgI-A45/468, and rhArgI-A45/l 68/303 is subjected to reduction treatment and gel electrophoresis detection results
  • Figure 7 shows the results of non-reducing SDS-polyacrylamide gel electrophoresis of PEGylated human arginase.
  • Example 1 Construction and expression of human arginase I recombinant plasmid pET30a(+)-rharginase-V without His-tag
  • PCR A nucleic acid fragment was obtained and confirmed by agarose gel electrophoresis.
  • the amplified fragment obtained above and the commercially available pET30a(+) expression vector were separately used in a reaction medium containing restriction endonucleases Ndel and Xhol (purchased from promega). After 1.5 hours of C treatment, the digested fragment was reacted with T4 DNA ligase at 16 ° C overnight. The ligated plasmid was then transformed into competent cells DH5a E. coli cells. Screening was performed on LB nutrient agar plates containing 3 ( ⁇ g/ml kanamycin.
  • a plasmid with the correct insert was obtained by restriction endonuclease analysis, called pET30a(+)-rharginase-V, the plasmid The insert was confirmed by sequencing.
  • the insert contained 969 bases, as shown in Figure 1, and its nucleic acid sequence was SEQ ID No. 2.
  • Example 2 Expression of human arginase I recombinant plasmid pET30a(+)-rharginase-V without His-tag and purification of the target protein
  • a single colony of the transformed cell culture plate was picked and transferred to 25 mL of LB medium.
  • the cell is at 37. C, 150 rpm shaking culture to OD600nm reached 0.6-0.8, and the final concentration was 0.2 mM IPTG to induce expression for 3 hours.
  • SDS-P AGE electrophoresis was performed to detect whether the selected clone had the expression of the target protein.
  • the clones with higher expression were selected to prepare the sweet oil bacteria for preservation as engineering bacteria.
  • E. coli engineering bacteria were cultured in a 15 L fermentor in a fed-batch manner, and when the OD600nm reached 12-13, the expression was induced by adding 0.2 mM IPTG to a final concentration of 3-4 hours. The obtained cells were centrifuged and centrifuged, and the supernatant was taken for further separation and purification.
  • Figure 2 is a result of gel electrophoresis detection of human arginase expression.
  • the samples for each lane are:
  • Figure 3 is a liquid chromatogram of human arginase expression.
  • the purity of the arginase obtained by the above chromatographic method was 90% or more.
  • PCR polymerase chain reaction
  • the polymerase chain reaction was carried out according to the method provided in the kit instructions, and then the template plasmid was digested with restriction endonuclease Dpn1, and competent cells were transformed.
  • the obtained recombinant mutant plasmid was sequenced and confirmed.
  • the sequencing results were successfully mutated to the alanine codon (GCT) clone after selection of the cysteine codon (TGT), and inoculated in LB liquid medium for amplification.
  • the mutant recombinant plasmid was extracted using the Wizard Plus Minipreps kit.
  • the recombinant human arginase I of the mutated human arginase I (rhArgl) sequence substituted with alanine at position 303 is represented by rhArgI-A303; the cysteine at position 168 is replaced with C.
  • the recombinant human arginase I of the amino acid is represented by rhArgI-A168; the recombinant human arginase I substituted with alanine at position 45 is represented by rhArgI-A45; cysteine substitution at positions 168 and 303
  • the recombinant human arginase I which is alanine is represented by rhArgI-A168/303; the recombinant human arginase I substituted for cysteine at positions 45 and 303 to alanine is represented by rhArgI-A45/303;
  • Recombinant human arginase I substituted with 168 cysteine for alanine is represented by rhArgI-A45/168; recombinant human arginase with 45, 168 and 303 cysteine replaced by alanine I denoted as rhArgI-A45/l 68/303; the recombinant mutant plasmid containing the human arginas
  • the transformed cell culture plate was selected for a single colony and transferred to 25 mL of LB medium.
  • the cell is at 37. C, 150 rpm shaking culture to OD600nm reached 0.6-0.8, and the final concentration was 0.2 mM IPTG to induce expression for 3 hours.
  • the selected clones were examined for the expression of the protein of interest by polyacrylamide gel electrophoresis (SDS-PAGE). The clones with higher expression were selected to prepare glycerol bacteria for preservation as engineering bacteria.
  • Example 4 Expression and purification of a plasmid containing a site-directed mutant arginase I (rhArgl)
  • the Escherichia coli engineered bacteria including pET30a(+)-rhArgI-A303, pET30a(+)-rhArgI-Al 68/303, pET30a(+) transformed with the mutant human arginase I (rhArgl) plasmid described above -rhArgI- A45/303 , pET30a(+)-rhArgI- A45/168 , or pET30a(+)-rhArgI- A45/168/303 ) was cultured in a 15L fermentor in batches, when the OD600nm reached 12 At -13 hours, expression was induced to a final concentration of 0.2 mM IPTG for 3-4 hours. The obtained cells were centrifuged and centrifuged, and the supernatant was taken for further separation and purification.
  • the collected samples were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and high pressure liquid chromatography to analyze the purity.
  • the purity of the arginase obtained by the above chromatography method is 90% or more.
  • the activity of arginase was determined by measuring the absorbance value of NADPH coupled to urease and glutamate dehydrogenase.
  • the arginase activity unit is defined as: 1 unit (U) of arginase can be at 30. C, pH 8.3 release ⁇ ⁇ urea.
  • the human arginase I (rhArgI) sequence is located at 168/303, 45/303, 45/168 and 45/168/303.
  • the cysteine belonging to the non-ionized polar amino acid is mutated to be non-
  • the post-alanine enzymatic activity of polar amino acid properties is significantly increased.
  • Example 6 SDS-PAGE electrophoresis analysis of wild type and site-directed mutant arginase I (rhArgl)
  • FIG. 4 is a result of gel electrophoresis detection of wild-type arginase I after reduction and non-reduction treatment.
  • the samples in each lane are: 1 , 3 : bacteriostatic supernatant; 2. purified His-tag-free human arginase 1 (non-reducing treatment); M: protein molecular weight standard; 4. purified His-tag-free human arginase I (reduced treatment).
  • Figure 5 shows the results of gel electrophoresis after purification and non-reduction treatment of purified arginase I mutants (rhArgI-A303, rhArgl-Al 68/303, and rhArgI-A45/303).
  • the samples in each lane are: 1. Arginase I mutant rhArgI-A303 (non-reducing treatment); 2. Arginase I mutant rhArgI-A168/303 (non-reducing treatment); 3. Fine Lysin I mutant rhArgI-45/A303 (non-reducing treatment); M: protein molecular weight standard; 4. arginase I mutant rhArgI-A303 (reduced treatment); 5.
  • FIG. 6a shows the results of gel electrophoresis after purification of the purified arginase I mutant (rhArgI-A45/468, and rhArgI-A45/l 68/303).
  • the samples in each lane are: M: protein molecular weight standard; 1. arginase I mutant rhArgI-A45/l 68/303; 2. arginase I mutant rhArgI-A45/168
  • Figure 6b shows the results of gel electrophoresis after purification of the purified arginase I mutants (rhArgI-A45/468, and rhArgI-A45/l 68/303).
  • the samples in each lane are: M: protein molecular weight standard; 1. arginase I mutant rhArgI-A45/l 68/303; 2. arginase I mutant rhArgI-A45/168
  • Example 7 Site-directed PEGylation of mutant arginase I (rhArgl) and purification of PEGylated protein
  • the above-mentioned site-modified PEGylated human arginase I was isolated and purified by using a cation exchange column of Macrocap SP to remove residual PEG and a small amount of unreacted protein.
  • the equilibration solution was a phosphate buffer, and the eluent was a phosphate buffer containing 1 M NaCl.
  • the modified protein sample is first diluted with purified water so that the conductance of the sample is the same as that of the equilibrium solution, and the diluted sample is used as a sample for loading. After equilibrating the column with 5 times column volume with an equilibration solution, the sample was loaded.
  • a site-directed mutagenized arginase I (rhArgI-A303) was reacted with a decyloxy polyethylene glycol maleimide (mPEG-MAL-30K) and purified by a Macrocap SP cation exchange column.
  • Site-directed PEGylation of human arginase I wherein the human arginase (presented by A303-M30K(2)) with two molecular weight 30K oxime ethoxylate maleimides was formed. That is, the ratio of all the cysteine groups at positions 45 and 168 to the decyloxy polyethylene glycol maleimide accounts for more than 70% of the total reaction product.
  • a site-directed mutagenized arginase I (rhArgI-A168/303) was reacted with a Y-type polyethylene glycol maleimide (Y-MAL-40K) via a Macrocap SP cation exchange column.
  • Y-MAL-40K Y-type polyethylene glycol maleimide
  • Purified fixed-point PEGylated human arginase I wherein the human arginase (with A168/303-Y40K) with a molecular weight of 40K Y-type polyethylene glycol maleimide was formed.
  • the ratio of the 45-position cysteine group coupled to the decyloxy polyethylene glycol maleimide accounts for more than 85% of the total reaction product.
  • the activity of arginase was determined by measuring the absorbance value of NADPH coupled with urease and glutamate dehydrogenase by the foregoing method, and the fixed-point PEG was calculated at various molecular weights and at different sites.
  • the enzymatic activities of the modified human arginase I A303-M20K (2), A303-M30K (2), A168/303-Y40K and A45/303-Y40K.
  • the results showed that the above-mentioned site-directed PEGylation-modified human arginase I retained arginase activity, and its activity was more than 400 U/mg, ranging from 400-800 U/mg.
  • Example 7 In vivo pharmacokinetic detection of site-directed PEGylated human arginase I
  • Serum arginase I was detected using a double antibody sandwich £1 ⁇ 18 method (the kit was purchased from Shanghai Yikesai Biological Products Co., Ltd.).
  • the anti-human arginase I monoclonal antibody was coated on the ELISA plate, and the sample and the different concentration standards were added to the wells ( ⁇ /well), and the reaction wells were sealed with sealing paper, 37.
  • C incubation for 90 minutes, human arginase I in the sample or standard will bind to the monoclonal antibody to form an immune complex; wash the plate 5 times; add rabbit anti-human arginase I polyclonal antibody ( ⁇ / well), Seal the reaction well with a sealing tape, 37.
  • the present invention modifies the 45, 168 and 303 cysteines in the human arginine sequence by site-directed mutagenesis, and one, two or three codons encoding a cysteine (TGT)
  • TGT cysteine
  • GCT codon
  • the present invention provides a novel site-directed PEGylated human arginase that removes potentially potentially immunogenic his-tag protein tag sequences and ranks 45, 168 and 303 in the human arginine sequence.
  • the polyethylene glycol maleimide was subjected to a coupling reaction.
  • the site-specific PEGylated human arginase synthesized by this method reduces the glomerular filtration rate, and the position of PEG coupled with human arginase is determined, and the formed product is uniform, easy to purify, and is favorable for large scale. Quality control of production.
  • the fixed-point PEGylated human arginase provided by the present invention has a long half-life in mammals, for example, a half-life in serum of rats can reach 15-28 hours.

Abstract

本发明提供了一种点突变的精氨酸酶及其制备方法，以及所述点突变的精氨酸酶在制备治疗与精氨酸酶相关的疾病的药物中的用途。本发明还提供了一种位点特异性聚乙二醇化的精氨酸酶及其制备方法，以及所述聚乙二醇化的精氨酸酶在制备治疗与精氨酸酶相关的疾病的药物中的用途。

Description

AJlt氨酸酶和定点聚乙二醇化人精氨酸酶及其应用
[0001]本申请要求了 2011年 11月 27日提交的、申请号为 201110445965.8、发明名称为 "人 精氨酸酶和聚乙二醇化人精氨酸酶及其应用"的中国专利申请的优先权以及 2012年 3月 16 日提交的、 申请号为 201210069605.7、发明名称为 "人精氨酸酶和定点聚乙二醇化人精氨 酸酶及其应用" 的中国专利申请的优先权， 其全部内容通过引用结合在本申请中。 技术领域
[0002]本发明涉及一种精氨酸酶， 以及其制备方法和在治疗与精氨酸酶相关的疾病中的 应用。 具体的， 本发明提供了一种点突变的精氨酸酶及其制备方法和在治疗与精氨酸酶 相关的疾病中的应用。 本发明还提供了一种位点特异性聚乙二醇化的精氨酸酶及其制备 方法和应用。 背景技术
[0003】精氨酸是一种哺乳动物包括人的重要氨基酸， 参与各种生理过程， 包括细胞增殖 和生长。 例如， 精氨酸可作为潜在信号分子氧化氮（NO )合成的直接前体。 NO作为一 种精神递质， 平滑肌松弛剂和血管扩张剂而起作用。 NO的生物合成涉及由氧化氮合酶催 化的 Ca++及 NADPH-依赖性反应。精氨酸的另一个作用是作为多胺、亚精胺或精胺的前体， 参与不同的生理过程。
[0004】有研究发现， 精氨酸低于 8μΜ水平时， 癌细胞发生不可回复的细胞死亡 （ Srorr & Burton, 1974, The effects of arginine deficiency on lymphoma cells. Br. J. Cancer 30, 50 ) 。 通 过研究对细胞生长的影响发现， 将精氨酸移除后正常细胞系会由细胞周期的 G0期进入静 止状态， 且能在这种环境下存活几周而无明显损伤； 当精氨酸浓度恢复正常时， 细胞又 能开始恢复正常的生理周期； 而对于肿瘤细胞来说， 精氨酸的匮乏会导致其经过细胞周 期 G1期的' R'点进入 S期， 并很快凋亡。 这种由于精氨酸匮乏而导致肿瘤细胞的凋亡是不 可逆的。 因此， 科学家们开始考虑通过控制机体中精氨酸的水平来对肿瘤， 尤其是营养 缺陷型肿瘤如肝癌和黑素瘤进行治疗。现今这种方式已被用来研究各种肿瘤细胞的抑制。
[0005]精氨酸酶是催化水解 L-精氨酸生成鸟氨酸与尿素的反应的酶， 一般含于产生尿素 的动物 （哺乳类、 板鳃鱼类、 两柄类、 海龟类） 的肝脏、 肾脏、 精巢中， 作为尿素循环 的一个环节而起作用。精氨酸酶是催化哺乳动物尿素循环途径中尿素形成最终步骤的酶， 将精氨酸转化为鸟氨酸和尿素。 在大多数哺乳动物中， 人精氨酸酶家族包括精氨酸酶 I和 精氨酸酶 II。 精氨酸酶 I主要在肝脏细胞表达， 精胺酸酶 II在肾脏和红细胞表达为主。
[0006】通过两种方法可以得到精氨酸酶， 一是从产生它的生物体中分离出来， 另一种是 通过基因工程技术重组得到。 通过基因工程技术重组生产精氨酸酶存在其优越性。 例如， 实验证明， 大肠杆菌可以生产大量的精氨酸酶。 然而， 通过基因工程技术重组生产精氨 酸酶有时会存在技术难题， 例如， 酶活性较低， 或在体内的稳定性差及半衰期短， 无法 应用于实际临床。
[0007] US7951366 B2公开了一种利用精氨酸剥夺来治疗人恶性肿瘤的药物组合物及方 法。 其中使用了带有 his-tag的重组人精氨酸酶 I, 通过分子量为 5,000 (MW 5,000)的聚乙 二醇对精氨酸酶 N-端或表面氨基酸中带有氨基的基团共价偶联聚乙二醇修饰， 修饰后的 人精氨酸酶的稳定性增加， 在人血清中的半衰期为 3天。
[0008] US20100247508A1中对带有 his-tag的人精氨酸酶进行了改造，将 168和 303位半胱氨 酸替换为丝氨酸， 并利用分子量为 20KDa的聚乙二醇对人精氨酸酶进行修饰。 但是， 与 US7951366 B2公开的重组人精氨酸酶 I相同， 该精氨酸酶带有额外的多肽片段如 His-tag。 而大部分国家的药物管理机构不建议使用这些多肽片段， 例如中国国家食品药品监督管 理局在 "人用重组 DNA制品质量控制技术指导原则"中提出以简化生产工艺为目的在产品 中 I入额外的多肽片段如 His-tag，在最终产品中应尽可能去除。
[0009] WO 2011/008495A2公开了在保留人精氨酸酶本身所带有的三个半胱氨酸的基础 上，在 N端第三个氨基酸的位置加入了一个半胱氨酸残基， 然后利用分子量为 20KDa的曱 氧基聚乙二醇马来酰亚胺对加入一个半胱氨酸残基的人精氨酸酶进行修饰。 但该人精氨 酸酶本身所带有的三个半胱氨酸仍然存在， 形成的 PEG化人精氨酸酶的产物不均一且产 率低， 难于分离纯化。
[0010】因此， 本领域需要一种更好的精氨酸酶或其衍生物， 用于治疗与精氨酸相关的疾 病或异常。 发明内容
[0011]本发明提供一种分离和基本纯化的精氨酸酶。 精氨酸酶是催化哺乳动物尿素循环 途径中尿素形成最终步骤的酶， 将精氨酸转化为鸟氨酸和尿素。 在大多数哺乳动物中， 精氨酸酶家族包括精氨酸酶 I和精氨酸酶 II。 对各种来源的精氨酸酶 I进行了测序和活性研 究发现， 不同来源的精氨酸酶 I的序列可能有所区别， 但也具有很多保守的区域和活性位 点。 如 Haraguchi, Y.等， 1987, Proc. Natl. Acad. Sci. 84， 412-415中报道的， 野生型人精氨 酸酶 I具有 SEQ ID NO. 1的氨基酸序列， 其中具有三个半胱氨酸， 分别位于 SEQ ID NO. 1 氨基酸序列的第 45 , 168和 303位， 其基因具有如 SEQ ID NO. 2的核酸序列。
[0012]本发明中，术语"分离"是指非天然的形式。术语"基本上纯化"是指可能存在用于生 产和 /或修饰蛋白质的其它成分， 但所述其它成分占整个产物基本不存在或比例较小。
[0013]本发明中对氨基酸序列位点的描述是本领域技术人员常用的。 例如， "在序列为 SEQ ID SEQ x上的第 45位"或"在序列为 SEQ ID SEQ x上的第 45位 Cys (即半胱氨酸） "或 表述" Cys45"具有相同或相近的含义， 都是表示在氨基酸序列上第 45位氨基酸， 或第 45位 的半胱氨酸。
[0014】在本发明的一个方面， 本发明提供了一种突变的精氨酸酶， 其特征在于， 所述精 氨酸酶为人精氨酸酶 I, 其具有
(1) 对应于 SEQ ID NO 1的氨基酸序列， 并且其中第 45 , 168和 303位的半胱氨酸中的一 个、 两个或三个发生突变， 或
(2) 在上述氨基酸序列中还引入了一个或几个氨基酸的取代、缺失、插入、增加或倒位后 所得的氨基酸序列， 并具有精氨酸酶的活性。
[0015]在本发明的一个方面， 本发明的精氨酸酶中所述半胱氨酸突变为非极性氨基酸。 氨基酸根据侧链基团的性质可分为极性氨基酸和非极性氨基酸。 氨基酸的侧链基团不带 电荷或极性极微弱的属于非极性氨基酸， 如： 甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮 氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸、 脯氨酸。
[0016】在本发明的其中一个方面， 本发明的精氨酸酶中所述半胱氨酸突变为甘氨酸、 丙 氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸。 优选的， 本 发明的精氨酸酶中所述半胱氨酸突变为丙氨酸。 在本发明中， 出乎意料地发现， 人精氨 酸酶 I序列中属于非电离极性氨基酸的半胱氨酸突变成属于非极性氨基酸性质的氨基酸 (例如丙氨酸）后， 酶活性明显增加。 其中一种可能是突变的精氨酸酶与底物之间的结 合能力增强。
[0017]在本发明的一个方面，本发明的精氨酸酶在 SEQ ID NO. 1的氨基酸序列上的第 45 ,
168和 303位的半胱氨酸中的一个发生突变。 在本发明的其中一个方面， 本发明的精氨 酸酶在 SEQ ID NO. 1的氨基酸序列上的第 303位半胱氨酸突变， 所述半胱氨酸优选突变为 甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸， 更优选的， 突变为丙氨酸。
[0018】在本发明的一个方面， 本发明的精氨酸酶在所述精氨酸酶的第 45 , 168和 303位 的半胱氨酸中的任意两个发生突变。 在本发明的其中一个方面， 本发明的精氨酸酶在 SEQ ID NO. 1的氨基酸序列上的第 45位和第 303位半胱氨酸突变， 所述半胱氨酸优选突变 为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸， 更优选的， 突变为丙氨酸。在本发明的其中一个方面，本发明的精氨酸酶在 SEQ ID NO. 1 的氨基酸序列上的第 168位和第 303位半胱氨酸突变， 所述半胱氨酸优选突变为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸， 更优选的， 突变为丙氨酸。 在本发明的其中一个方面， 本发明的精氨酸酶在 SEQ ID NO. 1的氨基酸 序列上的第 45位和第 168位半胱氨酸突变， 所述半胱氨酸优选突变为甘氨酸、 丙氨酸、 缬 氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸， 更优选的， 突变为丙 氨酸。
[0019]在本发明的其中一个方面， 本发明的精氨酸酶在 SEQ ID NO. 1的氨基酸序列上的 第 45位、 第 168位和第 303位半胱氨酸突变， 所述半胱氨酸优选突变为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸， 更优选的， 突变为 丙氨酸。
[0020]本发明的精氨酸酶具有比野生型精氨酸酶更强的活性。 本发明的精氨酸酶的特异 性活性一般为至少大约 500 U/mg, 优选至少大约 700 U/mg, 最优选至少大约 800 U/mg。 本发明的精氨酸酶一般具有比野生型精氨酸酶更强的活性。
[0021]在本发明的一个方面， 本发明的精氨酸酶具有 SEQ ID NO. 2的碱基序列的多核苷 酸， 并且其中至少一个编码对应于 SEQ ID NO. 1氨基酸序列第 45 , 168和 303位的半胱氨 酸的碱基序列发生替换。 优选的， 其中的一个、 两个或三个编码半胱氨酸的碱基序列发 生替换。 氨基酸是由多核苷酸编码的。 多核苷酸中的信使 R A链上决定一个氨基酸的相 邻的三个碱基叫做一个"密码子"， 亦称三联体密码。 在本发明的一个方面， 所述碱基的 替换是由 TGT替换为编码甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙 氨酸、 色氨酸、 脯氨酸的碱基序列， 优选的， 替换为编码丙氨酸的碱基序列。
[0022]在本发明的其中一个方面，编码丙氨酸的碱基序列为0«\ 00，0〔 ，0〔0,优选 为 GCT。
[0023】在本发明的一个方面， 本发明提供了制备上述精氨酸酶的方法， 其包括将精氨酸 酶基因或突变后的精氨酸酶基因在可表达蛋白的宿主中进行表达。 本发明的制备精氨酸 酶的方法通常包括以下主要步骤。 通过 PCR或合成的方法获得目的基因或目的核苷酸片 段。将带有目的基因的 DNA片段连接到能够独立复制并具有选择标记的载体上，如质粒、 噬菌体和病毒等， 以形成重组 DNA分子。 DNA片断与载体的连接方式主要有同聚末端连 接、 粘性末端连接、 平齐末端连接及人工接头分子连接。 重组 DNA必须进入宿主细胞中， 才能得到扩增和表达。 根据载体的性质不同， 可釆用转染、 转化、 转导等方式， 将重组 DNA分子导入宿主细胞内， 并使其大量繁殖。 合适的基因工程宿主是本领域公知的， 可 以是大肠杆菌、 酵母、 昆虫细胞等。
[0024】本发明还提供一种分离和基本纯化的聚乙二醇化（Pegylation ) 的精氨酸酶， 其特 征在于， 所述聚乙二醇化是位点特异性的。 本发明聚乙二醇化的精氨酸酶中聚乙二醇分 子与精氨酸酶分子上的特定氨基酸残基发生共价结合， 达到位点特异性聚乙二醇化的目 的。 在本发明的聚乙二醇化的精氨酸酶中， 每个精氨酸酶分子与至少一个聚乙二醇分子 结合， 形成聚乙二醇化的精氨酸酶。
[0025]在本发明的一个方面， 本发明聚乙二醇化的精氨酸酶中的精氨酸酶是前文描述的 突变的精氨酸酶。
[0026】在本发明的一个方面， 本发明提供了一种聚乙二醇化的精氨酸酶， 其特征在于， 所述精氨酸酶为人精氨酸酶 I, 其具有
(1) 对应于 SEQ ID NO 1的氨基酸序列， 并且其中第 45 , 168和 303位的一个或两个半胱 氨酸发生突变， 或
(2) 在上述氨基酸序列中还引入了一个或几个氨基酸的取代、缺失、插入、增加或倒位后 所得的氨基酸序列， 并具有精氨酸酶的活性。
[0027]在本发明的一个方面， 本发明的聚乙二醇化的精氨酸酶中所述半胱氨酸点突变为 非极性氨基酸。 20种蛋白质氨基酸在结构上的差别取决于侧链基团的不同。 氨基酸的侧 链基团不带电荷或极性极微弱的属于非极性氨基酸， 如： 甘氨酸、 丙氨酸、 缬氨酸、 亮 氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸、 脯氨酸。
[0028]在本发明的其中一个方面， 所述聚乙二醇化的人精氨酸酶 I的序列不具有用于纯化 此蛋白的标签序列， 例如在其 C末端或 N末端加入 His-tag序列。 His-Tag融合蛋白是目前最 常见的表达方式， 它的优点是纯化方便而且基本不影响蛋白的活性， 无论是表达的蛋白 是可溶性的或者包涵体都可以用固定金属离子亲和色谱纯化。 但是由于其潜在的免疫原 性， 中国国家食品药品监督管理局在"人用重组 DNA制品质量控制技术指导原则"中提出 以简化生产工艺为目的在产品中引入额外的多肽片段如 His-tag，在最终产品中应尽可能去 除。
[0029】在本发明中， 对精氨酸酶的聚乙二醇化可以通过化学修饰的方法实现， 所述化学 修饰可以通过带有偶联剂的聚乙二醇衍生物 （也称为聚乙二醇修饰剂） ， 将聚乙二醇分 子与精氨酸酶上的基团共价结合。 所述化学修饰可以是特异性的， 即通过使用与特定基 团结合的偶联剂， 令 PEG只能与精氨酸酶上的特定氨基酸结合。 定点 PEG化修饰需遵循 以下原则： （ 1 )尽量避免蛋白质活性部位的修饰， 以防对蛋白质活性发生影响； （2 ) 尽量简化整个过程， 是其具有可控性。 普通的聚乙二醇分子两端各有一个羟基， 若一端 以曱基封闭则得到曱氧基的聚乙二醇（mPEG )。 在多肽和蛋白质的聚乙二醇修饰中研究 最多的聚乙二醇修饰剂是 mPEG的衍生物。其中一种常见的聚乙二醇修饰方式是将聚乙二 醇分子 /聚乙二醇修饰剂与蛋白表面赖氨酸或蛋白 N-端的 ε-ΝΗ2或 α-ΝΗ2结合， 例如 mPEG-琥珀酰亚胺丁酸酯 （mPEG-SBA ) , mPEG-丙酸琥珀酰亚胺酯 （ mPEG-SPA ) , mPEG-琥珀酰亚胺琥珀酸酯（ mPEG-SS ) , mPEG-琥珀酰亚胺碳酸酯（ mPEG-SC ) , mPEG- 戊二酸单酯 -羟基琥珀酰亚胺酯（ mPEG-SG ) , mPEG-酰胺-琥珀酸亚胺酯（ mPEG-NHS ) , mPEG-三氟乙基磺酸酯 (mPEG tresylate)和 mPEG-醛 (mPEG aldehyde)„ 另外常见的 PEG修 饰方式是利用 5基的高亲核性， 选取能够特异性与蛋白质的 S基偶联的聚乙二醇分子 /聚 乙二醇修饰剂， 如 mPEG-马来酰亚胺， mPEG-邻-吡啶-二硫醚， mPEG-乙烯基砜， mPEG- 碘乙酰胺等来修饰蛋白质或多肽的疏基基团。 由于蛋白质或多肽的疏基比较少， 较易形 成均一性产物。
[0030】人精氨酸酶 I单体有三个半胱氨酸残基， 分别位于氨基酸序列当中的 45 , 168和 303 位。 在本发明的一个方面， 所述聚乙二醇化的精氨酸酶在其第 45 , 168和 303位的一个或 两个半胱氨酸位点发生定点聚乙二醇化。
[0031]在本发明的一个方面， 所述定点聚乙二醇化通过对精氨酸酶的半胱氨酸进行基因 工程定点突变， 然后进行半胱氨酸特异性聚乙二醇化修饰实现。 在本发明的又一个方面， 所述半胱氨酸特异性聚乙二醇化修饰是通过使用曱氧基聚乙二醇马来酰亚胺偶联剂将 PEG与精氨酸酶上的半胱氨酸的 S基基团共价结合。 [0032]在本发明的一个方面， 把本发明的精氨酸酶中不需要 PEG化的一个或两个半胱氨 酸点突变， 优选突变为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨 酸、 色氨酸、 脯氨酸， 更优选突变为丙氨酸。
[0033] PEG分子量的选择要综合考虑生物活性和药代动力学两方面的因素。 已有的研究 发现， 修饰的蛋白药物在体内的作用时间与偶联的 PEG数量和分子量有一定关系。 用于 本发明的 PEG, 其分子量范围可以在 5K-40K道尔顿之间， 可能是线性的或者含支链的分 子， 以及可能是本领域述及的 PEG衍生物。 本发明中共价结合至精氨酸酶分子的 PEG分 子并非限定于特定类型。 在本发明的一个方面， 对精氨酸酶进行 PEG化的 PEG分子的平 均分子量为 20K、 30Κ或 40K。 PEG化蛋白通过静脉注射的方式进入体内， 聚乙二醇的平 均分子量大小会影响其在血清的半衰期。 研究发现， 肾脏对 PEG分子的清除取决于肾小 球的滤过速率。对于蛋白质， 肾小球可以滤过分子量小于 70KDa的蛋白质分子； 对于 PEG 来说， 肾小球可以滤过分子量小于 30KDa的 PEG分子。 PEG的分子量其对要结合的蛋白的 半衰期和活性的影响通常是不可预测的， 与蛋白的分子量、 作用方式、 活性部位、 PEG 结合位点等密切相关。
[0034]在本发明的其中一个方面， 所述聚乙二醇化的精氨酸酶在 SEQ ID NO. 1定义的氨 基酸序列第 45位和第 168位两个位点发生定点聚乙二醇化， 优选的， 其中所述定点聚乙二 醇化通过将所述精氨酸酶的第 303位半胱氨酸突变实现， 其中所述 PEG的平均分子量为 20K或 30K。 其中， 所述半胱氨酸优选突变为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨 酸、 蛋氨酸、 苯丙氨酸、 色氨酸、 脯氨酸， 更优选突变为丙氨酸。
[0035】在本发明的一个方面， 所述的聚乙二醇化的精氨酸酶在 SEQ ID NO. 1定义的氨基 酸序列第 168位发生定点聚乙二醇化，其中所述定点聚乙二醇化通过将所述精氨酸酶的第 45位和第 303位半胱氨酸突变实现， 其中所述 PEG的平均分子量为 40K。 其中， 所述半胱 氨酸优选突变为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色 氨酸、 脯氨酸， 更优选突变为丙氨酸。
[0036]在本发明的一个方面， 所述聚乙二醇化的精氨酸酶在 SEQ ID NO. 1定义的氨基酸 序列第 45位发生定点聚乙二醇化，其中所述定点聚乙二醇化是通过将精氨酸酶的第 168位 和第 303位半胱氨酸突变实现， 其中所述 PEG的平均分子量为 40K。 其中， 所述半胱氨酸 优选突变为甘氨酸、 丙氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸、 脯氨酸， 更优选突变为丙氨酸。 [0037]本发明提供的聚乙二醇化的精氨酸酶的纯度通常超过超过 70% , 优选超过 80% , 最优选为 90%。 本发明中所述"聚乙二醇化的精氨酸酶的纯度"是指在对精氨酸酶进行聚 乙二醇化的化学修饰生产中， 产物里 PEG化精氨酸酶（即共价结合 PEG的精氨酸酶） 占 总的精氨酸酶（共价结合 PEG的精氨酸酶， 未共价结合 PEG的精氨酸酶和多聚的 PEG化精 氨酸酶） 的比例。 通常， 由于 PEG化反应后产物中还含有未共价结合 PEG的精氨酸酶和 多聚的 PEG化精氨酸酶， 因此需要对产物进行纯化。 常用的纯化手段是本领域公知的技 术， 包括阳离子交换柱等。
[0038]本发明提供的聚乙二醇化的精氨酸酶在血液或血清中的半衰期为至少 0.5天， 优选 至少 2.5天， 最优选至少 3.5天。 精氨酸酶的半衰期可以通过本领域公知和常用的方法进行 测量。 精氨酸酶在血液或血清中的半衰期的测量数据与釆用的动物模型有一定关系。 在 代谢速率较快的动物模型 （例如大鼠） 中获得的半衰期数据通常比在代谢速率较快的动 物模型 （例如人体） 中获得的数据短。
[0039】本发明还提供了制备聚乙二醇化（Pegylation ) 的精氨酸酶的方法。 所述聚乙二醇 化是位点特异性的。 本发明聚乙二醇化的精氨酸酶中聚乙二醇分子与精氨酸酶分子上的 特定氨基酸残基结合， 达到位点特异性聚乙二醇化的目的。 在本发明的聚乙二醇化的精 氨酸酶中， 每个精氨酸酶分子与至少一个聚乙二醇分子结合， 形成聚乙二醇化的精氨酸 醉。
[0040】在本发明中， 对精氨酸酶的定点聚乙二醇化可以通过化学修饰的方法实现， 所述 化学修饰是通过使用偶联剂将 PEG与精氨酸酶上的基团共价结合。 所述化学修饰可以是 特异性的， 即通过使用与特定基团结合的偶联剂， 令 PEG只能与精氨酸酶上的特定氨基 酸结合。
[0041]本发明还提供了利用上述本发明的任意一种精氨酸酶或聚乙二醇化的精氨酸酶或 其药物组合物在治疗与精氨酸酶有关的疾病的药物中的应用 /用途。 本领域已知所述与精 氨酸酶有关的疾病包括， 例如哺乳动物中与体内精氨酸相关的病症 /疾病 /紊乱。 此类病症 /疾病 /紊乱包括： 高精氨酸血症。 由于精氨酸酶活性的缺乏， 患者体内的精氨酸不能裂解 为尿素并加入鸟氨酸代谢循环， 血中精氨酸含量可高出正常 7 ~ 10倍， 同时脑脊液和尿中 精氨酸也增多， 尿中肌酐排出量增高。 另外， 此类病症 /疾病 /紊乱还包括精氨酸依赖性的 细胞增生或肿瘤， 例如肝癌， 黑色素细胞瘤， 乳腺癌， 小细胞肺癌， 前列腺癌， 淋巴癌 和白血病。 。 通过研究对细胞生长的影响发现， 将精氨酸移除后正常细胞系会由细胞周 期的 GO期进入静止状态， 且能在这种环境下存活几周而无明显损伤， 当精氨酸浓度恢复 正常时， 细胞又能开始恢复正常的生理周期； 而对于增生的细胞或肿瘤来说， 精氨酸的 匮乏会导致其经过细胞周期 G1期的' R'点进入 S期， 并很快凋亡。 这种由于精氨酸匮乏而 导致增生的细胞或肿瘤是不可逆的。 因此， 科学家们开始考虑通过控制机体中精氨酸的 水平来治疗细胞增生或肿瘤。
[0042]本发明还提供了一种药物组合物， 此药物组合物的活性成分是上述本发明的精氨 酸酶或聚乙二醇化的精氨酸酶。 所述药物组合物的配方可以以固体、 溶液、 乳剂、 分散 体、 胶束、 脂质体等形式应用， 其中所得配方含有一或多种本发明的人精氨酸酶或聚乙 二醇化的精氨酸酶作为活性成分， 并与适于肠道或胃肠外应用的有机或无机物载体或赋 形剂混合。 另外， 可以使用佐剂、 稳定剂、 增稠剂、 着色剂及香料。 一或多种分离的并 基本上纯化的人精氨酸酶或聚乙二醇化的精氨酸酶的活性成分也包括在足够量的药物配 方中以对目的过程、 条件或疾病产生所希望的作用。 上述药物组合物可以制成适于口服 的药剂形式， 例如， 片剂、 药片、 止咳糖、 水化或油化悬液、 分散的粉末或颗粒、 乳剂、 硬或软的胶嚢、 或糖浆。 口服的配方可以按照本领域已知技术进行包嚢化以减緩分解以 及在胃肠道的吸收， 因而提供了较长时间的持续作用。 所述配方还可以是无菌的可注射 溶液或悬液的形式。所述悬液可以是按照已知方法使用分散或润湿剂及悬浮剂进行配制。
[0043】本发明的药物组合物可进一步被制备成固体、 液体、 悬浮液、 微胶粒、 或微脂体 的形式。 在本发明的一个方面， 所述药物组合物的配方是适用于口服或注射的形式。 附图说明
[0044]图 1示出野生型人精氨酸酶 I的核酸序列。
[0045]图 2示出人精氨酸酶表达的还原性 SDS-聚丙烯酰胺凝胶电泳检测结果。
[0046]图 3示出人精氨酸酶表达的液相色谱图。
[0047]图 4示出野生型精氨酸酶 I经还原及非还原处理后凝胶电泳检测结果。
[0048]图 5示出纯化后的精氨酸酶 I突变体 （ rhArgI-A303 , rhArgl-Al 68/303 , 及 rhArgI-A45/303 )经还原及非还原处理后凝胶电泳检测结果。
[0049]图 6示出纯化后的精氨酸酶 I突变体（rhArgI-A45/468 , 及 rhArgI-A45/l 68/303 )经 还原及非还原处理后凝胶电泳检测结果。
[0050]其中 6a为 rhArgI-A45/303 , 及 rhArgI-A45/l 68/303经非还原处理后凝胶电泳检测结 果。
[0051]其中 6b为 rhArgI-A45/468 , 及 rhArgI-A45/l 68/303经还原处理后凝胶电泳检测结果
[0052]图 7示出聚乙二醇化人精氨酸酶的非还原性 SDS-聚丙烯酰胺凝胶电泳检测结果。 [0053]具体实施方式
[0054】本发明将在下面的实施例中进一步说明。 应当理解， 尽管这些实施例说明了本发 明的优选实施方案， 但仅是以例证的方式给出的。 根据上面的论述和这些实施例， 本领 域的技术人员可以确定本发明的基本特征， 并在不脱离本发明的精神和范围的情况下， 可对本发明做出多种改变和修饰， 以使其适用于多种用法和条件。 因此， 除了那些本文 所示和描述的那些之外， 根据前文所述， 本发明的各种修改形式对本领域的技术人员来 说将是显而易见的。 这些修改形式也旨在属于所附权利要求书的范围内。
[0055]实施例一:不含 His-tag的人精氨酸酶 I重组质粒 pET30a(+)- rharginase - V的构建及 表达
[0056]人精氨酸酶 I的基因序列公布于 1987年 (Haraguchi, Y.等， 1987, Proc. Natl. Acad. Sci. 84, 412-415)。 以人肝脏 5' stretch plus cDNA文库 (clontech)为模板， 基于上述人精氨酸酶 I 的基因序列设计引物进行聚合酶链式反应（PCR)获得人精氨酸酶 I核酸片段。 引物 ARG-V(+)含有一个 Ndel限制性内切酶位点，引物 ARG-V(-)含有一个 Xhol限制性内切酶位 点， 按照分子生物学领域普通实验技术进行聚合酶链式反应 (PCR)得到核酸片段， 并以琼 脂糖凝胶电泳确认。 将上述扩增所得片断和市购获得的 pET30a(+)表达载体分别用含有限 制性内切酶 Ndel和 Xhol (购自 promega)的反应介质中于 37。C处理 1.5小时， 已酶解的片断 与 T4DNA连接酶在 16°C反应过夜。 然后将所述已连接的质粒转化入感受态细胞 DH5a大 肠杆菌细胞内。在含有 3(^g/ml卡那霉素的 LB营养琼脂平板上进行筛选。通过限制性内切 酶分析获得具有正确插入片断的质粒， 称为 pET30a(+)-rharginase-V, 所述质粒进行测序 确认其插入片段。 插入片段包含 969个碱基， 如图 1所示， 其核酸序列为 SEQ ID No. 2。 ARG-V(+):
5* GGAATTCCATATGAGCGCCAAGTCCAGAACCATAG 3*
Ndel
ARG-V (-)： Xhol
[0057]实施例二：不含 His-tag的人精氨酸酶 I重组质粒 pET30a(+)- rharginase -V的表达和目 的蛋白纯化
[0058]将 1 ΟΟμΙ BL21 (DE3)感受态细胞置于水上融化，向感受态细胞悬液中加入 1 μΐ所述已 构建的 pET30a(+)-rharginase-V质粒， 混匀后水浴放置 30分钟， 然后将混合物于 42。C水浴 放置 90秒， 再快速转移至水浴中放置 2分钟。 加入 500μ1 Ι^Β液体培养基， 于 37°C, 150rpm 震荡培养 1小时。 取 200μ1涂布含有卡那霉素的 LB营养琼脂平板， 37°C倒置培养 16小时。
[0059]挑选所述转化细胞培养平板单一菌落并转移到 25mL LB培养基。 所述细胞在 37。C, 150rpm震荡培养至 OD600nm达到 0.6-0.8 , 加入终浓度为 0.2mM IPTG诱导表达 3小时。 SDS-P AGE电泳检测所挑选克隆是否有目的蛋白的表达。 挑选表达量较高的克隆制备甘 油菌保存作为工程菌。
[0060]将上述大肠杆菌工程菌在 15L发酵罐中以分批补料的方式培养， 当 OD600nm达到 12-13时， 加入终浓度为 0.2mM IPTG诱导表达 3-4小时。 将所获得的菌体进行破菌离心， 取上清液进行下一步的分离纯化。
[0061]蛋白的纯化釆用 CM阳离子交换层析。 先用 Tris-HCl平衡液平衡层析柱， 然后将所 述离心得到的上清液上样， 上样完毕后继续用 3倍柱体积的平衡液冲洗弱结合的杂质。 待 UV280nm检测值稳定后， 用含有 Tris-HCl及 NaCl的洗脱液进行洗脱， 收集目的峰。 对收 集的样品进行聚丙烯酰胺凝胶电泳 (SDS-PAGE)和高压液相色谱分析纯度。
[0062]实验结果如图 2， 3所示。
[0063]图 2是人精氨酸酶表达的凝胶电泳检测结果。 其中各泳道的样品为：
[0064] 1 : 蛋白分子量标准； 2. 破菌上清液； 3. 纯化的不含 His-tag的人精氨酸酶 I； 4. CM 阳离子交换柱流出液。
[0065]图 3是人精氨酸酶表达的液相色谱图。
[0066]根据图 2和图 3可见， 经上述层析方法所获得的精氨酸酶的纯度为 90%以上。
[0067】实施例三： 人精氨酸酶 I (rhArgl)的定点突变
[0068]以野生型人精氨酸酶质粒 pET30a-rharginaseI-V为模板， 利用 Stratagene公司的 QuikChange Site-directed Mutagenesis kit对人精氨酸酶 I序列中 45 , 168 , 和 303位编码半胱 氨酸的密码子分别进行单点， 双点或三点定点突变， 将 45 , 168 , 和 303位编码半胱氨酸 的密码子 (TGT)替换成编码丙氨酸的密码子 (GCT)。 引入 45 , 168 , 和 303位替代酸密码子 (GCT) 的引物分别为：
ARG-m45(+):
5* GAGAAAC
ARG-m45(-):
ARG-ml68(+):
5* GATTCTCC
ARG-ml68(-):
5* CAATATCC
ARG-m303(+):
5* GTTGCAATAACCTTGGCTGCTTTCGGACTTGCTCGGG 3*
ARG-m303(-):
5* CCCGAGCAAGTCCGAAAGCAGCCAAGGTTATTGCAAC 3* [0069]按照试剂盒说明书所提供的方法进行聚合酶链式反应 (PCR)， 然后以限制性内切酶 Dpn l消化模板质粒， 及转化感受态细胞。 将获得的重组突变质粒测序确认。 测序结果经 比对后选择半胱氨酸的密码子 (TGT)成功突变成丙氨酸的密码子 (GCT)的克隆，接种于 LB 液态培养基扩增。 以 Wizard Plus Minipreps 试剂盒， 抽提突变重组质粒。
[0070]突变的人精氨酸酶 I(rhArgl)序列中 303位半胱氨酸替换为丙氨酸的重组人精氨酸酶 I表示为 rhArgI-A303 ; 168位半胱氨酸替换为丙氨酸的重组人精氨酸酶 I表示为 rhArgI-A168; 45位半胱氨酸替换为丙氨酸的重组人精氨酸酶 I表示为 rhArgI-A45 ; 168和 303位半胱氨酸替换为丙氨酸的重组人精氨酸酶 I表示为 rhArgI-A168/303; 45和 303位半胱 氨酸替换为丙氨酸的重组人精氨酸酶 I表示为 rhArgI-A45/303； 45和 168位半胱氨酸替换为 丙氨酸的重组人精氨酸酶 I表示为 rhArgI-A45/168; 45 , 168和 303位半胱氨酸替换为丙氨 酸的重组人精氨酸酶 I表示为 rhArgI-A45/l 68/303; 含有上述突变的人精氨酸酶 I(rhArgl)序 歹' J的重组突变质粒分别称为 pET30a(+)-rhArgI-A303 , PET30a(+)-rhArgI-A168 , pET30a(+)-rhArgI-A45 , pET30a(+)-rhArgI-Al 68/303 , pET30a(+)-rhArgI- A45/303 , pET30a(+)-rhArgI- A45/168 , 或 pET30a(+)-rhArgI- A45/168/303。
[0071]将 1 ΟΟμΙ BL21 (DE3)感受态细胞置于水上融化，向感受态细胞悬液中加入 1 μΐ所述已 构建的突变重组质粒， 混勾后水浴放置 30分钟， 然后将混合物于 42。C水浴放置 90秒， 再 快速转移至水浴中放置 2分钟。 加入 500μ1 LB液体培养基， 于 37。C , 150rpm震荡培养 1小 时。 取 200μ1涂布含有卡那霉素的 LB营养琼脂平板， 37°C倒置培养 16小时。
[0072]挑选所述转化细胞培养平板单一菌落并转移到 25mL LB培养基。 所述细胞在 37。C, 150rpm震荡培养至 OD600nm达到 0.6-0.8 , 加入终浓度为 0.2mM IPTG诱导表达 3小时。 以 聚丙烯酰胺凝胶电泳 (SDS-PAGE)检测所挑选克隆是否有目的蛋白的表达。挑选表达量较 高的克隆制备甘油菌保存作为工程菌。
[0073]实施例四： 含定点突变精氨酸酶 I (rhArgl)质粒的表达和纯化
[0074]将上述转化了突变人精氨酸酶 I(rhArgl)质粒的大肠杆菌工程菌 （ 包括 pET30a(+)-rhArgI-A303 , pET30a(+)-rhArgI-Al 68/303 , pET30a(+)-rhArgI- A45/303 , pET30a(+)-rhArgI- A45/168 , 或 pET30a(+)-rhArgI- A45/168/303 )在 15L发酵罐中以分批补 料的方式培养， 当 OD600nm达到 12-13时， 加入终浓度为 0.2mM IPTG诱导表达 3-4小时。 将所获得的菌体进行破菌离心， 取上清液进行下一步的分离纯化。
[0075】 目的蛋白的纯化釆用 CM阳离子交换层析。 先用 Tris-HCl平衡液平衡层析柱， 然后 将所述离心得到的上清液上样， 上样完毕后继续用 3倍柱体积的平衡液冲洗弱结合的杂 质。 待 UV280nm检测值稳定后， 用含有 Tris-HCl及 NaCl的洗脱液进行洗脱， 收集目的峰， 由此获得定点突变的精氨酸酶 I rhArgI-A303， rhArgI-A168/303, rhArgI-A45/303, rhArgI-A45/168, 或 rhArgI-A45/l 68/303。
[0076]对收集的样品进行聚丙烯酰胺凝胶电泳 (SDS-PAGE)和高压液相色谱分析纯度。 经 上述层析方法所获得的精氨酸酶的纯度为 90%以上。
[0077]实施例五： 定点突变精氨酸酶 I (rhArgl)的活性
[0078]通过测定与尿素酶和谷氨酸脱氢酶相偶联的 NADPH的吸光度值测定精氨酸酶的 活性， 其原理如下所示， NADPH被氧化成 NADP+, 前者在 340 nm处有最大吸收峰， 测定 在该波长下吸光率的降低， 再根据 NADPH的摩尔消光系数 ΔΕ340 = 6220 M-lcm-1 , 即可 计算出精氨酸酶的活性。
精氨酸 +¾0 精氨酸酶 鸟氨酸 +尿素 尿素 + 2H20 尿素酶 HC03—+2NH4+ ΝΗ4++α-酮戊二酸 + NADPH 谷氨酸脱氢酶 谷氨酸 +NADP++ Η20
[0079]精氨酸酶活性单位定义为： 1单位 （U) 的精氨酸酶能够在 30。C， pH 8.3的条件下释 放 Ι μηιοΐ尿素。
[0080】精氨酸酶比活的计算釆用下述方程：
[0081]比活 (U/mg) = [(ΔΑ/Δΐ) χ (1/ε) χ 106 χ (1/2)] I [Ε]
ΔΑ = 340 nm吸光度的差值
ε = NADPH Michaelis-Menten 常数 (Km) {622< ~xzm x)
[E] =反应体系中精氨酸酶浓度 (mg/mL)
[0082]利用上述方法计算的在不同位点上进行了突变的人精氨酸酶 I rhArgI-A303 , rhArgI-A168/303 , rhArgI-A45/303 , rhArgI-A45/168以及 rhArgI-A45/168/303的酶活性， 其活性分别为 747 ± 63 U/mg, 814 ± 91 U/mg, 786 ± 58 U/mg, 782 ± 19 U/mg, 759士 68 U/mg。 而未突变的人精氨酸酶 I活性为 491 ± 42 U/mg。 由此可见， 人精氨酸酶 I(rhArgI)序 列中位于 168/303 , 45/303 , 45/168及 45/168/303位属于非电离极性氨基酸的半胱氨酸突变 成属于非极性氨基酸性质的丙氨酸后酶活性明显增加。
[0083]实施例六： 野生型与定点突变精氨酸酶 I (rhArgl)的 SDS-PAGE电泳分析
[0084]以传统方法进行聚丙烯酰胺凝胶的配制， 分离胶浓度为 12%。 将纯化的野生型精 氨酸酶 I及其各种不同类型的突变体分别进行还原处理（即在样品緩冲液中加入 基乙 醇以破坏分子内或分子间的二硫键 )和非还原处理以分析野生型精氨酸酶 I及其各种不同 类型的突变体的分子内或分子间二硫键的形成情况。 结果如图 4 , 图 5 , 图 6a及图 6b所示：
[0085]图 4是野生型精氨酸酶 I经还原及非还原处理后凝胶电泳检测结果。 其中各泳道的 样品为： 1 , 3 : 破菌上清液； 2. 纯化的不含 His-tag的人精氨酸酶 1 (经非还原处理）； M: 蛋 白分子量标准； 4. 纯化的不含 His-tag的人精氨酸酶 I (经还原处理） 。
[0086]图 5是纯化后的精氨酸酶 I突变体 （ rhArgI-A303 , rhArgl-Al 68/303 , 及 rhArgI-A45/303 )经还原及非还原处理后凝胶电泳检测结果。 其中各泳道的样品为： 1. 精 氨酸酶 I突变体 rhArgI-A303 (经非还原处理） ； 2. 精氨酸酶 I突变体 rhArgI-A168/303 (经 非还原处理）；3. 精氨酸酶 I突变体 rhArgI-45/A303 (经非还原处理） ； M: 蛋白分子量标 准； 4. 精氨酸酶 I突变体 rhArgI-A303(经还原处理）； 5. 精氨酸酶 I突变体 rhArgl-Al 68/303 (经还原处理）；6. 精氨酸酶 I突变体 rhArgI-45/A303 (经还原处理） [0087]图 6a是纯化后的精氨酸酶 I突变体（ rhArgI-A45/468 , 及 rhArgI-A45/l 68/303 )经非 还原处理后凝胶电泳检测结果。 其中各泳道的样品为： M: 蛋白分子量标准； 1. 精氨酸 酶 I突变体 rhArgI-A45/l 68/303; 2. 精氨酸酶 I突变体 rhArgI-A45/168
[0088]图 6b是纯化后的精氨酸酶 I突变体（rhArgI-A45/468 , 及 rhArgI-A45/l 68/303 )经还 原处理后凝胶电泳检测结果。 其中各泳道的样品为： M: 蛋白分子量标准； 1. 精氨酸酶 I 突变体 rhArgI-A45/l 68/303 ; 2. 精氨酸酶 I突变体 rhArgI-A45/168
[0089]通过对样品经还原处理和非还原处理在聚丙烯酰胺凝胶电泳的差别可以看出， 在 本发明实验条件下， 野生型人精氨酸酶 I有其他构象的存在（图 4 )。 在此条件下， 当 303 位点的半胱氨酸突变成丙氨酸后， 也显示有其他构象的精氨酸酶的存在（图 5 )。 而当 45 , 168 , 303位半胱氨酸中的任意两个发生突变 （如 rhArgl- A 45/303 , rhArgl- A 168/303 , 或 rhArgl- A 45/168 )或三个全部发生突变， 在非还原性 SDS-聚丙烯酰胺凝胶电泳中则只 表现出一种构象（图 5 , 图 6a ) 。 不受任何现有理论的限制， 发明人认为， 野生型人精 氨酸酶 I由于其氨基酸序列中 45 , 168 , 和 303位点半胱氨酸的存在， 有机会在某种条件下 于分子内部形成二硫键而有异构体的出现， 当 303位点的半胱氨酸突变成丙氨酸后， 当样 品未经还原处理时， 在 SDS-聚丙烯酰胺凝胶电泳中依然显示有其他构象的精氨酸酶的存 在， 可能是剩下的两个未突变的 45和 168位半胱氨酸形成了分子内的二硫键。 当 45 , 168 , 303位半胱氨酸中的任意两个发生突变（如 rhArgl- A 45/303 , rhArgl- A 168/303 ,或 rhArgl- A 45/168 )或三个全部发生突变，令到产生分子内二硫键的机会丧失，只表现出一种构象。
[0090]实施例七： 突变精氨酸酶 I (rhArgl)的定点 PEG化修饰和 PEG化蛋白的纯化
[0091】在 20mM PBS緩冲液（pH7.0 ) 中， 将进行了定点突变的精氨酸酶 I rhArgI-A303 , rhArgI-A168/303或 rhArgI-A45/303分别与曱氧基聚乙二醇马来酰亚胺按照摩尔比为 1 :5 - 1 : 10相混合。 所述曱氧基聚乙二醇马来酰亚胺分子量分别是约 20K， 30K, 40K, 其中分子 量是 40K的曱氧基聚乙二醇马来酰亚胺为 Y-型， 包含两个直链曱氧基聚乙二醇。 在室温 下反应 2-4小时。 反应结束后将反应产物置于 4°0水箱保存。
[0092]釆用介质为 Macrocap SP的阳离子交换柱对上述定点修饰的 PEG化人精氨酸酶 I进 行分离纯化， 去除残留的 PEG和少量未反应的蛋白。 平衡液为磷酸盐緩冲液， 洗脱液为含 lMNacl的磷酸盐緩冲液。 先用纯化水将修饰后的蛋白样品进行稀释， 使样品的电导与平 衡液的电导相同， 稀释后的样品作为上样样品。 用平衡液平衡层析柱 5倍柱体积后上样， 上样结束后继续用平衡液淋洗 5倍柱体积， 然后用 35%洗脱液进行洗脱， 收集洗脱峰。 对 收集后的洗脱峰分别用 G-25脱盐柱进行脱盐， 收集目的蛋白进行 SDS-PAGE电泳和精氨 酸酶活性分析。 如图 7所示：
[0093]将进行了定点突变的精氨酸酶 I(rhArgI-A303)与曱氧基聚乙二醇马来酰亚胺 (mPEG-MAL-20K)反应， 经 Macrocap SP的阳离子交换柱纯化后的定点 PEG化人精氨酸酶 I； 其中所形成的带有 2个分子量为 20K的曱氧基聚乙二醇马来酰亚胺的人精氨酸酶（以 A303-M20K(2)表示） ， 即 45和 168位半胱氨酸基团全部偶联曱氧基聚乙二醇马来酰亚胺 的比例占全部反应产物的 70%以上。
[0094]将进行了定点突变的精氨酸酶 I(rhArgI-A303)与曱氧基聚乙二醇马来酰亚胺 (mPEG-MAL-30K)反应， 经 Macrocap SP的阳离子交换柱纯化后的定点 PEG化人精氨酸酶 I； 其中所形成的带有 2个分子量为 30K的曱氧基聚乙二醇马来酰亚胺的人精氨酸酶（以 A303-M30K(2)表示） ， 即 45和 168位半胱氨酸基团全部偶联曱氧基聚乙二醇马来酰亚胺 的比例占全部反应产物的 70%以上。
[0095]将进行了定点突变的精氨酸酶 I(rhArgI-A168/303)与 Y-型聚乙二醇马来酰亚胺 (Y-MAL-40K)反应， 经 Macrocap SP的阳离子交换柱纯化后的定点 PEG化人精氨酸酶 I； 其 中所形成的带有 1个分子量为 40K的 Y-型聚乙二醇马来酰亚胺的人精氨酸酶 （以 A168/303-Y40K表示） ， 即 45位半胱氨酸基团偶联曱氧基聚乙二醇马来酰亚胺的比例占 全部反应产物的 85%以上。
[0096]将进行了定点突变的精氨酸酶 I(rhArgI-A45/303)与 Y-型聚乙二醇马来酰亚胺 (Y-MAL-40K)反应， 经 Macrocap SP的阳离子交换柱纯化后的定点 PEG化人精氨酸酶 I； 其 中所形成的带有 1个分子量为 40K的 Y-型聚乙二醇马来酰亚胺的人精氨酸酶 （以 A45/303-Y40K表示） ， 即 168位半胱氨酸基团偶联曱氧基聚乙二醇马来酰亚胺的比例占 全部反应产物的 85%以上。
[0097]利用前述方法通过测量与尿素酶和谷氨酸脱氢酶相偶联的 NADPH的吸光度值来 测定精氨酸酶的活性， 计算各种不同分子量大小以及不同位点上进行了定点 PEG化修饰 的人精氨酸酶 I A303-M20K(2), A303-M30K(2), A168/303-Y40K以及 A45/303-Y40K的酶 活性。 测试结果发现上述定点 PEG化修饰的人精氨酸酶 I保持了精氨酸酶的活性，其活性 都大于 400U/mg , 范围在 400-800U/mg之间。
[0098]实施例七： 定点 PEG化修饰的人精氨酸酶 I的体内药代动力学检测
[0099]将上述以不同分子量大小以及不同位点上进行了定点 PEG化修饰的人精氨酸酶 I的 蛋白溶液送彭立生物医药科技（上海）有限公司进行药代动力学研究， 以单剂量尾静脉 注射方式给予 SpragueDawley大鼠， 剂量为 3mg/kg。 给药前及给药后 2分钟， 1, 4, 24, 72, 120, 168和 240小时眼眶静脉取血， 检测血清内人精氨酸酶 I和精氨酸的浓度。 每个 时间点取血 0.4mL, 转移至 2mL离心管， 样品在室温放置 30分钟， 然后再 4。C, 4000g条件 下离心 15分钟。
[0100] 血清精氨酸酶 I的检测釆用双抗体夹心 £1^18 法（试剂盒购自上海依科赛生物制品 有限公司） 。 抗人精氨酸酶 I单抗包被于酶标板上， 分别将样品及不同浓度标准品加入孔 中 （ΙΟΟμΙ/孔）， 用封板胶纸封住反应孔， 37。C孵育 90分钟， 样品或标准品中的人精氨酸 酶 I会与单抗结合成免疫复合物； 洗板 5次；加入兔抗人精氨酸酶 I多克隆抗体（ ΙΟΟμΙ/孔）， 用封板胶纸封住反应孔， 37。C孵育 60分钟； 洗板 5次； 加入辣根过氧化物酶偶联的羊抗兔 IgG ( ΙΟΟμΙ/孔）共同孵育 30分钟； 洗板 5次； 加入显色底物， 避光孵育 10-15分钟， 最后 加入终止液， 混匀后测量 OD450值。 以二次方程拟合方式绘制标准曲线， 通过样品的 OD 值计算其浓度。 使用 WinnoLin软件的非房室模型分析药代动力学参数。 如下表所示各种 不同分子量大小以及不同位点上进行了定点 PEG化修饰的人精氨酸酶 I A303-M20K(2), A303-M30K(2), A168/303-Y40K以及 A45/303-Y40K的主要药代动力学参数半衰期丁1/2分 别为： 28.0±4.5, 28.4±8.4, 27.2±3.8, 15.1 ±0.6小时。
[0101] 表 1: 定点 PEG化修饰的人精氨酸酶 I在大鼠体内单剂量注射药代动力学参数（平 均值士标准偏差； n=6)
11/2 c AUCLAST AUCJNF
(h) ( g/mL) (h* g/mL) (h* g/mL)
A303-M20K(2) 28.0 ±4.5 41.5 ± 9.5 1603.5 ± 187.5 1614.5 ± 188.6
A303-M30K(2) 28.4 ± 8.4 32.4 ± 3.1 1113.6± 116.8 1192.9 ± 121.2
A168/303-Y40K 27.2 ± 3.8 45.2 ± 5.1 2077.5 ± 160.6 2083.9 ± 163.3
A45/303-Y40K 15.1 ± 0.6 105.2 ± 66.9 548.1 ± 50.3 564.5 ± 50.8
[0102】本发明利用定点突变的方法对人精氨酸序列当中 45, 168和 303位半胱氨酸进行了改 造， 将其中一个，两个或三个编码半胱氨酸的密码子 (TGT)替换成编码其它氨基酸， 特别 是丙氨酸的密码子 (GCT),得到了活性增强的人精氨酸。另外，本发明提供了新的定点 PEG 化的人精氨酸酶，去除了可能具有潜在的有免疫原性的 his-tag蛋白标签序列，并在人精氨 酸序列当中 45, 168和 303位半胱氨酸进行定点 PEG化， 分别与 20, 30或 40KDa的曱氧基 聚乙二醇马来酰亚胺进行偶联反应。 以此方法合成的定点 PEG化的人精氨酸酶减少了肾 小球的滤过率， 而且 PEG与人精氨酸酶偶联的位置确定， 形成的产物均一， 易于纯化， 有利于大规模生产的质量控制。 本发明所提供的定点 PEG化人精氨酸酶在哺乳动物体内 的半衰期长， 例如在大鼠体内血清中的半衰期可达到 15-28小时。
[0103】除非另外指出，本发明的实践将使用生物技术、有机化学、无机化学等的常规技术， 显然除在上述说明和实施例中所特别描述之外， 还可以别的方式实现本发明。 其它在本 发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导， 许多改变和变化是可行的， 因此其在本发明的范围之内。 本文所提到的所有专利、 专利 申请与科技论文均据此通过引用结合到本文。

Claims

权 利 要 求
1. 一种精氨酸酶， 其特征在于， 所述精氨酸酶为人精氨酸酶 I , 其 由 SEQ I D NO. 1定义的氨基酸序列中第 45 , 168和 303位的半胱氨酸中的 一个、 两个或三个突变为非极性氨基酸。
2. 权利要求 1所述的精氨酸酶， 其中所述第 45 , 168和 303位的半胱 氨酸独立优选的， 突变为丙氨酸、 甘氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸或脯氨酸， 最优选的， 突变为丙氨酸。
3. 权利要求 1或 2所述的精氨酸酶， 其特征在于所述精氨酸酶的第 45 , 168和 303位的半胱氨酸中的一个发生突变， 优选的， 第 303位半胱氨 酸突变， 更优选的， 突变为丙氨酸。
4. 权利要求 1-3中任一项所述的精氨酸酶， 其特征在于所述精氨酸 酶的第 45 , 168和 303位的半胱氨酸中的任意两个发生突变， 例如第 45位 和第 303位半胱氨酸突变， 第 168位和第 303位突变， 或第 168位和第 45位 突变， 优选突变为丙氨酸。
5. 权利要求 1-3中任一项所述的精氨酸酶， 其特征在于所述精氨酸 酶的第 45位、 第 168位和第 303位半胱氨酸突变， 优选突变为丙氨酸。
6. 一种聚乙二醇化的精氨酸酶， 其特征在于， 所述精氨酸酶为人精 氨酸酶 I , 所述聚乙二醇化是位点特异性的， 在由 SEQ I D NO. 1定义的氨 基酸序列中的第 45 , 168和 303位的一个或两个半胱氨酸位点发生聚乙二 醇化。
7. 权利要求 6所述的聚乙二醇化的精氨酸酶， 其中所述定点聚乙二 醇化是通过对精氨酸酶上不进行聚乙二醇化的一个或两个半胱氨酸位点 对半胱氨酸进行基因工程定点突变为其它氨基酸， 然后对突变后的精氨 酸酶进行半胱氨酸特异性聚乙二醇化修饰实现。
8. 权利要求 7所述的聚乙二醇化的精氨酸酶， 其中所述点突变是将 精氨酸酶的半胱氨酸突变为非极性氨基酸， 优选的， 突变为丙氨酸、 甘 氨酸、 缬氨酸、 亮氨酸、 异亮氨酸、 蛋氨酸、 苯丙氨酸、 色氨酸、 脯氨 酸， 最优选的， 突变为丙氨酸。
9. 权利要求 6-8中任一项所述的聚乙二醇化的精氨酸酶， 其中所述 PEG的平均分子量为 2 0K、 30Κ或 4 0Κ。
1 0. 权利要求 6-9中任一项的聚乙二醇化的精氨酸酶， 其中所述精氨 酸酶在血液或血清中的半衰期为至少 0. 5天， 优选至少 2. 5天， 最优选至 少 3. 5天。
1 1. 制备权利要求 1 -5中任一项的精氨酸酶或权利要求 6-1 0中任一 项的聚乙二醇化的精氨酸酶的方法。
1 2. 权利要求 1 -5中任一项的精氨酸酶或权利要求 6- 1 0中任一项的 聚乙二醇化的精氨酸酶在制备与精氨酸酶有关的疾病的药物中的用途， 优选的， 所述疾病选自高精氨酸血症和精氨酸依赖性的细胞增生或肿瘤， 例如肝癌， 黑色素细胞瘤， 乳腺癌， 小细胞肺癌， 前列腺癌， 淋巴癌或白血 病。
1 3. 一种治疗与精氨酸酶有关的疾病的药物组合物， 其中含有权利 要求 1 -7中任一项的精氨酸酶或权利要求 6-1 0中任一项的聚乙二醇化的 精氨酸酶， 其中所述疾病选自高精氨酸血症和精氨酸依赖性的细胞增生 或肿瘤， 例如肝癌， 黑色素细胞瘤， 乳腺癌， 小细胞肺癌， 前列腺癌， 淋巴 癌或白血病。
14. 治疗与精氨酸酶有关的疾病的方法， 包括给予有需要的个体权利要 求 1-5中任一项的精氨酸酶或权利要求 6-1 0中任一项的聚乙二醇化的精氨酸 酶，优选的，所述疾病选自高精氨酸血症和精氨酸依赖性的细胞增生或肿瘤， 例如肝癌， 黑色素细胞瘤， 乳腺癌， 小细胞肺癌， 前列腺癌， 淋巴癌或白血 病。
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BR112014015820B1 (pt) 2022-08-09
JP2015503912A (ja) 2015-02-05
EP2799539A1 (en) 2014-11-05
EP2799540A1 (en) 2014-11-05
BR112014015803A2 (pt) 2020-10-27
CN103184209B (zh) 2015-09-16
KR102076364B1 (ko) 2020-02-11
CN103184208B (zh) 2015-09-16
KR102076348B1 (ko) 2020-02-11
CN103184208A (zh) 2013-07-03
JP2015503333A (ja) 2015-02-02
BR112014015803B1 (pt) 2023-03-07
EP2799539B1 (en) 2017-09-13
US20150010522A1 (en) 2015-01-08
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BR112014015820A2 (pt) 2020-10-27
EP2799540A4 (en) 2015-08-26
CN103184209A (zh) 2013-07-03
KR20140108577A (ko) 2014-09-11
KR20140107616A (ko) 2014-09-04
WO2013097658A1 (zh) 2013-07-04

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