CN104745565A - Polyethyleneglycol-modified recombinant metarginase - Google Patents
Polyethyleneglycol-modified recombinant metarginase Download PDFInfo
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- CN104745565A CN104745565A CN201510140645.XA CN201510140645A CN104745565A CN 104745565 A CN104745565 A CN 104745565A CN 201510140645 A CN201510140645 A CN 201510140645A CN 104745565 A CN104745565 A CN 104745565A
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- arginine deiminase
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- C12Y305/03006—Arginine deiminase (3.5.3.6)
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Abstract
The invention relates to the field of biomedicine, particularly a polyethyleneglycol-modified recombinant metarginase. According to the polyethyleneglycol-modified recombinant metarginase compound, 1-30 branched polyethyleneglycols are utilized to modify the metarginase, wherein the branched polyethyleneglycol is a Y-type methoxypolyethylene glycol containing single active functional group, and the single active functional group is arranged at the terminal point. Under the same condition of lowering immunogenicity, the number of modified sites is smaller, and the possibility for influencing the protein active center is lower, so the specific activity of the proteinase is higher. The polyethyleneglycol-modified metarginase can decompose and exhaust the exogenous arginines of cells more effectively than the past metarginase, has lower immunogenicity, and can be used for treating multiple related tumor diseases.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to a kind of polyethyleneglycol modified restructuring arginine deiminase.
Background technology
Arginine is the important amino acid that supply organism synthesizes most polypeptide and protein.Arginine needed for normal cell can ornithine cycle in body by argininosuccinate synthetase (argininosuccinate synthetase, ASS) with argininosuccinate lyase (argininosuccinate lyase, ASL) acting in conjunction catalysis citrulline and generating.And some tumour cells, arginine self normally cannot be synthesized because it lacks ASS, arginine must be absorbed to carry out growth and breeding from outside atmosphere, this class cell is referred to as arginine-auxotrophic type tumour cell, such as liver cancer cell (hepatocellularcarcinomas, HCCs) and melanoma cells (melanomas).
Malignant melanoma and liver cancer are the fatal disease killing Most patients in diagnosis in a year.Its sickness rate just promptly increases, and in other countries, its rate of rise is even higher.The place of hepatitis epidemic in the world, the sickness rate of liver cancer is higher.Be badly in need of effectively treating these diseases.
Have and treat some cancer by the method for selective removal indispensable amino acid.As by decomposition arginine, tumour cell is carried out to the arginase of nutrient deprivation.Arginase anti tumor activity in vitro has been reported (Bach from nineteen sixty, S.J., R.A.Hawkins, and D.Swaine. " A short method for the purification of arginase from oxliver. " Biochemical Journal 89.2 (1963): 263.).Although the external tumor-inhibiting action of arginase is clear and definite, but use it in body and attempt not achieved the desired result (Storr by the multinomial research of arginine depletion Therapeutic cancer, J.M., and A.F.Burton. " The effects of arginine deficiency on lymphoma cells. " Britishjournal of cancer 30.1 (1974): 50.), this is because body reply amino acid starvation time body in amino acid homeostatic mechanism (amino acid homeostatic mechanism), decompose approach by activator matter when amino acid is deficient and arginine is discharged into participation circulation in blood, supplement the arginine depletion that arginase metabolism causes.
Arginine deiminase (arginine deiminase, EC 3.5.3.6 is called for short ADI) is a kind of PTS being used for the treatment of arginine-deficient type tumour of great potential.Arginine can be resolved into citrulline and ammonia by it in cell, makes arginine-deficient type cancer cells dead because arginic supply is blocked, thus reaches the object of Therapeutic cancer.2002, Ensor etc. find that mycoplasma ADI recombinant expressed in intestinal bacteria has activity in vitro and in vivo to the melanoma of 23 of arginine-auxotrophic type kinds of people and the hepatoma of 16 kinds of people, improve the ADI transformation period in vivo by carrying out polyoxyethylene glycol (PEG) change to ADI.2004, AD1-PEG-20 was used for the clinical experiment of liver cancer by them, and successfully to make in patients blood plasma arginic can reduce to zero by detectable level for result.At present, the ADI-PEG-20 preparation in the mycoplasma source of U.S. phoenix company exploitation is used for II phase of hepatocellular carcinoma and melanoma (nomenclature of drug is respectively Hepacid and Melanocid) and III clinical trial phase carries out at global multiple center.
Summary of the invention
In order to solve above-mentioned technical problem, the object of this invention is to provide a kind of arginine deiminase (PEGadi) of polyoxyethylene glycol covalent modification, this PEGadi immunogenicity reduces, and activity decrease is few, Increased Plasma Half-life.Be applicable to clinical application.
In order to realize above-mentioned object, present invention employs following technical scheme:
A kind of polyethyleneglycol modified restructuring arginine deiminase compound, this compound adopts 1 ~ 30 branched polyethylene glycol to modify arginine deiminase, branched polyethylene glycol is the Y type methoxy poly (ethylene glycol) containing single-activity functional groups, and single-activity functional groups is arranged on end points, structural formula is as follows:
Described n1, n2, n3 are respectively the positive integer being more than or equal to 1, and the value of n1+n2+n3 is 80 ~ 2000, and wherein the value of n3 is preferably 5 ~ 100; Described R is the activity functional groups modifying arginine deiminase.
As preferably, described Y type methoxy poly (ethylene glycol) molecular weight is 2000 ~ 10000Da.
As preferably, described activity functional groups is selected from succinimide ester, nitro phenyl ester, pendent succinic acid succinate, pendent succinic acid carbonic ether, maleimide and iodo-acetamide and combination thereof.As preferred again, described activity functional groups is selected from succsinic acid carbonic ether.
As preferably, described arginine deiminase adopts 3 ~ 12 Y type methoxy poly (ethylene glycol)s to modify.As preferred again, described arginine deiminase adopts 5 ~ 9 Y type methoxy poly (ethylene glycol)s to modify.
Owing to this invention takes above-mentioned technical scheme, compared with the product polyethyleneglycol modified with using straight chain, in the immunogenic situation of same reduction, the number of decorating site is less, the possibility affecting protein-active center is lower, and therefore proteolytic enzyme specific activity is higher.The polyethyleneglycol modified arginine deiminase of the present invention more effectively decomposes the exogenous arginine exhausting cell, lower immunogenicity than arginine deiminase in the past, can be used for treating relevant kinds of tumors disease.
Accompanying drawing explanation
Fig. 1 represents the part electrophoresis result of the polyethyleneglycol modified arginine deiminase product of different group and molecular weight, the ADI that the mPEG10000 being wherein followed successively by unmodified ADI, the YPEG5000 of maleimide base group, the YPEG5000 of pendent succinic acid carbonate group and pendent succinic acid carbonate group from left to right modifies.
Fig. 2 represents unmodified ADI, and YPEG modifies the immunogenicity that ADI and PEG modifies ADI.
Embodiment
1, the synthesis of arginine deiminase gene, cloning and expressing
Arginine deiminase is cloned and at expression in escherichia coli from mycoplasma arginini.See Misawa, Satoru, etal. " High-level expression of Mycoplasma arginine deiminase in Escherichia coli andits efficient renaturation as an anti-tumor enzyme. " Journal of biotechnology 36.2 (1994): 145-155.
2, the abstraction and purification of expression product
Thalline is suspended in 40mM TrisCl, 0.2M NaCl, 0.5mM EDTA, pH8.0 (in damping fluid).Cell homogenates crusher machine thalline.Described homogenate at 4 DEG C, the centrifugal 30min of 10000g.Collecting precipitation.Centrifugation is dissolved in denatured lysis buffer (50mM Tris by the mass volume ratio of 1:20,6M Guanidinium hydrochloride, 20mM beta-mercaptoethanol (2-ME), pH8.5), in, vibrate (150rpm) hydrotropy in 37 DEG C of shaking tables, after 1hr 4 DEG C centrifugal (13,500rpm, 25min), discard precipitation, supernatant is stored in 4 DEG C of refrigerators.Denatured lysis albumen is diluted to renaturation dilution buffer (50mM PB, 4M urea, 1mM EDTA, 5mM 2-ME, pH7.0) in, whole operating process is carried out under ice bath environment, controls final diluent protein concentration and is about 0.05mg/ml, renaturation diluent is carried out gradient dialysis renaturation in the ratio of 1:10 under 4 DEG C of conditions.The outer transparent liquid of renaturation is 50mMPB, 1M urea, and 1mM EDTA, 5mM 2-ME, pH7.0, progressively reduce wherein urea concentration, and every 12hr changes outer transparent liquid.The renaturation time amounts to 48hr.Renaturation diluent DEAE ion exchange chromatography is purified.Flow velocity 10ml/min loading.Balance 30min with level pad with flow velocity 10ml/min after loading, use elution buffer with flow velocity 10ml/min wash-out afterwards, collect elution peak and namely obtain target product ADI.Through SDS-PAGE electrophoresis detection, the ADI purity that above-mentioned purge process obtains is more than 95%.
3, arginine deiminase is polyethyleneglycol modified
In the phosphoric acid buffer of 100mM, pH 8.0, polyoxyethylene glycol is connected with ADI covalency.Described polyoxyethylene glycol comprises above-mentioned YPEG and straight chain mono methoxy polyethylene glycol (mPEG).YPEG linking group comprises succinimide ester, nitro phenyl ester, pendent succinic acid succinate, pendent succinic acid carbonic ether, maleimide and iodo-acetamide, and PEG linking group comprises pendent succinic acid carbonic ether.Wherein the total weight average molecular weight of YPEG be 2000,5000,8000,10000 and the total weight average molecular weight of 20000, PEG be 10000.
A) isolation and purification of modified outcome
Above-mentioned modified outcome is splined on molecular sieve gel chromatography column, carries out wash-out with 40mM phosphoric acid buffer, collect elution peak respectively.Electrophoresis detection the results are shown in Figure 1
B) enzyme assay
With Folin-phenol reagent box, using bovine serum albumin as standard detection protein concentration.With reference to Boyde, T.R.C., the activity of the ADI that the ADI that the method for andMohammed Rahmatullah. " Optimization of conditions for the colorimetric determinationof citrulline, using diacetyl monoxime. " in Analytical biochemistry 107.2 (1980): 424-431. detects unmodified ADI, the YPEG of molecular weight 5000 modifies and the mPEG of molecular weight 10000 modifies.The sample of 100 μ l 10 μ g/ml is added in 1.9ml 10mM arginine solution, 3ml mixed acid solution termination reaction is added after 37 DEG C of reaction 10min, add 500 μ l colouring reagentss again, boiling water bath 20min under lucifuge condition, detects light absorption value after cool to room temperature under 490nm.37 DEG C, under the environment of pH 6.8, it is a unit of enzyme activity U that per minute transforms the arginine enzyme amount generated needed for 1 μm of ol citrulline.
Find that unmodified ADI activity is about ADI activity that 18, YPEG modifies and is about the ADI activity that 11, PEG modifies and is about 7.
C) immunogenicity determining
Weekly to injecting unmodified ADI, the ADI of YPEG modification of molecular weight 5000 and the ADI of the mPEG modification of molecular weight 10000 in rabbit vein, dosage is about 6U/kg, totally 8 times.After the 2nd, 4,8 week, get blood to described animal, separation of serum is stored in-70 DEG C.Antibody titer is detected by ELISA method.In every hole of 96 hole microwell plates, add 50 μ g ADI, wrap by 1 hour under room temperature.Wash plate with PBS, then use bovine serum albumin (1mg/ml) to close at 4 DEG C and spend the night.Next day dilutes rabbit anteserum, and is added in hole.37 DEG C hatch 1 hour after, wash plate with PBS, and the goat anti-rabbit igg of peroxidase is added in hole by coupling.Plate incubation 30 minutes, then measures the absorbancy of gained by microplate reader.Potency unit is defined as the most high dilution of serum causing comparatively Background absorbance (about 0.50) to exceed twice.
As shown in Figure 2, after 2 injections, the ADI of two kinds of PEG modifications does not all produce antibody to result, and the antibody titers that the ADI of 8 injection latter two PEG modification produces all only has unmodified ADI to produce about 1/10th of antibody titers.
Claims (6)
1. a polyethyleneglycol modified restructuring arginine deiminase compound, it is characterized in that: this compound adopts 1 ~ 30 branched polyethylene glycol to modify arginine deiminase, branched polyethylene glycol is the Y type methoxy poly (ethylene glycol) containing single-activity functional groups, and single-activity functional groups is arranged on end points, structural formula is as follows:
Described n1, n2, n3 are respectively the positive integer being more than or equal to 1, and the value of n1+n2+n3 is 80 ~ 2000, and wherein the value of n3 is preferably 5 ~ 100; Described R is the activity functional groups modifying arginine deiminase.
2. a kind of polyethyleneglycol modified restructuring arginine deiminase compound according to claim 1, is characterized in that: Y type methoxy poly (ethylene glycol) molecular weight is 2000 ~ 10000Da.
3. a kind of polyethyleneglycol modified restructuring arginine deiminase compound according to claim 1, is characterized in that: activity functional groups is selected from succinimide ester, nitro phenyl ester, pendent succinic acid succinate, pendent succinic acid carbonic ether, maleimide and iodo-acetamide and combination thereof.
4. a kind of polyethyleneglycol modified restructuring arginine deiminase compound according to claim 3, is characterized in that: activity functional groups is selected from succsinic acid carbonic ether.
5. a kind of polyethyleneglycol modified restructuring arginine deiminase compound according to Claims 1 to 4 any one claim, is characterized in that: arginine deiminase adopts 3 ~ 12 Y type methoxy poly (ethylene glycol)s to modify.
6. a kind of polyethyleneglycol modified restructuring arginine deiminase compound according to claim 5, is characterized in that: arginine deiminase adopts 5 ~ 9 Y type methoxy poly (ethylene glycol)s to modify.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108324951A (en) * | 2018-04-16 | 2018-07-27 | 薛剑峰 | A kind of arginine deiminase injection and preparation method thereof |
| CN109438568A (en) * | 2018-11-30 | 2019-03-08 | 湖南华腾制药有限公司 | The preparation and application of the interleukin I L-12 prodrug of monodisperse poly glycol monomethyl ether modification |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103131687A (en) * | 2013-02-04 | 2013-06-05 | 杭州北望生物技术有限公司 | Polyethylene glycol covalent modification lysostaphin and prepared method and application thereof |
| CN104130996A (en) * | 2013-05-03 | 2014-11-05 | 上海医药工业研究院 | Arginine deiminase mutant from arthritis-type mycoplasma and application thereof |
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- 2015-03-27 CN CN201510140645.XA patent/CN104745565A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103131687A (en) * | 2013-02-04 | 2013-06-05 | 杭州北望生物技术有限公司 | Polyethylene glycol covalent modification lysostaphin and prepared method and application thereof |
| CN104130996A (en) * | 2013-05-03 | 2014-11-05 | 上海医药工业研究院 | Arginine deiminase mutant from arthritis-type mycoplasma and application thereof |
Non-Patent Citations (1)
| Title |
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| 张加慧 等: "新型Y 型聚乙二醇-尿酸氧化酶修饰物的制备及免疫原性和生物活性评价", 《中国药学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108324951A (en) * | 2018-04-16 | 2018-07-27 | 薛剑峰 | A kind of arginine deiminase injection and preparation method thereof |
| CN109438568A (en) * | 2018-11-30 | 2019-03-08 | 湖南华腾制药有限公司 | The preparation and application of the interleukin I L-12 prodrug of monodisperse poly glycol monomethyl ether modification |
| CN109438568B (en) * | 2018-11-30 | 2022-01-11 | 湖南华腾制药有限公司 | Preparation and application of monodisperse polyethylene glycol monomethyl ether modified interleukin IL-12 prodrug |
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