CN102191224A - Heavy targeted modification method for herpes simplex viruses and application thereof - Google Patents
Heavy targeted modification method for herpes simplex viruses and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicinal biotechnology and in particular relates to a heavy targeted modification method for herpes simplex viruses (HSV) and application thereof. In the method, surface glycoprotein of the HSV is chemically modified by using an allogenic targeted substance, particularly pegylation folic acid, so that the natural cell tropism is deprived and specifically transduced into tumor cells or tissues and does not infect other normal cells or tissues. By the method, the capacity of the HSV of specifically infecting the tumor cells can be effectively improved; immunoreaction and in vivo neutralization of antibodies are avoided; toxic or side effect is reduced; and safety is improved.
Description
Technical field
The invention belongs to the medical biotechnology field, be specifically related to a kind of heavy targeting modification method of hsv, use this method and can effectively improve the hsv ability of infected tumor's cell specifically, evade neutralizing antibody in immune response and the body, reduce toxic side effect, improve security.
Background technology
(Herpes simplex virus HSV) is the coating double-stranded DNA virus to hsv, is divided into I type (HSV-1) and II type (HSV-2) according to antigenic difference usually.I type bleb disease mainly is by the close contact transmission of respiratory tract, skin and mucous membrane, infects the mucocutaneous and organ of waist with the upper part.II type simplexvirus mainly is present in penis, urethra of women's uterine neck, vagina, skin of vulva and the male sex etc. to be located, and is the arch-criminal who causes sexual organ inflammation and bleb.The I type mainly causes the infection of skin, mucous membrane (oral mucosa) and organ (brain) beyond the sexual organ, and the II type mainly causes the genital area mucocutaneous infections.Virus enters in the body through respiratory tract, oral cavity, sexual organ mucous membrane and damaged skin, dives to occupy in human body normal mucosa, blood, saliva and the sensory nerve ganglion cell.When Abwehrkraft des Koepers descended, during as heating, gastrointestinal dysfunction, menstruation, gestation, focal infection and mood change, the HSV that hides in the body was activated and falls ill.The people is the unique natural reservoir (of bird flu viruses) of simple scar exanthema virus, mainly passes through immediate contagion.
At present, HSV-1 has become important therapy of tumor virus vector and oncolytic virus, its major cause is: 1. HSV-1 is the coating double-stranded DNA virus of gene group leader for 152kb, its capacity is big, can insert the big fragment of external source, because in the gene of HSV-1 genome encoding half being arranged is dispensable gene, can be replaced by the external source therapeutic gene; 2. the host cell scope of HSV-1 is wide, can infect quiescent stage and non-quiescent stage cell, the efficiency of infection height, even under the very low situation of infection multiplicity (MOI), HSV is at the external cell colony that still can infect about 70%; 3. in the host cell that infects, whole replication cycle processes of HSV-1 can finish in 20 hours, discharged the progeny virion of thousands of meters; 4. the HSV-1 virion can merge by cytolemma and carry out direct cell-cell propagation, also can propagate by extracellular space (extracellular space), this is particularly useful for oncolytic virus, because the virus of low dosage just can realize in solid tumor that virus is invaded (Viral Penetration) efficiently; 5. there is the medicine (as acycloguanosine, Famciclovir etc.) of multiple anti-HSV-1 to be used for the treatment of the infection of HSV-1 clinically, and HSV-1 expresses antitumor suicide gene---thymidine kinase (HSV thymidine kinasa, HSV-TK), this just provides a security mechanism for the antitumor action of HSV-TK, because available medicine is closed the replication cycle of HSV-1; 6. HSV-1 can effectively infect the kinds of experiments animal, helps setting up animal model, also is convenient to the preclinical study result is moved in (translation) clinical trial.Therefore, there is the scheme of more or less a hundred relevant HSV to enter each clinical study stage phase in the worldwide at present.
Yet as other any virus type seemingly, the host cell scope of HSV-1 is wide, is not single-minded infected tumor cell.How to make oncolytic HSV from the external tumour cell that imports specifically, promptly the target of cell transduction (Cell TransductionalTargeting) is the therapy of tumor of HSV-1 mediation and the problem that oncolytic HSV viral therapy needs to be resolved hurrily.Present solution mainly is to depend on intratumor injection, but the clinical limitation of this method is bigger, especially can't be used for the patient that tumour has spread.Moreover also there is stronger immunogenicity in oncolytic HSV, and this also is a serious challenge for repeat administration.Thereby need carry out the cell transduction targeting modification to HSV-1, promptly heavy target (Re-targeting).
Receptor-mediated target administration is the important channel of targeted therapy.Found from 1986 folic acid (folic acid, folate, FA) be enter cell by receptor-mediated endocytic pathway since, (Folate Receptor, FR) Jie Dao targeting drug delivery system is just obtained considerable progress to folacin receptor.Compare with other receptor-mediated gene transfer mode, there is its distinct advantages in the folacin receptor system: 1. FR is only at tumor cell membrane surface high expression level, and in most healthy tissuess, expresses hardly, so have good tumor tissues specificity; 2. the part of FR is a folic acid, and FA is the VITAMIN of needed by human, but human body can not synthesize FA, needs the exogenous FA of picked-up.Folic acid easily combines with other molecules by-carboxyl, and can not cause its avidity reduction; In addition folic acid also have, reduced immunogenicity, be easy to modify, volume is little, height chemical stability and biological stability, with advantages such as the physiological compatibility of organic solvent, low cost; 3. FA and FR have high-affinity, by the FR mediated endocytosis, can be effectively will take the photograph people's cell in conjunction with the folic acid of FR, folate conjugate etc.At present, folacin receptor mediated targeting drug delivery system is widely used in genomic medicine, protein drug, chemotherapeutics, toxin medicine, radiopharmaceuticals, monoclonal antibody etc., and has obtained good progress.
Usually utilize the folacin receptor mediated genomic medicine targeted delivery need be with part and cationic polymer chemically crosslinked, genomic medicine combines by static with conjugate again, form part-cationic polymer-genomic medicine mixture, effect by receptor-ligand imports target cell with genomic medicine.For improving receptor-mediated gene transfering efficiency, need usually that (polyethylene glycol PEG) is joint, so that more effectively offer and mediate folic acid and folacin receptor combining at tumor cell surface with polyoxyethylene glycol.
Usually, medicine polyethyleneglycol modified, promptly PEGization (PEGylation) is that activated polyglycol is coupled on protein, polypeptide, small molecules organic drug and the liposome etc. by chemical process.Since polyoxyethylene glycol in 1977 was used to modify bovine serum albumin first, the Pegylation technology developed rapidly, had now become the important means of improving pharmaceutical grade protein and nonprotein clinical drug effect.This is because PEG safety, nontoxic has profit two parent's property.PEG links to each other with medicine, can increase the relative molecular weight of medicine, reduce the enzymolysis of medicine, avoid very fast elimination in the metabolism of kidney, the transformation period of prolong drug increases the water-soluble of medicine, strengthens stability of drug, and the PEG chain is wrapped in the antigenic determinant that medical surfaces can cover medicine, reduces the immunogenicity of medicine; Thereby the molecular structure that has changed medicine has simultaneously improved the character of pharmacokinetics and pharmacodynamics, has improved the Plasma Concentration of site of action.The medicine (as Interferon, rabbit, adenosine deaminase, Zorubicin etc.) of the existing a plurality of Pegylations approval that obtains FDA at present comes into the market, and the medicine that is in the PEGization of preclinical study and clinical experimental stage has tens of kinds more than.
In recent years, the existing research PEGization that begins to attempt virus (as adenovirus) is used for genetic treatment of tumor.Welsh MJ just attempted PEG parcel adenovirus to escape in it and antibody in 1998, Jame M.Wilson etc. has significantly strengthened its transduction efficiency and stability to adenovirus PEGization, the adenovirus of discovery PEGization such as Michael A.Barry can effectively reduce the natural immunity reaction of adenovirus, and the adenovirus that Pastore L. etc. also show PEGization has significantly improved its security.The RGD small peptide PEGization modification adenovirus of usefulness target tumor new vesseles such as hinsaku Nakagawal significantly strengthens its cell transduction efficient and antibody is escaped ability, and the PEGization adenovirus of discovery folic acid immobilizations (Folate Immobilized) such as In Kyung Oh can weigh the target tumor cell.Maria A.Croyle etc. finds that also the lentiviral vectors of PEGization can prevent its inactivation in serum in addition, You-Kyoung Kim etc. also finds the external in vivo equal transduction efficiency of regulating and control of baculovirus vector of PEGization, and the adeno-associated virus of discovery PEGization such as Hong T.Le can be used as carrier of gene conveying or the like.Improve the ability of virus-specific ground infected tumor cell, effectively evade the immune response of body to HSV-1 simultaneously, reduce toxic side effect, increasing preceding security etc. is this area researchist's the focus of attention.
Summary of the invention
The purpose of this invention is to provide a kind of new hsv heavy targeting modification method and should.The present invention makes the n cell preferendum of HSV be replaced by the external source targeting substance, thereby can be gone into tumour cell or tissue by transduction specifically, and do not infect normal cell or tissue, also therefore make HSV before arriving tumour cell, effectively evade the removing of the neutralizing antibody of body simultaneously.
Particularly, the heavy targeting modification method of a kind of hsv of the present invention, it is characterized in that, mainly be to use external source targeting substance folic acid-polyoxyethylene glycol nanoparticle (folate-PEGylated Nanoparticles, FPN) chemical modification or modification HSV-1, realize that by folacin receptor external source targeting substance specificity imports tumour cell, avoid HSV-1 before arriving tumour cell, just to be removed simultaneously by body immune system.
The hsv of heavy target mainly is selected from the I type among the present invention, secondly is the II type.
The hsv of described heavy target mainly is a recombinant herpes simplex virus, secondly is non-recombinant herpes simplex virus (wild-type).Wherein, recombinant herpes simplex virus comprises the oncolytic hsv of the recombinant herpes simplex virus and the condition rf of replication defect type.
Among the present invention, the n cell preferendum of hsv is replaced by the external source targeting substance, thereby makes hsv be gone into tumour cell or tissue by transduction specifically, and does not infect normal cell or tissue.
Described external source targeting substance mainly is a folic acid, secondly is the ligand molecular of other tumour cell high expression level acceptor, includes but not limited to: the apoptosis induction ligand that Urogastron, vascular endothelial growth factor, oestrogenic hormon, tumour necrosis factor are correlated with.
Among the present invention, the external source targeting substance combines with hsv, mainly is the chemical coupling mode by gentleness, secondly is by biological mode and physics mode.
Among the present invention, the target cell of the hsv of heavy target mainly is a tumour cell, secondly is endothelial cells in tumor neogenetic blood vessels.
The heavy targeting modification method of hsv of the present invention comprises the steps:
1.HSV cultivation and purifying
Adopt I type (HSV-1), or II type (HSV-2) hsv, no matter be HSV-1 or HSV-2, all comprise recombinant herpes simplex virus and non-recombinant herpes simplex virus (wild-type).Wherein, hsv mainly is a recombinant herpes simplex virus, comprises the oncolytic hsv (as MGH1, G207, G47, NV1020, AE618,7134 etc.) of the recombinant herpes simplex virus and the condition rf of replication defect type.
The cultivation of hsv is preferentially carried out in Africa green hair monkey-kidney cells (Vero cell) and derived cell thereof (as 2-2,911 etc.), secondly increases in 293 cells, Chinese hamster ovary celI, Hela cell etc.
Conventional Cultivation of Vero, hsv are 0.01 to inoculate by infection multiplicity (MOI), continue cultivation 72 hours, cleer and peaceful cell on the conventional centrifugal collection culture of difference, conventional freezing cracking process lysing cell and centrifugal collection virus, above-mentioned virus merging is stored in-80 ℃, in order to purifying.
The virus stock solution used of above-mentioned preservation carries out purifying with conventional sucrose density gradient centrifugation, and is resultant that the purified virus particle is stored in-80 ℃, to be used for follow-up targeting modification.
Above-mentioned repeatedly production and purge process are to required virus quantity, and common minimum virus quantity is 1 * 10
10Individual virion.
2.FA-PEG and the synthetic and sign of polycation-PEG-FA
The external source targeting substance mainly is a folic acid among the present invention, next is the ligand molecular of other tumour cell high expression level acceptor, include but not limited to: the apoptosis induction ligand that Urogastron, vascular endothelial growth factor, oestrogenic hormon, tumour necrosis factor are correlated with (TNF-related apoptosis-inducing ligand, TRAIL).
The external source targeting substance combines with HSV-1's, mainly is the chemical coupling mode by gentleness, to keep viral vigor.Usually, the external source targeting substance needs can carry out gentle chemical coupling with HSV-1 after the PEGization, mainly is to use folic acid-polyoxyethylene glycol nanoparticle (FPN) chemical modification among the present invention or modifies HSV-1.Therefore at first synthetic FA-PEG (its building-up process and chemical formula structure are as shown in Figure 1), use then infrared spectra,
1H NMR (Nuclear Magnetic Resonance, nucleus magnetic resonance) structure and the purity of the above-mentioned synthetic couplings of technical measurement such as, (matrix-assisted time-of flight mass spectrometer, MALDI-TOF) technology is carried out further phenetic analysis to available in case of necessity matrix time-of-fight mass spectrometry.Purity can be used for the targeting modification of follow-up HSV above 95% FA-PEG conjugate.
The external source targeting substance combines with HSV-1's, can also be by biological mode and physics mode.At first synthetic polycation-PEG-FA, (chitosan, CS) as the polycation component, institute's synthetic CS-PEG-FA chemical formula structure is seen Fig. 2 to select natural glycosaminoglycan-chitosan among the present invention.Synthetic CS-PEG-FA infrared spectra,
1Technology such as H NMR and/or MALDI-TOF are confirmed its structure and substitution value.Purity can be used for the targeting modification of follow-up HSV above 95% CS-PEG-FA conjugate.
3. the FA-PEGization of hsv modification
On the basis of preparation and purifying hsv, hsv is carried out chemical coupling with FA-PEG respectively modifies (Fig. 3) and carries out packing modification (Fig. 4) with CS-PEG-FA, determine the degree of modification of hsv with the fluorescamine method, with dynamic light scattering (Dynamic light scattering instrument, DLS) size of the virus of mensuration modification is measured the surface charge that FA-PEG modifies hsv with zeta potential instrument.Measure the titre of the HSV that is modified simultaneously with conventional plaque counting process.
4. cell in vitro experiment
After the modification of HSV process PEGization, its natural cell transduction approach is blocked, and can modified HSV be taken in by cell, and have the transduction specificity of tumour cell, and can it effectively reduce the immunogenicity of HSV with the cell in vitro examination.
Among the present invention, cultivate folacin receptor and cross the epithelial cancer cells KB of expression and the lung cell A549 of folacin receptor defective, infect respectively with FA-PEG-HSV, PEG-HSV, the HSV with unmodified compares simultaneously.The gradient of infection of virus is with virogenetic cell plaque number, the cell of immunofluorescence cell chemical process marker expression viral protein, with flow cytometer infected cells is counted, relatively the similarities and differences between the above-mentioned cell of infection of the HSV of FA-PEGization modification and other HSV.The result shows that the HSV of FA-PEGization modification (FA-PEG-HSV) can significantly improve target transduction tumour cell ability 3-10 doubly.
Simultaneously, HSV with FA-PEG-HSV, PEG-HSV and unmodified infects scavenger cell RAW 264.1 respectively, (Interleukin 6 to detect IL-6 with ELISA, IL-6) secretory volume (but immunoreactive degree of secretory volume direct reaction of scavenger cell secrete cytokines IL-6), relatively its difference.In addition, also can be by measuring the direct immune response difference of the genomic dna content indirect reaction of HSV different virus of cells infected.Total DNA of the scavenger cell RAW 264.1 of different virus has been infected in extraction, with HSV-TK gene fragment in PCR and the quantitative pcr amplification HSV-1 genome, by the electrophoresis similarities and differences between them relatively.The result shows that the HSV of FA-PEGization modification (FA-PEG-HSV) can effectively reduce the immunogenicity 50%-80% of hsv.
Experimental technique that the present invention relates to and method can be with reference to technology known in the art, and to virus strain and experiment all can buy with cell strain by the commercial channel.
The present invention uses the external source targeting substance especially to pass through Pegylation folic acid chemical modification or modifies the surface glycoprotein of hsv HSV, make its natural cell tropism forfeiture, but gone into tumour cell or tissue by transduction specifically, to other normal cell or organize and do not infect.Use present method and can effectively improve the hsv ability of infected tumor's cell specifically, evade neutralizing antibody in immune response and the body, reduce toxic side effect, improve security.
For the ease of understanding, below will the present invention be described in detail by concrete drawings and Examples.It needs to be noted, specific examples and accompanying drawing only are in order to illustrate, obviously those of ordinary skill in the art can illustrate according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.
Description of drawings
Synthetic and the chemical formula structure diagrammatic sketch of Fig. 1 FA-PEG.
The chemical formula structure diagrammatic sketch of Fig. 2 CS-PEG-FA.
The HSV synoptic diagram is modified in Fig. 3 FA-PEG chemical coupling.
Fig. 4 CS-PEG-FA packing is modified the HSV synoptic diagram.
Embodiment
Embodiment 1
The cultivation of HSV-1 and purifying
The Vero cell inoculation is in the Tissue Culture Dish of 12.5cm in diameter, treats that cell grows to 80% when full, and cell culture medium is replaced by the DMEM substratum that contains 5% newborn calf serum, and with MOI=0.05 virus inoculation HSV.At 37 ℃, contain 5%CO
2Incubator in continue to cultivate 72 hours.Cleer and peaceful cell debris on the cell culture medium is collected in the aseptic frozen pipe-80 ℃ of preservations.
With cleer and peaceful cell multigelation on the above-mentioned virus of collecting three times, thereby abundant lysing cell discharges the HSV virion in the cell.The viral liquid of freeze thawing three times is put into centrifuge tube, 5000rpm, 4 ℃ centrifugal 10 minutes, collect viral supernatant.12000rpm, 4 ℃ centrifugal 30 minutes, further remove the cell debris in the viral supernatant.The viral supernatant liquor 25ml that preliminary purification is good joins (30ml, Beckman Coulter) in the aseptic ultracentrifugation pipe.The HBSS solution that 5ml is contained 25% sucrose with transfer pipet careful join ultracentrifugation pipe pipe at the bottom of, 25000rpm, 4 ℃ of ultracentrifugations 4 hours (the centrifuge rotor model is a Beckman SW28 rotor).After centrifugal, outwell supernatant liquor, with the HBSS precipitation of the resuspended ultracentrifugation pipe of 250l HBSS bottom.Resuspended G207 virus solution is placed 4 ℃ of shaking tables, softly shake and spend the night.3000rpm, 4 ℃ centrifugal 10 minutes, further remove cell debris.Virus behind the taking-up partial purification is carried out titer determination, remaining virus-80 ℃ preservation.
Embodiment 2
Synthetic and the sign of FA-PEG
Folic acid and NHS are formed Acibenzolar; be about to folic acid and be dissolved in methyl-sulphoxide (DMSO), with dicyclohexylcarbodiimide (DCC), the n-hydroxy-succinamide is 1: 2: 2 ratio reaction with mol ratio; stirred 18 hours, entire reaction is carried out under the condition of nitrogen protection.With centrifugal 30 minutes of gained suspended matter 15000rpm, remove white insolubles, the extracting yellow supernatant liquor.
To be 1: 8 NH with above-mentioned folic acid activator mol ratio
2-PEG-COOH is dissolved in DMSO, under the condition of nitrogen protection,, the gained material was dialysed respectively under 4 ℃ of conditions 3 days and 2 days in DMSO and ultrapure water respectively with folic acid activator reaction 4 hours, with the finished product-20 ℃ of lyophilizes.
Product F A-PEG after the freeze-drying is got sample segment (about 5mg) be dissolved in deuterium, detect, confirm response situation and structure by nucleus magnetic resonance among the DMSO.
Embodiment 3
The FA-PEGization modification of hsv
Branch is got methoxyl group-PEG-NHS (mPEG) and is dissolved in 2ml HBSS, and (ratio of mPEG and HSV is that the HSV of 1PFU adds 1 * 10 to add HSV
7Individual mPEG molecule), ice bath reacted 4 hours down.The PEG-FA of reaction back gained is dialysis 2 days under 4 ℃ of conditions in ultrapure water, with the finished product-80 ℃ of preservations.
FA-PEG-COOH is dissolved among the 2ml HBSS, add EDC and NHS and activate, its mol ratio is 1: 10: 10, room temperature reaction 1 hour, the FA-PEG-COOH that gets after the activation is dissolved in 2ml HBSS, and (ratio of HSV and FA-PEG-COOH is the HSV adding 1 * 10 of 1PFU to add HSV
7Individual FA-PEG-COOH molecule), under ice bath, reacted 4 hours.The PEG-FA of reaction back gained is dialysis 2 days under 4 ℃ of conditions in ultrapure water, with the finished product FA-PEG-HSV-80 ℃ of preservations.
Further detect the purity of FA-PEG-HSV, specifically: fluorescence ammonia dyestuff is dissolved in the acetone, is configured to the fluorescence ammonia solution of 7mg/mL.Add HSV or the FA-PEG-HSV of 900l in the fluorescence ammonia dyestuff of 100l, mix at once, under dark surrounds, hatched 15 minutes then.By spectrophotometer fluorescence intensity (excitation wavelength is 390nm, and emission wavelength is 475nm), with PEG-diamines drawing standard curve.
Embodiment 4
The HSV virus particle diameter and the zeta current potential thereof of targeting modification detect
HSV, PEG-HSV and FA-PEG-HSV are dissolved in ultrapure water, measure the size of each papova particle diameter with dynamic light scattering (Dynamic lightscattering instrument, DLS, Malvern company) respectively.
With HSV, PEG-HSV and FA-PEG-HSV be dissolved in respectively 2mL damping fluid HBSS (Hank ' s Balanced SaltSolution, its composition is 0.137M NaCl, 5.4mM KCl, 0.25mM Na
2HPO
4, 0.44mM KH
2PO
4, 1.3mM CaCl
2, 1.0mM MgSO
4, 4.2mM NaHCO
3, pH7.4) in, measure the surface zeta potential current potential size of each papova respectively with laser light scatterometer (ZetasizerNano System, Malvern company).
Embodiment 5
The titre of HSV virus detects
With the Vero cell with 3 * 10
5The density of cells/well is seeded in 6 orifice plates.Inoculate after 24 hours, treat that the Vero cell grows to 100% when full, remove cell culture medium, PBS washes cell one time.Then will be in advance with the DMEM substratum 10 times of dilutions HSV virus well or viral the adding in every hole of HSV of PEG modified successively.At 37 ℃, contain 5%CO
2Incubator in hatch made in 2 hours virus fully combine with cell.After 2 hours, inhale with transfer pipet and to go not and cell bonded virus, it is 5% DMEM substratum that every hole adds 2ml newborn calf serum content, at 37 ℃, contains 5%CO
2Incubator in continue to cultivate 68 hours.After 68 hours, remove cell conditioned medium with the transfer pipet suction, every hole adding concentration is 10% formaldehyde fixed liquid 1ml, and incubated at room was inhaled and gone 10% formaldehyde solution after 30 minutes, 10% Viola crystallina (worker is given birth in the Shanghai) solution of every hole adding 1ml-and with the Vero cell dyeing.After the incubated at room 30 minutes, with the Vero cell in flushing with clean water 6 orifice plates, number goes out the plaque quantity in every hole, and calculates the PFU value of virus according to following formula.
PFU=plaque quantity * viral dilution multiple
Embodiment 6
The HSV virus target detection of targeting modification
KB and A549 cell are respectively with 1 * 10
5Cells/well is inoculated in carries out the routine cultivation in 24 orifice plates.After one day, infect two kinds of clones with HSV, PEG-HSV and FA-PEG-HSV with MOI=1 respectively,, contain 5%CO at 37 ℃
2Incubator in cultivated 72 hours, collecting cell and cell culture medium, thus three the cracking cells of thawing discharge the progeny virion in the cell, detect filial generation PFU number behind each papova cells infected.
Embodiment 7
The immunogenic mensuration of HSV virus
Scavenger cell RAW 264.1 is 1 * 10 with cell density
6Cells/well is inoculated in carries out the routine cultivation in 6 orifice plates, after one day, comprise 2 * 10 respectively with 2ml
8The fresh culture of the HSV of individual virion, PEG-HSV and FA-PEG-HSV continues to cultivate 24 hours.Measure the secretory volume of the interleukin 6 (IL-6) in the cell culture with conventional euzymelinked immunosorbent assay (ELISA) (ELISA).The difference of IL-2 expression amount between each group of statistical, thus judge its immunogenicity.
Claims (10)
1. the heavy targeting modification method of a hsv, it is characterized in that, use external source targeting substance chemical modification or modify hsv HSV, realize that by the external source targeting substance specificity imports tumour cell, removed by body immune system before avoiding hsv HSV to arrive tumour cell, by following step:
1) cultivate and the purifying hsv, obtaining minimum virus quantity is 1 * 1010 virion;
2) the external source targeting substance combines with hsv, obtains purity and surpasses 95% conjugate;
3) the external source targeting substance of hsv-PEGization modification;
4) cell in vitro experimental verification.
2. method according to claim 1 is characterized in that, the hsv of described heavy target is I herpes simplex virus type or II herpes simplex virus type.
3. method according to claim 1 is characterized in that, described hsv is recombinant herpes simplex virus or non-recombinant herpes simplex virus.
4. method according to claim 3 is characterized in that, described recombinant herpes simplex virus comprises the oncolytic hsv of the recombinant herpes simplex virus and the condition rf of replication defect type.
5. method according to claim 1 is characterized in that, described external source targeting substance is selected from the ligand molecular of folic acid or other tumour cell high expression level acceptor.
6. method according to claim 1 is characterized in that, described external source targeting substance is a folic acid.
7. method according to claim 5, it is characterized in that the ligand molecular of described other tumour cell high expression level acceptor includes but not limited to: the apoptosis induction ligand that Urogastron, vascular endothelial growth factor, oestrogenic hormon, tumour necrosis factor are correlated with.
8. method according to claim 1 is characterized in that, described external source targeting substance passes through chemical coupling mode or biological mode or physics mode and combines with hsv.
9. method according to claim 1 is characterized in that, the target cell of the hsv of described heavy target is tumour cell or endothelial cells in tumor neogenetic blood vessels.
The hsv of the heavy target of claim 1 preparation improve its specifically infected tumor's cell ability, evade immune response and body inner virus neutralizing antibody, reduce toxic side effect and improve purposes in the security preparation.
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| WO2022033467A1 (en) * | 2020-08-14 | 2022-02-17 | 上海行深生物科技有限公司 | Method for constructing oncolytic virus |
| CN115624524A (en) * | 2022-10-25 | 2023-01-20 | 中国医科大学附属第一医院 | Preparation and application of PEG albumin modified oncolytic virus intravenous delivery preparation |
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| WO2022033467A1 (en) * | 2020-08-14 | 2022-02-17 | 上海行深生物科技有限公司 | Method for constructing oncolytic virus |
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