CN108165536A - A kind of recombination oncolytic vaccinia virus and preparation method and application - Google Patents
A kind of recombination oncolytic vaccinia virus and preparation method and application Download PDFInfo
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- CN108165536A CN108165536A CN201711310440.7A CN201711310440A CN108165536A CN 108165536 A CN108165536 A CN 108165536A CN 201711310440 A CN201711310440 A CN 201711310440A CN 108165536 A CN108165536 A CN 108165536A
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
- A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
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- C12N2740/00—Reverse transcribing RNA viruses
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- C12N2740/10011—Retroviridae
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Abstract
The invention discloses a kind of recombination oncolytic vaccinia virus and preparation method and application, thymidine kinase (TK) area of the virus includes the coded sequence of the PD1 full length antibodies shown in SEQ ID NO.1.The present invention effectively combines the tumor killing effect of gene therapy with the oncolytic effect of viral therapy, be prepared for it is a kind of can high efficient expression PD1 full length antibody genes oncolytic vaccinia virus.While oncolytic virus plays oncolytic effect cracking tumour cell, great expression PD1 full length antibodies inhibit the activity of T cell surface PD1, activate T cell immune response, play dual antitumous effect.Its killing ability to malignant B cell lympha tumour is enhanced relative to simple gene therapy or virus therapy.By the way of virus replication related gene missing, vaccinia virus gene Zu TK areas are lacked, it is ensured that virus is replicated in the specifically inside tumor cell of abnormality proliferation, and the then not reproducible in normal cell, greatly strengthens the safety of oncolytic vaccinia virus carrier.
Description
Technical field
The invention belongs to genetic engineering, immunology and oncology are related to a kind of recombination oncolytic vaccinia virus and its preparation
Method and application.
Background technology
The incidence of malignant lymphoma accounts for the 3-5% of entire cancer patient, there is 452,000 newly-increased patient every year.It is more than
90% adult patient from ripe B cell, prompt B cell lympha tumour be threaten human health important diseases it
One.
Immunization therapy is another treatment of cancer new method after operation, radiotherapy, chemotherapy, molecular targeted therapy.Various
In cancer immunotherapy method, immunologic test point inhibitor is one of drug for obtaining prominent curative effect in recent years.Immunologic test point
It is the inhibitory pathway of epidemic prevention system excessive activation, on the immunocyte surface being activated there are some albumen such as CTLA4,
For PD1 when playing the role of " braking " when excessive immune reacts, however when tumour occurs, these albumen are lasting due to overexpression
Ground sends out inhibition signal for immune system, so as to promote the immunologic escape of tumour.Therefore immunologic test point inhibitor is exempted from cancer
It plays an important role in epidemic disease treatment.
PD-1/PD-L1 signal paths are one of mostly important target spots of blocking immunity checkpoint strategy.Occur in tumour
When T cell can be caused to express PD1, the tumor-infiltrating lymphocytes (TIL) of multiple types cancer also express PD-1.Study table simultaneously
Bright a variety of cancer cell height expression PD-L1, including lymthoma, leukaemia, malignant mela noma and non-small cell lung cancer (NSCLC)
Deng.The PD-1 ligands of PD-1 and cancer cell surfaces (PD-L1 or PD-L2) combine the activation for inhibiting T cell, lead to specificity T
Cell depletion or disability.
On B cell lympha tumour, researches show that expression high on the TIL and periphery blood T cell of follicular lymphoma (FL)
PD1, and T cell disables.On the other hand, a variety of B cell tumours, including diffusing big B lymthomas (DLBCL), the primary vertical big B leaching of diaphragm
Great expression PD-L1 in bar knurl (PMBCL) and the big B lymthomas of testis.Therefore, effectively PD-1/PD-L1 signals is blocked to lead to
Road can enhance killing of the T cell to B cell lympha tumour.
Recently, PD-1 the and PD-L1 monoclonal antibodies of blocking immunity checkpoint and inhibitor have been applied to I-III phase clinical researches.
In June, 2015, U.S.'s food and Drug Administration (FDA) approval PD-1 monoclonal antibodies nivolumab are used for as a medicine
The previously advanced NSCLC patients Jing Guo chemotherapy.I phase clinical researches show that nivolumab treatment NSCLC patients' is average total raw
It is 42% and 18% to deposit the survival rate that the phase is 14.9 months, 1 year and 3 years.
But immunologic test point inhibitor also has its limitation：(1) most patients cannot obtain lasting complete reaction；
(2) about there are seriously immune related side effects (irAEs) in 25-30% patient after the treatment of PD-1 monoclonal antibodies；(3) PD-1/PD-L1 accesses
The characteristics of in tumor microenvironment, affects the effect of PD-1 monoclonal antibodies.Combination therapy is to overcome immunologic test point inhibitor limitation
Main direction of studying.The strategy currently studied includes that anti-PD-1 monoclonal antibodies and chemicotherapy, small molecule targeted drug, other exempt from
Epidemic disease checkpoint inhibitor or oncolytic virus joint.
A kind of tumor biotherapy that oncolytic virus (Oncolytic virus, OV) therapy is this year to quickly grow is new
Method.In October, 2015, the OV products T-Vec of FDA approvals Amgen are used to treat malignant mela noma.In December, T-Vec is again
Granted European Food drug administration, it was demonstrated that OV therapies are a kind of very promising new methods for the treatment of cancer.Oncolytic bovine vaccine
Viral (Oncolytic vaccinia virus, OVV) is a kind of oncolytic virus being concerned in recent years.It the advantage is that：
Virus stability is good, it is pathogenic it is low, gene Transfection efficiencies are high, good security.Oncolytic vaccinia virus can selective infected tumor
Cell simultaneously replicates, kill tumour cell in the cell, and the toxicity very little of normal tissue and cell.JX-594 is first use
In the oncolytic vaccinia virus of clinical research, it has lacked thymidine kinase (TK) area and has carried allogenic gene GM-
CSF, JX-594 can be replicated in the tumour cell of fast breeding, while the GM-CSF carried can further stimulate antitumor exempt from
Epidemic disease.The landmark result of study such as Breitbach CJ is shown：JX-594 is injected intravenously 23 refractory, recurrence entities and swells
Knurl, 13 patients are effective, and 1 patient obtains part and alleviates；Show that OVV can be used for being injected intravenously, play systemic anti-tumor and make
With.
Invention content
It was found by the inventors of the present invention that the monoclonal antibody of existing PD1 is in B cell lympha tumour is treated, there are limitations.For
This, the present invention provides a kind of recombination oncolytic vaccinia virus and preparation method and application.
The purpose of the present invention is what is be achieved through the following technical solutions：A kind of recombination oncolytic vaccinia virus, the virus
Thymidine kinase (TK) area includes the coded sequence of the PD1 full length antibodies shown in SEQ ID NO.1.
A kind of preparation method of recombination oncolytic vaccinia virus, which is characterized in that include the following steps：
(1) people's overall length PD-1 monoclonal antibodies are synthesized, gene order is as shown in SEQ ID NO.1；
(2) overall length PD-1 monoclonal antibodies are expanded, are subcloned after digestion to the TK areas of vaccinia virus shuttle plasmid (pTK)
In, construct recombinant plasmid pCB-aPD1.Wherein, aPD1 genes are controlled by the early late phase promoter (P-se/l) of vaccinia virus.
(3) by the way of homologous recombination, by pCB-aPD1 plasmid transfections to being infected wild type vaccinia
In the cell of virus, make the two homologous recombination, generate vaccinia virus recombinant VV-aPD1.After screening, obtain the TK areas and include
The recombination oncolytic vaccinia virus of the coded sequence of PD1 full length antibodies shown in SEQ ID NO.1.
A kind of application of recombination oncolytic vaccinia virus in the drug for treating treatment B cell lympha tumour is prepared.
The beneficial effects of the invention are as follows：
1st, the present invention effectively combines the tumor killing effect of gene therapy with the oncolytic effect of viral therapy, is prepared for one
Kind can high efficient expression PD1 full length antibody genes oncolytic vaccinia virus.Oncolytic effect cracking tumour cell is played in oncolytic virus
While, great expression PD1 full length antibodies inhibit the activity of T cell surface PD1, activate T cell immune response, play dual
Antitumous effect.It is enhanced relative to simple gene therapy or virus therapy to kill malignant B cell lympha tumour
Hinder ability.
2nd, the present invention lacks vaccinia virus gene Zu TK areas by the way of virus replication related gene missing, it is ensured that
Virus is replicated in the specifically inside tumor cell of abnormality proliferation, and the then not reproducible in normal cell, is greatly strengthened molten
The safety of knurl vaccinia virus vector.
Description of the drawings
Fig. 1 a are pCB-aPD-1 DNA gel electrophoresis results after ECORI and BglII double digestions, and 1b is part sequencing knot
Fruit.
Fig. 2 is that the flow cytometer detection pCB-aPD-1 and PD-1 after the T cell incubation of activation is expressed, and then detects PD-1 antibody
Activity, wherein, left figure is the Jurkat cell control without stimulation, and right figure is expresses PD1 antigens after ConA is stimulated
Jurkat cell.
Fig. 3 is pCB-aPD1-GFP and fluorograms of the WT in 293A cells after homologous recombination.
Fig. 4 is after OVV-aPD-1 is recombinated, and extraction virus genom DNA passes through PCR testing goals gene and wild virus gene
In the presence of.
Fig. 5 is the expression efficiency of flow cytometer detection GFP after OVV-aPD-1 infects Raji cell strains with concentration gradient.
Fig. 6 is after OVV-aPD-1 infects Raji cell strains with concentration gradient, by detecting Colony forming and CCK8 decoration methods
The cytotoxicity of OVV-aPD-1 is detected, wherein, b is the data analysis to a.
Specific embodiment
Present invention combination attached drawing and example are further described.Unless otherwise indicated, it is normal that this field can be used in the present invention
Rule technology.
1st, the synthesis of people's overall length PD-1 monoclonal antibodies：
The single-stranded variable region sequences of PD-1 monoclonal antibodies pass through with reference to the sequence for applying your treasured PD-1 monoclonal antibodies nivolumab
Amino acid is deduced and codon optimization obtains the single-stranded variable region sequences of PD-1 of our designs, and the Fc sections of monoclonal antibody are with reference to human IgG 4
Fc sections of sequences, using the heavy chain and light chain of method synthesis people's PD-1 antibody of chemical synthesis, the heavy chain and light chain gene are by linking
Sub- IRES connections.The full-length gene order of PD-1 monoclonal antibodies is as shown in SEQ ID NO.1.
2nd, the structure of the oncolytic vaccinia virus shuttle plasmid pCB-aPD1 of carrier PD-1 monoclonal antibodies and identification：
The Specific PCR primers for containing ECORI and BglII restriction enzyme sites using end amplify PD1 full length antibody sequences
(containing heavy chain and light chain), using subclone after ECORI and BglII double digestions to the vaccinia virus shuttle matter for carrying GFP genes
In the TK areas of grain (pTK-GFP), pCB-aPD1 is constructed.DNA gel electrophoresis result after digestion as shown in Figure 1a, can from figure
The aPD1 full length sequences of 2700bp are generated to find out pCB-aPD1 after digestion；1b is part sequencing result, as a result proves institute gram
Grand aPD1 gene orders are correct.APD1 genes are controlled by the early late phase promoter (P-se/l) of vaccinia virus.The sun constructed
Property clone using digestion, sequencing identified, selection identification correctly clone carry out plasmid largely extract, prepare>4 μ g's is high-purity
Plasmid is spent, -80 DEG C save backup.
Wherein, vaccinia virus shuttle plasmid, which can refer to room, honor, Li Hongyu, Chen Keda, Zhou Wenshuo, Yan Hui (2012)
.Zeocin with the structure international epidemic pestology magazines of the bis- selection markers vaccinia virus recombinant carriers of gfp, 39 (3),
148-152。
3rd, pCB-aPD-1 expresses the detection of PD-1 antibody
Jurkat cell is transduceed pCB-aPD-1 after ConA is stimulated, and passes through stream using PE-anti Human IgG4 (Fc)
Formula detection pCB-aPD-1 expression PD-1 antibody abilities, the results are shown in Figure 2, illustrates that pCB-aPD-1 can express 11.9% PD-1
Antibody.
4th, the packaging of OVV-a PD1 viruses and identification
By the way of homologous recombination, by liposome transfection by pCB-aPD1-GFP plasmid transfections to being felt
In the 293A cells for having contaminated Wild-type vaccinia strain, make the two homologous recombination, generate VV-aPD-1-GFP.The virus of packaging is through 3-
5 wheel medicines sieves (mycophenolic acid, xanthine and female xanthine) combines GFP developments and are screened, and thermophilic spot purifies, continuously at least three-wheel " choosing spot ",
PCR is identified, filters out correct positive restructuring vaccinia virus.Fig. 3 results illustrate pCB-aPD1-GFP successes in 293A cells
It is recombinated with Wild-type vaccinia strain, generates virus plaque.Fig. 4 is the result shows that the recombinant virus generated through more wheel medicine sieves carries PD1
The heavy chain (1300bp) of full length antibody, light chain (704bp) and sub- IRES (650bp) sequence of link, and without wild poison pollution.
5th, OVV-aPD1 is to the Infection in Vitro of B cell lymphoma cell strain
It is to verify Infection in Vitro of the OVV-aPD1 to B cell lymphoma cell strain, chooses Raji cell strains, OVV-aPD1 divides
Raji cell strains are not infected with 5,10,20MOI concentration gradient, using flow cytomery OVV-aPD1 to Raji cell strains
Efficiency of infection, the results are shown in Figure 5, and OVV-aPD1 reaches 24.28% to the efficiency of infection of Raji cell strains in 20MOI.
6th, OVV-a PD1 are to the Cytotoxicity in vitro of B cell lymphoma cell strain
It is to verify OVV-a PD1 to the Cytotoxicity in vitro of B cell lymphoma cell strain, OVV-aPD1 is respectively with 5,10,20MOI
Concentration gradient infection Raji cell strains 72 hours, utilize Colony forming experiment and CCK8 decoration methods detection OVV-a PD1 couple
The growth inhibition effect of Raji cell strains, as a result as shown in figure 6 a and 6b, OVV-a PD1 make the growth inhibition of Raji cell strains
With in concentration dependant, in 20MOI, cell reaches 65% in small and dispersion colony growth, cytotoxic effect, seriously affects thin
Born of the same parents are proliferated.
Sequence table
<110>Zhejiang University
<120>A kind of recombination oncolytic vaccinia virus and preparation method and application
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<170> SIPOSequenceListing 1.0
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<211> 2702
<212> DNA
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gaattctcac ttgcccaggg acagggacag gcttttctga gtataatggt tgtgcagtgc 60
ttcgtgcatc acggagcagg agaacacgtt gccctcctgc catctagact tatccacggt 120
cagccggcta tacagaaaga agctgccatc ggagtccagc acgggtggtg tggtcttgta 180
attgttctcg ggctggccat tggactccca ctccacagcg atgtcagatg gatagaagcc 240
cttcaccaga caggtcaggc tcacctggtt ctttgtcatc tcctcctggg aagggggcag 300
ggtgtacacc tgtggctccc taggctggcc cttggcctta gagattgtct tctcgatgct 360
ggatggcagg cccttattgc tcaccttgca cttatactcc ttgccgttca gccagtcctg 420
atgcagcacg gtcagcacgc tcaccacccg gtatgtggag ttaaactgct cctccctagg 480
cttggtctta gcattgtgca cctccacgcc atccacatac cagttgaact gcacctctgg 540
atcctcctgg gacacgtcca ccaccacgca ggtcacctca ggtgtccgag agatcatcag 600
tgtgtcctta ggctttggag gaaacaggaa cacggaaggt cctcccagga actcaggagc 660
aggacaaggt gggcaaggag gtccgtactt agactccacc ctcttatcca ccttggtatt 720
gctaggctta tggtccacgt tgcaggtata tgtcttggtg cccagagagc tggagggcac 780
tgtcaccaca gagctcaggc tgtacaggcc ggaagactgc agcacagcag gaaaggtgtg 840
cacgccggat gtcagggcgc cggagttcca gctcacggtc actggctcag ggaaataatc 900
cttcaccaga cagcccagag cggcggtgct ctcggatgta gacctgctgc agggagccag 960
tgggaacacg gaggggccct ttgtgctggc gctggacacg gtcaccagtg tgccctggcc 1020
ccagtaatcg tcattggtag cacaatagta cacggctgta tcctcggccc tcagagagtt 1080
catctgcaga aacagggtat tcttggagtt gtccctagag attgtgaatc tgcccttcac 1140
ggaatcggca tagtacctct tagagccgtc gtaccagatc acagccaccc actccagtcc 1200
cttgccagga gcctgtctca cccagtgcat gccggagtta gagaaggtga tgccgctggc 1260
cttgcagtcc agccgcaggg acctgcctgg ctgcaccacc ccccctcctg attcgaccag 1320
ctgcacctgt gagtggacac cagttgcggt tgcgaccaga aacagaatga tacatgacca 1380
tcccatggaa ggtcgtctcc ttgtgggttg tggcaagctt atcatcgtgt ttttcaaagg 1440
aaaaccacgt ccccgtggtt cggggggcct agacgttttt ttaacctcga ctaaacacat 1500
gtaaagcatg tgcaccgagg ccccagatca gatcccatac aatggggtac cttctgggca 1560
tccttcagcc ccttgttgaa tacgcttgag gagagccatt tgactctttc cacaactatc 1620
caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta tacacgtggc 1680
ttttggccgc agaggcacct gtcgccaggt ggggggttcc gctgcctgca aagggtcgct 1740
acagacgttg tttgtcttca agaagcttcc agaggaactg cttccttcac gacattcaac 1800
agaccttgca ttcctttggc gagaggggaa agacccctag gaatgctcgt caagaagaca 1860
gggccaggtt tccgggccct cacattgcca aaagacggca atatggtgga aaataacata 1920
tagacaaacg cacaccggcc ttattccaag cggcttcggc cagtaacgtt aggggggggg 1980
gagggagagg ggcgtcaaca ttctccacgg ttgaaacttt ttgtgactgg tgaggacaga 2040
ccctggtgag tgacttcaca agcatacacc ttgtgcttct cgtaatcggc cttggacagt 2100
gtcagggtgc tggacagaga atatgtgcta tccttggagt cctgctcggt cacagactcc 2160
tggctattgc cggactgcag agcgttatcc accttccact gcaccttggc ctcccgaggg 2220
tagaaattgt tcagcaggca caccacgctg gctgtgccgg acttcagctg ctcgtcggaa 2280
gggggaaaga tgaacacgga gggagcggcc acggttctct tgatctccac ctttgtgccc 2340
tggccaaagg tccgtggcca attagagctc tgctggcaat agtacacggc gaaatcctca 2400
ggctccaggg aagagattgt cagggtaaag tctgtgccag atccgcttcc ggagaaccta 2460
gcggggatgc cggtagctct gttagaggcg tcgtagatca gcagcctggg agcctggcct 2520
ggcttctgct gataccaggc caggtaggag gacacggact ggctagcccg gcagctcagg 2580
gtggccctct ctcctgggga cagagacaga gttgcggggc tctgagtcag cacaatctct 2640
gaatgcacac cagttgcggt tgcgaccagg aacaggatga tacaggacca tcccatagat 2700
ct 2702
Claims (3)
1. a kind of recombination oncolytic vaccinia virus, which is characterized in that thymidine kinase (TK) area of the virus includes SEQ
The coded sequence of PD1 full length antibodies shown in ID NO.1.
2. a kind of preparation method of recombination oncolytic vaccinia virus described in claim 1, which is characterized in that include the following steps：
(1) people's overall length PD-1 monoclonal antibodies are synthesized, gene order is as shown in SEQ ID NO.1；
(2) overall length PD-1 monoclonal antibodies are expanded, after digestion in subclone to the TK areas of vaccinia virus shuttle plasmid (pTK), structure
Build out recombinant plasmid pCB-aPD1.Wherein, aPD1 genes are controlled by the early late phase promoter (P-se/l) of vaccinia virus.
(3) by the way of homologous recombination, by pCB-aPD1 plasmid transfections to being infected Wild-type vaccinia strain
Cell in, both make homologous recombination, generate vaccinia virus recombinant VV-aPD1.After screening, obtain the TK areas and include SEQ
The recombination oncolytic vaccinia virus of the coded sequence of PD1 full length antibodies shown in ID NO.1.
3. a kind of recombination oncolytic vaccinia virus described in claim 1 is in the drug for treating treatment B cell lympha tumour is prepared
Application.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957551A (en) * | 2019-04-03 | 2019-07-02 | 浙江省医学科学院 | Express vaccinia virus recombinant and its application of people's beta-alexin 2 |
| CN111150748A (en) * | 2019-12-27 | 2020-05-15 | 杭州荣谷生物科技有限公司 | Application of recombinant oncolytic virus in preparation of medicine for treating digestive tract cancer |
| WO2020173123A1 (en) * | 2019-02-26 | 2020-09-03 | 南京大学 | Replication oncolytic adenovirus having activated immune co-stimulatory signal pathway and blocking immune checkpoint and application thereof |
| CN111763660A (en) * | 2020-08-07 | 2020-10-13 | 南京大学 | Recombinant oncolytic vaccinia virus and preparation method and application thereof |
| CN112094823A (en) * | 2020-07-21 | 2020-12-18 | 南京大学 | Novel recombinant oncolytic vaccinia virus with immune checkpoint activation and immune co-stimulation and construction method and application thereof |
| CN113583979A (en) * | 2021-08-03 | 2021-11-02 | 杭州荣谷生物科技有限公司 | Recombinant oncolytic vaccinia virus, preparation method and application thereof |
| EP3552615B1 (en) * | 2014-07-16 | 2022-01-26 | Transgene SA | Oncolytic virus for expression of immune checkpoint modulators |
| WO2022122026A1 (en) * | 2020-12-11 | 2022-06-16 | Genesail (Shanghai) Co., Ltd. | A modified oncolytic virus, composition and use thereof |
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| WO2020173123A1 (en) * | 2019-02-26 | 2020-09-03 | 南京大学 | Replication oncolytic adenovirus having activated immune co-stimulatory signal pathway and blocking immune checkpoint and application thereof |
| CN109957551A (en) * | 2019-04-03 | 2019-07-02 | 浙江省医学科学院 | Express vaccinia virus recombinant and its application of people's beta-alexin 2 |
| CN111150748A (en) * | 2019-12-27 | 2020-05-15 | 杭州荣谷生物科技有限公司 | Application of recombinant oncolytic virus in preparation of medicine for treating digestive tract cancer |
| CN112094823A (en) * | 2020-07-21 | 2020-12-18 | 南京大学 | Novel recombinant oncolytic vaccinia virus with immune checkpoint activation and immune co-stimulation and construction method and application thereof |
| CN111763660A (en) * | 2020-08-07 | 2020-10-13 | 南京大学 | Recombinant oncolytic vaccinia virus and preparation method and application thereof |
| WO2022122026A1 (en) * | 2020-12-11 | 2022-06-16 | Genesail (Shanghai) Co., Ltd. | A modified oncolytic virus, composition and use thereof |
| CN113583979A (en) * | 2021-08-03 | 2021-11-02 | 杭州荣谷生物科技有限公司 | Recombinant oncolytic vaccinia virus, preparation method and application thereof |
| CN113583979B (en) * | 2021-08-03 | 2022-11-22 | 杭州荣谷生物科技有限公司 | Recombinant oncolytic vaccinia virus, preparation method and application thereof |
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Application publication date: 20180615 |