CN114010666B - Application of oncolytic virus, PARP inhibitor and PD-1 antibody in preparation of antitumor drugs - Google Patents

Application of oncolytic virus, PARP inhibitor and PD-1 antibody in preparation of antitumor drugs Download PDF

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CN114010666B
CN114010666B CN202111233544.9A CN202111233544A CN114010666B CN 114010666 B CN114010666 B CN 114010666B CN 202111233544 A CN202111233544 A CN 202111233544A CN 114010666 B CN114010666 B CN 114010666B
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蔡宇伽
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Abstract

The invention provides an application of oncolytic virus, PARP inhibitor and PD-1 antibody in preparing antitumor drugs, through inserting miRT and hGGMCSF in I type herpes simplex virus, obtain oncolytic virus oHSV, make the security of oncolytic virus raise and strengthen the organism immune response at the same time; through combined use of oncolytic virus oHSV and Olaparib, the combined use of the oncolytic virus oHSV and Olaparib can increase the PD-1 expression quantity of CD4+ T cells and CD8+ T cells, so that an immune checkpoint inhibitor, namely a PD-1 antibody, is introduced, the curative effect of a treatment scheme in breast cancer is further improved, the combined use of oHSV, olaparib and the PD-1 antibody is further used for inhibiting the size of breast cancer in-situ tumor of a mouse, and on the basis of the combined use of the two, the number of lung metastases is further reduced, and the survival period of the mouse is remarkably prolonged.

Description

Application of oncolytic virus, PARP inhibitor and PD-1 antibody in preparation of antitumor drugs
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of oncolytic virus, PARP inhibitor and PD-1 antibody in preparation of antitumor drugs
Background
Oncolytic viruses with unique oncolytic mechanisms provide a new option for the treatment of tumors, particularly for tumors that do not respond to conventional therapies, replication of oncolytic viruses in tumor cells can lyse and kill tumor cells, and furthermore, oncolytic viruses and the tumor-associated antigens released by lysis can induce an immune response that further kills tumors.
Due to tumor heterogeneity and complexity of tumor microenvironment and the effects of the body's immune system, it is difficult to achieve good therapeutic results in certain tumors with oncolytic viruses alone, for example, the first FDA approved oncolytic virus T-VEC does not improve overall survival in melanoma patients, and only 16.3% of patients receiving treatment have tumor volumes that continue to shrink over 6 months. Indication of oncolytic viruses that are greatly affected.
The oncolytic virus and other medicaments can realize complementary advantages, thereby improving the tumor treatment effect. For example, the clinical results of the Amgen company and Bai-Shi Guibao co-operating to develop T-VEC and CTLA-4 antibody combination therapy for advanced melanoma stage I show that the overall response rate of the combination therapy is significantly higher than that of single-drug therapy. In addition, the document reports that histone deacetylase inhibitor, anti-tumor angiogenesis inhibitor and the like can synergistically enhance the anti-tumor treatment effect of the herpes simplex virus oncolytic virus.
Chinese patent 201711478207.X discloses that the combined use of an M1 oncolytic virus of alphavirus and a PARP inhibitor has the function of improving the killing of tumor cells at the cellular level. The patent kills tumor cells in vitro by using oncolytic viruses and PARP inhibitors, in vivo experiments are not performed, namely, the oncolytic viruses and the PARP inhibitors are verified in an animal model which can reflect the actual clinical situation, the use time of the number of times of use of the oncolytic viruses and the use time of the number of times of use of the PARP inhibitors are not explored, and after the oncolytic viruses and the PARP inhibitors are used, the change of the body immunity situation is not explored, and the problems are solved in the scheme. And the patent document does not mention the technical proposal of the combined use with PD-1 antibodies.
Disclosure of Invention
It is an object of the present invention to provide an anti-tumor pharmaceutical composition comprising an oncolytic virus selected from the group consisting of herpes simplex virus type I and a PARP inhibitor.
As an embodiment of the present invention, the PARP inhibitor is Olaparib and the molecular formula of Olaparib is C 24H23FN4O3; the structural formula of Olaparib is shown in formula I:
The PARP inhibitor Olaparib and Olaparib can simultaneously inhibit the activities of PARP1 and PARP2, and Olaparib is a medicament approved for clinical treatment, and the safety and the reliability of the medicament are verified. The composition further comprises a PD-1 antibody. The PD-1 antibody is a monoclonal or polyclonal antibody directed against human PD-1 and murine PD-1.
The oncolytic virus is prepared by inserting miRT124,124 and hGGMCSF into type I herpes simplex virus. The oncolytic virus is prepared by a method comprising the steps of:
s1, respectively amplifying sequences at two ends of HSV-1 and ICP34.5 genes;
S2, connecting hGM-CSF, CMV-GFP, 4xmiRT and ICP34.5 end sequences and plasmid pCMV-GFP by using a homologous recombination method to form a complete donor plasmid, namely pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4 xmiRT;
S3, designing sgRNA aiming at an ICP34.5 region, and connecting the sgRNA to a Lenti-CRISPR-V2 vector in an enzyme digestion connection mode, wherein the Lenti-CRISPR-ICP34.5 is marked;
S4, co-transfecting HEK293T cells with a donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells, observing the fluorescent plaque after a few hours, and picking the fluorescent plaque; HSV-1, in which hGGMCSF, CMV-GFP, 4xmiRT, 124 was inserted, was obtained and designated oHSV-GFP;
s5, using a plasmid with a Cre fragment as a template, amplifying the Cre fragment, and connecting the Cre fragment to pCCL-PGK-eGFP vector by an enzyme digestion connection method, and marking the plasmid as pCCL-PGK-Cre;
S6, transfecting pCCL-PGK-Cre plasmid prepared in the step S5 into HEK293T cells, adding the oHSV-GFP prepared in the step S4 at a dose of MOI=1.0, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells, observing no fluorescent plaque, and picking up the no fluorescent plaque; HSV-1, in which hGGMCSF and 4xmiRT124,124 were inserted, was obtained and designated oHSV.
Preferably, the step S4 specifically includes the following steps:
HEK293T cells were co-transfected with donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and with Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, after 24 hours, HSV-1 was added at a dose of MOI=1.0 and after complete lesions of the cells, the culture supernatant was recovered; ; adding the supernatant into Vero cells by 10-fold dilution, observing the fluorescent plaque after 48 hours, and picking the fluorescent plaque; the HSV-1, in which hGGMCSF, CMV-GFP, 4xmiRT124,124 were inserted, was obtained by several rounds of purification in the above-described manner, and was designated oHSV-GFP.
Preferably, the step S5 specifically includes the following steps:
Transfecting pCCL-PGK-Cre plasmid prepared in the step S5 into HEK293T cells, adding the oHSV-GFP prepared in the step S4 at the dose of MOI=1.0 after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells by 10-fold ratio dilution, observing no fluorescent plaque after 48 hours, and picking up the no fluorescent plaque; the HSV-1, intercalated with hGGMCSF and 4xmiRT124,124, was obtained after multiple rounds of purification in the manner described above and was designated oHSV.
Further, the oncolytic virus is used in an amount of 10X 10 7-100×107 PFU and the Olaparib is used in an amount of 100-1000mg/kg.
Further, the PD-1 antibody is used in an amount of 450 μg to 750 μg.
The application of the anti-tumor pharmaceutical composition in preparing anti-tumor drugs or preparations also belongs to the protection scope of the invention.
The tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer or gastric cancer.
It is another object of the present invention to provide an anti-neoplastic drug system comprising an oncolytic virus selected from the group consisting of herpes simplex virus type I and a PARP inhibitor.
The PARP inhibitor is a substance for inhibiting PARP activity, a substance for degrading PARP, a gene tool for reducing PARP level, or any combination thereof.
As an embodiment of the present invention, the PARP inhibitor is Olaparib and the molecular formula of Olaparib is C 24H23FN4O3; the structural formula of Olaparib is shown in formula I:
the preparation method of the oncolytic virus comprises the following steps:
s1, respectively amplifying sequences at two ends of HSV-1 and ICP34.5 genes;
S2, connecting hGM-CSF, CMV-GFP, 4xmiRT and ICP34.5 end sequences and plasmid pCMV-GFP by using a homologous recombination method to form a complete donor plasmid, namely pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4 xmiRT;
S3, designing sgRNA aiming at an ICP34.5 region, and connecting the sgRNA to a Lenti-CRISPR-V2 vector in an enzyme digestion connection mode, wherein the Lenti-CRISPR-ICP34.5 is marked;
S4, co-transfecting HEK293T cells with a donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, adding HSV-1 at a dose of MOI=1.0 after 24 hours, and collecting culture supernatant after complete lesions of the cells; adding the supernatant into Vero cells by 10-fold dilution, observing the fluorescent plaque after 48 hours, and picking the fluorescent plaque; the HSV-1 inserted with hGGMCSF, CMV-GFP and 4xmiRT124,124 was obtained by several rounds of purification in the above-mentioned manner, and was designated oHSV-GFP;
S5, using a plasmid with a Cre fragment as a template, amplifying the Cre fragment, and connecting the Cre fragment to pCCL-PGK-eGFP vector by an enzyme digestion connection method, and marking the plasmid as pCCL-PGK-eGFP;
S6, transfecting pCCL-PGK-Cre plasmid prepared in the step S5 into HEK293T cells, adding the oHSV-GFP prepared in the step S4 at the dose of MOI=1.0 after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells by 10-fold ratio dilution, observing no fluorescent plaque after 48 hours, and picking up the no fluorescent plaque; the HSV-1, intercalated with hGGMCSF and 4xmiRT124,124, was obtained after multiple rounds of purification in the manner described above and was designated oHSV.
The total amount of oncolytic virus is 10×10 7-100×107 PFU, and the total amount of Olaparib is 100-1000mg/kg.
Preferably, the total amount of oncolytic virus is 10X 10 7-100×107 PFU and the total amount of Olaparib is 100-1000mg/kg.
The anti-tumor drug system also comprises a PD-1 antibody, wherein the dosage of the PD-1 antibody is 450-750 mug.
Preferably, the total amount of the PD-1 antibody is 450-750 μg, wherein the PD-1 antibody is a monoclonal or polyclonal antibody directed against human PD-1 and murine PD-1.
The application of the anti-tumor drug system in preparing anti-tumor drugs also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the beneficial effects that:
1) The invention provides an oncolytic virus inserted with miRT124,124 and hGGMCSF, which can improve the safety of oncolytic virus and enhance the immune response of organism;
2) The inventor proves through a series of experiments that PARP inhibitors such as Olaparib can enhance the replication of oncolytic viruses, thereby enhancing the curative effect of oncolytic viruses;
3) The inventor finds that the combination of oncolytic virus oHSV and Olaparib can increase the PD-1 expression quantity of CD4+ T cells and CD8+ T cells when used together, so that an immune check point inhibitor, namely a PD-1 antibody is introduced, the curative effect of a treatment scheme in breast cancer is further improved, and oHSV, olaparib is used together, so that the number of lung metastases of the breast cancer of a mouse is reduced;
4) oHSV, olaparib is used in combination with PD-1 antibody to inhibit the size of breast cancer in-situ tumor of mice, and the number of lung metastasis is further reduced on the basis of the combination of the two, so that the survival period of mice is obviously prolonged;
5) oHSV, olaparib in combination with PD-1 antibodies, activate the immune system of the body, increasing the number of CD45 positive cells; up-regulating PD-1 expression levels without altering cd4+ and cd8+ T cell numbers is also responsible for enhanced efficacy in combination with immune checkpoint inhibitors.
Drawings
FIG. 1 is a graph showing a statistical analysis of the number of lung metastases of breast cancer in mice when oncolytic viruses are used in combination with Olaparib;
FIG. 2 is a graph showing analysis of the size results of tumor in situ in breast cancer in mice when oncolytic viruses Olaparib are used in combination with PD-1 antibodies;
FIG. 3 is a graph showing analysis of the number of lung metastases from breast cancer in mice when oncolytic viruses Olaparib are used in combination with PD-1 antibodies;
fig. 4: analysis of the number of mouse CD45+ cells when oncolytic viruses Olaparib were used in combination with PD-1 antibodies;
Fig. 5: analysis of the expression level of mouse CD4+ T cells PD-1 when oncolytic virus Olaparib is combined with PD-1 antibody;
Fig. 6: analysis of the expression level of mouse CD8+ T cells PD-1 when oncolytic virus Olaparib is combined with PD-1 antibody;
fig. 7: analysis of the survival results of mice using oncolytic viruses Olaparib in combination with PD-1 antibodies significantly extended.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
In the following examples, PARP inhibitors refer to compound Olaparib (Chinese name: olaparib) and related compounds capable of inhibiting PARP binding activity, catalytic activity; olaparib has the formula C 24H23FN4O3; the structural formula is shown in the following figure:
PD-1 antibodies are monoclonal or polyclonal antibodies directed against human PD-1 and murine PD-1.
Herpes simplex virus type I (HSV-1) is from the university of Denmark Ois.
The technical scheme and technical effects of the present invention are described in further detail below with reference to examples.
Example 1
Preparation of oncolytic Virus oHSV
Oncolytic virus oHSV is obtained by modifying type I herpes simplex virus (HSV-1), and the specific preparation steps are as follows:
1) Amplifying the sequences at the two ends of HSV-1 and ICP34.5 genes respectively by using a PCR method; wherein HSV-1
(GenBank: JQ 780693.1); the upstream base sequence of ICP34.5 is shown as SEQ ID NO.1 in the sequence table;
the downstream base of ICP34.5 is shown as SEQ ID NO.2 in the sequence table;
2) The hGM-CSF, CMV-GFP, 4xmiRT and ICP34.5 sequences, and plasmid pCMV-GFP were then ligated together using homologous recombination to form a complete donor plasmid, designated
PUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124; wherein the sequence of hGM-CSF is shown as SEQ ID NO.3 in the sequence table; the sequence of CMV-GFP is shown as SEQ ID NO.4 in the sequence table;
3) The sgRNA aiming at the ICP34.5 region (SEQ ID NO.1 in the sequence table) is designed (SEQ ID NO.6 in the sequence table), and is connected to a Lenti-CRISPR-V2 carrier by Esp I enzyme digestion and T4DNA ligase is marked as Lenti-CRISPR-ICP34.5;
4) HEK293T cells were co-transfected with donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and with Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, after 24 hours, HSV-1 was added at a dose of MOI=1.0 and after complete lesions of the cells, the culture supernatant was recovered; adding the supernatant into Vero cells by 10-fold dilution, observing the fluorescent plaque after 48 hours, and picking the fluorescent plaque; the hGGMCSF, CMV-GFP, inserted into the purified liquid are obtained by the above method through multiple rounds of purification,
4XmiRT124,124 HSV-1, designated oHSV-GFP;
5) Amplifying the Cre fragment by taking a plasmid with the Cre fragment as a template, and connecting the Cre fragment to a pCCL-PGK-eGFP vector by a method of BamHI and XhoI double digestion and T4DNA ligase connection, wherein the plasmid is marked as pCCL-PGK-Cre;
6) Transfecting pCCL-PGK-Cre plasmid into HEK293T cells, adding oHSV-GFP at the dose of MOI=1.0 after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells by 10-fold ratio dilution, observing no fluorescent plaque after 48 hours, and picking up the no fluorescent plaque;
The HSV-1, intercalated with hGGMCSF and 4xmiRT124,124, was obtained after multiple rounds of purification in the manner described above and was designated oHSV.
Example 2
Oncolytic Virus oHSV, olaparib in combination with test experiments
(1) Experimental materials:
BALB/C mice, mice breast cancer cell line 4T1, RPMI1640 medium, oncolytic virus oHSV, olaparib at 6 weeks of age.
(2) The experimental method comprises the following steps:
A. 4T1 cells of 5 x 10 5 were injected in situ into the right fat pad of the mice;
B. When the tumor size reaches 100mm 3, starting treatment, wherein the oncolytic virus is injected in situ every other day, and the dosage of each injection is 5×10 7 PFU, and the total injection is 5 times; olaparib is injected intraperitoneally every day, the dosage of each time is 50mg/kg, and the total injection is 10 times;
C. The second day after the fifth injection of oncolytic virus, the in situ tumor is removed;
D. on the seventh day after the removal of the in situ tumor, mice were sacrificed, lungs were removed, and the number of lung metastases was observed and recorded under a microscope;
(3) Experimental results:
the results of fig. 1 demonstrate that oHSV in combination with Olaparib is capable of inhibiting the number of breast cancer lung metastases in mice.
Example 3
Oncolytic virus oHSV, olaparib was used in combination with the PD-1 antibody in a test experiment.
(1) Experimental materials:
BALB/C mice at 6 weeks of age, mouse breast cancer cell line 4t1, rpm 1640 medium, oncolytic virus, olaparib, dnase, collagenase, hyaluronidase, anti-mouse PD-1 antibody, anti-mouse CD45 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody.
(2) The experimental method comprises the following steps:
E. 4T1 cells of 5 x 10 5 were injected in situ into the right fat pad of the mice;
F. When the tumor size reaches 100mm 3, starting treatment, wherein the oncolytic virus is injected in situ every other day, the use amount of the oncolytic virus is 5 times 10 7 PFU each time, the total injection is 5 times, and the total use amount of the oncolytic virus is 25 times 10 7 PFU; olaparib are injected intraperitoneally every day, the dosage of each injection is 50mg/kg, 10 injections are performed in total, and the total dosage of Olaparib is 10mg; the anti-mouse PD-1 antibody is respectively injected into the abdominal cavity for the second day after the third and fifth injections of oncolytic viruses and the removal of in-situ tumors, the use amount is 200 mug each time, and the total injection is 3 times;
G. The second day after the fifth injection of oncolytic virus, the in-situ tumor is removed, the in-situ tumor is digested by DNase, collagenase and hyaluronidase, then the in-situ tumor is incubated by an anti-mouse PD-1 antibody, an anti-mouse CD45 antibody, an anti-mouse CD4 antibody and an anti-mouse CD8 antibody, and finally the relevant indexes are detected by using a flow cytometer;
H. The short term experiments were processed as follows: on the seventh day after the removal of the in situ tumor, mice were sacrificed, lungs were removed, and the number of lung metastases was observed and recorded under a microscope;
The survival experiment was processed as follows: after the in situ tumor is removed, the mice are not treated at all, the survival time of the mice is only recorded, and a survival curve is drawn.
(3) Experimental results
As shown in fig. 2, the use of oncolytic viruses Olaparib in combination with PD-1 antibodies significantly inhibited the size of breast cancer in-situ tumors in mice;
As shown in fig. 3, the use of oncolytic viruses Olaparib in combination with PD-1 antibodies significantly reduced the number of breast cancer lung metastases in mice;
as shown in fig. 4, the use of oncolytic viruses Olaparib in combination with PD-1 antibodies significantly increased the number of mouse cd45+ cells;
as shown in fig. 5, the use of oncolytic viruses Olaparib in combination with PD-1 antibodies significantly increased the expression level of mouse cd4+ T cells PD-1;
As shown in fig. 6, the use of oncolytic viruses Olaparib in combination with PD-1 antibodies significantly increased the expression level of mouse cd8+ T cells PD-1;
As shown in fig. 7, the use of oncolytic viruses Olaparib in combination with PD-1 antibodies significantly prolonged survival in mice.
In summary, the inventors have found that the combined use of the oncolytic viruses oHSV and Olaparib can increase the expression level of PD-1 in cd4+ T cells and cd8+ T cells, thereby introducing an immune checkpoint inhibitor, namely a PD-1 antibody, so that the curative effect of the treatment scheme in breast cancer is further improved, and the combined use of oHSV, olaparib reduces the number of lung metastases of breast cancer in mice;
oHSV, olaparib is combined with PD-1 antibody to inhibit the size of breast cancer in-situ tumor and the number of lung metastases of mice, and remarkably prolong the survival period of mice; simultaneously activating the immune system of the organism to increase the number of CD45 positive cells; and up-regulating the expression level of PD-1 to play a role in enhancing the curative effect of the immune checkpoint inhibitor; finally prolonging the survival time of the tumor-bearing mice. Wherein the dose of oHSV is 5x107 PFU/mouse, and the dose of Olaparib is 50mg/kg; the PD-1 antibody was used at a dose of 200. Mu.g/mouse.
The foregoing describes specific embodiments of the present application. It is to be understood that the application is not limited to the particular embodiments described above, and that various changes or modifications may be made by those skilled in the art within the scope of the appended claims without affecting the spirit of the application. The embodiments of the application and the features of the embodiments may be combined with each other arbitrarily without conflict.
Sequence listing
<110> Shanghai university of transportation
<120> A method for inhibiting tumor metastasis using oncolytic virus in combination with PARP inhibitor
<130> KAG45783
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<170> SIPOSequenceListing 1.0
<210> 1
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gcccgccgcc gccatcgcgg cccccgccgc ccccggccgc ccgggcccac gggcgcggtc 120
ccaaccgcac agtcccaggt aacctccacg cccaactcgg aacccgtggt caggagcgcg 180
cccgcggccg ccccgccgcc gccccccgcc agtgggcccc cgccttcttg ttcgctgctg 240
ctgcgccagt ggctccacgt tcccgagtcc gcgtccgacg acgacgacga cgactggccg 300
gacagccccc cgcccgagcc ggcgccagag gcccggccca ccgccgccgc cccccgcccc 360
cggtccccac cgcccggcgc gggcccgggg ggcggggcta acccctccca ccccccctca 420
cgccccttcc gccttccgcc gcgcctcgcc ctccgcctgc gcgtcaccgc agagcacctg 480
gcgcgcctgc gcctgcgacg cgcgggcggg gagggggcgc cgaagccccc cgcgaccccc 540
gcgacccccg cgacccccac gcgggtgcgc ttctcgcccc acgtccgggt gcgccacctg 600
gtggtctggg cctcggccgc ccgcctggcg cgccgcggct cgtgggcccg cgagcgggcc 660
gaccgggctc ggttccggcg ccgggtggcg gaggccgagg cggtcatcgg gccgtgcctg 720
gggcccgagg cccgtgcccg ggccctggcc cgcggagccg gcccggcgaa ctcagtc 777
<210> 2
<211> 716
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
cgttacaccc gaggcggcct gggtcttccg cggagctccc gggagctccg caccaagccg 60
ctctccggag agacgatggc aggagccgcg catatatacg cttggagccg gcccgccccc 120
gaggcgggcc cgccctcgga gggcgggact ggccaatcgg cggccgccag cgcggcgggg 180
cccggccaac cagcgtccgc cgagtcgtcg gggcccggcc cactgggcgg taactcccgc 240
ccagtgggcc gggccgccca cttcccggta tggtaattaa aaacttgcag aggccttgtt 300
ccgcttcccg gtatggtaat tagaaactca ttaatgggcg gccccggccg cccttcccgc 360
ttccggcaat tcccgcggcc cttaatgggc aaccccggta ttccccgcct cccgcgccgc 420
gcgtaaccac tcccctgggg ttccgggtta tgttaattgc ttttttggcg gaacacacgg 480
cccctcgcgc attggcccgc gggtcgctca atgaacccgc attggtcccc tggggttccg 540
ggtatggtaa tgagtttctt cgggaaggcg ggaagccccg gggcaccgac gcaggccaag 600
cccctgttgc gtcggcggga ggggcatgct aatggggttc tttgggggac accgggttgg 660
tcccccaaat cgggggccgg gccgtgcatg ctaatgatat tctttggggg cgccgg 716
<210> 3
<211> 429
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 3
tggctgcaga gcctgctgct cttgggcact gtggcctgca gcatctctgc acccgcccgc 60
tcgcccagcc ccagcacgca gccctgggag catgtgaatg ccatccagga ggcccggcgt 120
ctcctgaacc tgagtagaga cactgctgct gagatgaatg aaacagtaga agtcatctca 180
gaaatgtttg acctccagga gccgacctgc ctacagaccc gcctggagct gtacaagcag 240
ggcctgcggg gcagcctcac caagctcaag ggccccttga ccatgatggc cagccactac 300
aagcagcact gccctccaac cccggaaact tcctgtgcaa cccagattat cacctttgaa 360
agtttcaaag agaacctgaa ggactttctg cttgtcatcc cctttgactg ctgggagcca 420
gtccaggag 429
<210> 4
<211> 1520
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 4
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120
atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480
acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg 540
ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct 600
agaggatccg gtactcgagg aactgaaaaa ccagaaagtt aactggtaag tttagtcttt 660
ttgtctttta tttcaggtcc cgatccggtg gtggtgcaaa tcaaagaact gctcctcagt 720
ggatgttgcc tttacttcta ggcctgtacg gaagtgttac ttctgctcta aaagctgcgg 780
aattgtaccc gcgggccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc 840
ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg 900
gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc 960
tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc 1020
gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg 1080
tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga 1140
agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg 1200
acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca 1260
tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg 1320
acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg 1380
tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg 1440
agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca 1500
tggacgagct gtacaagtaa 1520
<210> 5
<211> 1035
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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atgtccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt 60
gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat 120
acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac 180
cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg 240
cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt 300
cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc 360
cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact 420
gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat 480
ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc 540
agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg 600
aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg 660
gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc 720
cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc 780
ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt 840
cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc 900
cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt 960
gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa 1020
gatggcgatt agtaa 1035
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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cggagccggc ccggcgaact 20

Claims (5)

1. An antitumor pharmaceutical composition, characterized in that: comprises an oncolytic virus and a PARP inhibitor, wherein the oncolytic virus is selected from herpes simplex virus I, the PARP inhibitor is Olaparib, and the molecular formula of Olaparib is C 24H23FN4O3; the structural formula of Olaparib is shown in formula I:
the oncolytic virus is prepared by inserting miRT124,124 and hGGMCSF into type I herpes simplex virus;
the oncolytic virus is prepared by a method comprising the steps of:
s1, respectively amplifying two end sequences of ICP34.5 genes of HSV-1;
S2, using a homologous recombination method, connecting the hGM-CSF, CMV-GFP, 4xmiRT and ICP34.5 end sequences and the plasmid pCMV-GFP to form a complete donor plasmid, which is named pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4 xmiRT;
S3, designing sgRNA aiming at an ICP34.5 region, and connecting the sgRNA to a Lenti-CRISPR-V2 vector in an enzyme digestion connection mode, wherein the Lenti-CRISPR-ICP34.5 is marked;
S4, co-transfecting HEK293T cells with a donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, adding HSV-1 at a dose of MOI=1.0 after 24 hours, and collecting culture supernatant after complete lesions of the cells; adding the supernatant into Vero cells by 10-fold dilution, observing the fluorescent plaque after 48 hours, and picking the fluorescent plaque; after multiple rounds of purification, HSV-1, in which hGGMCSF, CMV-GFP, 4xmiRT, 124 were inserted, was obtained and designated oHSV-GFP;
s5, using a plasmid with a Cre fragment as a template, amplifying the Cre fragment, and connecting the Cre fragment to pCCL-PGK-eGFP vector by an enzyme digestion connection method, and marking the plasmid as pCCL-PGK-Cre;
S6, transfecting pCCL-PGK-Cre plasmid prepared in the step S5 into HEK293T cells, adding the oHSV-GFP prepared in the step S4 at the dose of MOI=1.0 after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells by 10-fold dilution, observing no fluorescent plaque after 48 hours, and picking up the fluorescent plaque; the HSV-1, intercalated with hGGMCSF and 4xmiRT124,124, was obtained after multiple rounds of purification in the manner described above and was designated oHSV.
2. The antitumor pharmaceutical composition according to claim 1, characterized in that: the oncolytic virus is used in an amount of 10X 10 7-100×107 PFU and Olaparib is used in an amount of 100-1000mg/kg.
3. The antitumor pharmaceutical composition according to claim 1, characterized in that: the composition further comprises a PD-1 antibody.
4. An anti-tumor pharmaceutical composition according to claim 3, characterized in that: the dosage of the PD-1 antibody is 450 mug-750 mug.
5. Use of an anti-tumor pharmaceutical composition according to any one of claims 1-4 for the preparation of an anti-tumor drug or formulation; the tumor is breast cancer.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109985240A (en) * 2017-12-29 2019-07-09 广州威溶特医药科技有限公司 PARP inhibitor and oncolytic virus are in the application for preparing anti-tumor drug
CN113164589A (en) * 2018-06-29 2021-07-23 维西欧制药公司 Compositions and methods for modulating monocyte and macrophage inflammatory phenotype and immunotherapy uses thereof
CN113164484A (en) * 2018-11-29 2021-07-23 复诺健生物科技加拿大有限公司 HSV vectors with reduced neurotoxicity

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013526852A (en) * 2010-04-06 2013-06-27 カリス ライフ サイエンシズ ルクセンブルク ホールディングス Circulating biomarkers for disease
DK3184641T3 (en) * 2013-10-28 2020-08-03 Univ Of Pittsburgh - Of The Commonwealth System Of Higher Education ONCOLYTIC HSV VECTOR
WO2017143449A1 (en) * 2016-02-24 2017-08-31 Children's Hospital Of Eastern Ontario Research Institute Inc. Smc combination therapy for the treatment of cancer
US20200102361A1 (en) * 2017-05-24 2020-04-02 Universitat Autònoma De Barcelona Viral expression construct comprising a fibroblast growth factor 21 (fgf21) coding sequence
EP3658165A4 (en) * 2017-07-26 2021-09-01 Oncorus, Inc. Oncolytic viral vectors and uses thereof
CN108635380A (en) * 2018-04-13 2018-10-12 北京唯源立康生物科技有限公司 Recombination oncolytic virus composition and its application in preparing the drug for treating tumour

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109985240A (en) * 2017-12-29 2019-07-09 广州威溶特医药科技有限公司 PARP inhibitor and oncolytic virus are in the application for preparing anti-tumor drug
CN113164589A (en) * 2018-06-29 2021-07-23 维西欧制药公司 Compositions and methods for modulating monocyte and macrophage inflammatory phenotype and immunotherapy uses thereof
CN113164484A (en) * 2018-11-29 2021-07-23 复诺健生物科技加拿大有限公司 HSV vectors with reduced neurotoxicity

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