CN114010666B - Application of oncolytic viruses, PARP inhibitors and PD-1 antibodies in the preparation of anti-tumor drugs - Google Patents

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 viruses, PARP inhibitors and PD-1 antibodies in the preparation of anti-tumor drugs

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to the application of oncolytic viruses, PARP inhibitors and PD-1 antibodies in the preparation of anti-tumor drugs.

Background technique

Oncolytic viruses with unique oncolytic mechanisms provide a new option for tumor treatment, especially for some tumors that do not respond to conventional treatments. Oncolytic viruses can replicate in tumor cells and lyse and kill tumor cells. In addition, oncolytic viruses and tumor-associated antigens released by lysis can induce immune responses to further kill tumors.

Due to the heterogeneity of tumors, the complexity of the tumor microenvironment, and the influence of the body's immune system, it is difficult to achieve good therapeutic effects in certain tumors using only oncolytic viruses. For example, the first oncolytic virus approved by the FDA, T-VEC, cannot improve the overall survival rate of melanoma patients, and only 16.3% of the patients treated have a continuous reduction in tumor volume within 6 months. This greatly affects the indications of oncolytic viruses.

The combined use of oncolytic viruses and other drugs can complement each other's advantages, thereby improving the effect of tumor treatment. For example, Amgen and Bristol-Myers Squibb have cooperated to carry out a phase I clinical trial of T-VEC combined with CTLA-4 antibody for the treatment of advanced melanoma. The results showed that the overall response rate of combined treatment was significantly higher than that of single drug treatment. In addition, it has been reported in the literature that histone deacetylase inhibitors and anti-tumor angiogenesis inhibitors can synergistically enhance the anti-tumor therapeutic effect of herpes simplex virus-like oncolytic viruses.

Chinese patent 201711478207.X discloses the combined use of an M1 oncolytic virus of the genus Alphavirus and a PARP inhibitor, which has the function of improving the killing of tumor cells at the cellular level. This patent uses oncolytic viruses and PARP inhibitors to kill tumor cells in vitro, without conducting in vivo experiments, that is, verifying them in an animal model that best reflects the actual clinical situation. At the same time, the amount, frequency, and time of use of oncolytic viruses and the amount, frequency, and time of use of PARP inhibitors were not explored. In addition, the changes in the body's immune status after the use of oncolytic viruses and PARP inhibitors were not explored, and these problems have been solved in this solution. And the patent document does not mention the technical solution for combined use with PD-1 antibodies.

Summary of the invention

One of the objects of the present invention is to provide an anti-tumor pharmaceutical composition, which comprises an oncolytic virus and a PARP inhibitor, wherein the oncolytic virus is selected from herpes simplex virus type I.

As one embodiment of the present invention, the PARP inhibitor is Olaparib, and the molecular formula of Olaparib is C ₂₄ H ₂₃ FN ₄ O ₃ ; the structural formula of Olaparib is shown in Formula 1:

The PARP inhibitor used in the present invention is Olaparib, which can inhibit the activity of PARP1 and PARP2 at the same time, and Olaparib is a drug approved for clinical treatment, and its safety and reliability have been verified. The composition also includes PD-1 antibody. The PD-1 antibody is a monoclonal or polyclonal antibody against human PD-1 and mouse PD-1.

The oncolytic virus is prepared by inserting miRT124 and hGMCSF into herpes simplex virus type I. The oncolytic virus is prepared by a method comprising the following steps:

S1. Amplify the sequences at both ends of HSV-1 and ICP34.5 genes respectively;

S2. Using homologous recombination, the sequences at both ends of hGM-CSF, CMV-GFP, 4xmiRT124 and ICP34.5, as well as the plasmid pCMV-GFP, were connected to form a complete donor plasmid, which was recorded as pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124;

S3. Design sgRNA targeting the ICP34.5 region and connect it to the Lenti-CRISPR-V2 vector by enzyme digestion and ligation, recorded as Lenti-CRISPR-ICP34.5;

S4. Co-transfect HEK293T cells with the donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and the Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5. After the cells are completely diseased, collect the culture supernatant; add the supernatant to Vero cells, observe the fluorescent plaques after several hours, and pick the fluorescent plaques; obtain HSV-1 inserted with hGMCSF, CMV-GFP, and 4xmiRT124, which is recorded as oHSV-GFP;

S5. Using the plasmid with the Cre fragment as a template, amplify the Cre fragment and connect it to the pCCL-PGK-eGFP vector by restriction digestion and ligation, which is recorded as pCCL-PGK-Cre;

S6. The pCCL-PGK-Cre plasmid prepared in step S5 was transfected into HEK293T cells, and the oHSV-GFP prepared in step S4 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells, and the non-fluorescent plaques were observed and picked; HSV-1 inserted with hGMCSF and 4xmiRT124 was obtained, which was recorded as oHSV.

Preferably, step S4 specifically includes the following steps:

HEK293T cells were co-transfected with the donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and the Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5. After 24 hours, HSV-1 was added at a dose of MOI = 1.0, and the culture supernatant was collected after the cells were completely diseased; the supernatant was added to Vero cells at a 10-fold dilution method. After 48 hours, the fluorescent plaques were observed and picked; after multiple rounds of purification using the above method, HSV-1 inserted with hGMCSF, CMV-GFP, and 4xmiRT124 was obtained, which was recorded as oHSV-GFP.

Preferably, step S5 specifically includes the following steps:

The pCCL-PGK-Cre plasmid prepared in step S5 was transfected into HEK293T cells. After 24 hours, the oHSV-GFP prepared in step S4 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, non-fluorescent plaques were observed and picked; after multiple rounds of purification using the above method, HSV-1 inserted with hGMCSF and 4xmiRT124 was obtained, which was recorded as oHSV.

Furthermore, the dosage of the oncolytic virus is 10×10 ⁷ -100×10 ⁷ PFU, and the dosage of Olaparib is 100-1000 mg/kg.

Furthermore, the dosage of the PD-1 antibody is 450 μg-750 μg.

The use of the anti-tumor drug composition of the present invention in the preparation of anti-tumor drugs or preparations also falls within the protection scope of the present invention.

The tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer.

Another object of the present invention is to provide an anti-tumor drug system, comprising an oncolytic virus and a PARP inhibitor, wherein the oncolytic virus is selected from herpes simplex virus type I.

The PARP inhibitor is a substance that inhibits PARP activity, or a substance that degrades PARP, or a genetic tool that reduces the level of PARP, or any combination thereof.

As one embodiment of the present invention, the PARP inhibitor is Olaparib, and the molecular formula of Olaparib is C ₂₄ H ₂₃ FN ₄ O ₃ ; the structural formula of Olaparib is shown in Formula 1:

The method for preparing the oncolytic virus comprises the following steps:

S1. Amplify the sequences at both ends of HSV-1 and ICP34.5 genes respectively;

S2. Using homologous recombination, the sequences at both ends of hGM-CSF, CMV-GFP, 4xmiRT124 and ICP34.5, as well as the plasmid pCMV-GFP, were connected to form a complete donor plasmid, which was recorded as pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124;

S3. Design sgRNA targeting the ICP34.5 region and connect it to the Lenti-CRISPR-V2 vector by enzyme digestion and ligation, recorded as Lenti-CRISPR-ICP34.5;

S4. HEK293T cells were co-transfected with the donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and the Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5. After 24 hours, HSV-1 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, the fluorescent plaques were observed and picked; after multiple rounds of purification using the above method, HSV-1 inserted with hGMCSF, CMV-GFP, and 4xmiRT124 was obtained, which was recorded as oHSV-GFP;

S5. Using the plasmid with the Cre fragment as a template, amplify the Cre fragment and connect it to the pCCL-PGK-eGFP vector by restriction enzyme ligation, which is recorded as pCCL-PGK-eGFP;

S6. The pCCL-PGK-Cre plasmid prepared in step S5 was transfected into HEK293T cells. After 24 hours, the oHSV-GFP prepared in step S4 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, the non-fluorescent plaques were observed and picked; after multiple rounds of purification using the above method, HSV-1 inserted with hGMCSF and 4xmiRT124 was obtained, which was recorded as oHSV.

The total dosage of the oncolytic virus is 10×10 ⁷ -100×10 ⁷ PFU, and the total dosage of Olaparib is 100-1000 mg/kg.

Preferably, the total dosage of the oncolytic virus is 10×10 ⁷ -100×10 ⁷ PFU, and the total dosage of Olaparib is 100-1000 mg/kg.

The anti-tumor drug system also includes PD-1 antibody, and the dosage of the PD-1 antibody is 450-750 μg.

Preferably, the total dosage of the PD-1 antibody is 450-750 μg, wherein the PD-1 antibody is a monoclonal or polyclonal antibody against human PD-1 and mouse PD-1.

The application of the anti-tumor medication system of the present invention in the preparation of anti-tumor drugs also falls within the protection scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects:

1) The present invention provides an oncolytic virus into which miRT124 and hGMCSF are inserted, so that the safety of the oncolytic virus is improved while enhancing the body's immune response;

2) The inventors have demonstrated through a series of experiments that PARP inhibitors, such as Olaparib, can enhance the replication of oncolytic viruses, thereby enhancing the efficacy of oncolytic viruses;

3) The inventors used oncolytic virus oHSV and Olaparib together and found that the combination of the two can increase the expression of PD-1 in CD4+T cells and CD8+T cells, thereby introducing immune checkpoint inhibitors, namely PD-1 antibodies, to further improve the efficacy of the treatment in breast cancer. The combination of oHSV and Olaparib reduced the number of lung metastases in breast cancer mice;

4) oHSV, Olaparib and PD-1 antibody were used in combination to inhibit the size of in situ breast cancer tumors in mice, and further reduce the number of lung metastases based on the combination of the first two, thereby significantly prolonging the survival of mice;

5) The combination of oHSV, Olaparib and PD-1 antibodies activates the body's immune system and increases the number of CD45-positive cells. Without changing the number of CD4+ and CD8+ T cells, it upregulates the expression level of PD-1, which is why the combination with immune checkpoint inhibitors enhances the efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a statistical analysis of the number of lung metastases of breast cancer in mice when oncolytic virus and Olaparib are used in combination;

FIG2 is an analysis of the size of in situ breast cancer tumors in mice when oncolytic virus, Olaparib and PD-1 antibody are used in combination;

FIG3 is an analysis of the number of lung metastases of breast cancer in mice when oncolytic virus, Olaparib and PD-1 antibody are used in combination;

Figure 4: Analysis of the number of CD45+ cells in mice when oncolytic virus, Olaparib and PD-1 antibody were used in combination;

Figure 5: Analysis of the expression of PD-1 in mouse CD4+T cells when oncolytic virus, Olaparib and PD-1 antibody are combined;

Figure 6: Analysis of the expression of PD-1 in mouse CD8+T cells when oncolytic virus, Olaparib and PD-1 antibody are combined;

Figure 7: Analysis of the results of the combined use of oncolytic virus, Olaparib and PD-1 antibody significantly prolonging the survival of mice.

Detailed ways

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

In the following examples, PARP inhibitors refer to the compound Olaparib (Chinese name: Olaparib) and related compounds that can inhibit the binding activity and catalytic activity of PARP; the molecular formula of Olaparib is C ₂₄ H ₂₃ FN ₄ O ₃ ; the structural formula is shown in the figure below:

PD-1 antibody refers to monoclonal or polyclonal antibodies against human PD-1 and mouse PD-1.

Herpes simplex virus type 1 (HSV-1) was obtained from Aarhus University, Denmark.

The technical scheme and technical effects of the present invention are further described in detail below in conjunction with embodiments.

Example 1

Preparation of oncolytic virus oHSV

The oncolytic virus oHSV was obtained by modifying herpes simplex virus type I (HSV-1). The specific preparation steps are as follows:

1) The two end sequences of HSV-1 and ICP34.5 genes were amplified by PCR.

(GenBank: JQ780693.1); the upstream base sequence of ICP34.5 is shown in the sequence list as SEQ ID NO.1;

The downstream base of ICP34.5 is shown in SEQ ID NO.2 in the sequence list;

2) Then, using homologous recombination, the sequences at both ends of hGM-CSF, CMV-GFP, 4xmiRT124 and ICP34.5, as well as the plasmid pCMV-GFP, were connected to form a complete donor plasmid, which was recorded as

pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124; wherein the sequence of hGM-CSF is as SEQ ID NO.3 in the sequence list; the sequence of CMV-GFP is as SEQ ID NO.4 in the sequence list;

3) Designing an sgRNA (such as SEQ ID NO.6 in the sequence list) targeting the ICP34.5 region (such as SEQ ID NO.1 in the sequence list), and ligating it to the Lenti-CRISPR-V2 vector by Esp3I digestion and T4 DNA ligase, recorded as Lenti-CRISPR-ICP34.5;

4) HEK293T cells were co-transfected with the donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and the Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5. After 24 hours, HSV-1 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, the fluorescent plaques were observed and picked; after multiple rounds of purification using the above method, the hGMCSF, CMV-GFP,

HSV-1 of 4xmiRT124, denoted as oHSV-GFP;

5) Using the plasmid with the Cre fragment as a template, amplify the Cre fragment, and connect it to the pCCL-PGK-eGFP vector by double restriction digestion with BamHI and XhoI and ligation with T4 DNA ligase, which is recorded as pCCL-PGK-Cre;

6) The pCCL-PGK-Cre plasmid was transfected into HEK293T cells. After 24 hours, oHSV-GFP was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, non-fluorescent plaques were observed and picked;

After multiple rounds of purification using the above method, HSV-1 inserted with hGMCSF and 4xmiRT124 was obtained, which was recorded as oHSV.

Example 2

Oncolytic virus oHSV and Olaparib combined use test experiment

(1) Experimental materials:

6-week-old BALB/C mice, mouse breast cancer cell line 4T1, RPMI1640 medium, oncolytic virus oHSV, Olaparib.

(2) Experimental methods:

A. 5×10 ⁵ 4T1 cells were injected orthotopically into the right fat pad of mice;

B. When the tumor size reaches 100 mm ³ , treatment begins, with oncolytic virus injected orally every other day, with a dosage of 5×10 ⁷ PFU each time, for a total of 5 injections; Olaparib is injected intraperitoneally daily, with a dosage of 50 mg/kg each time, for a total of 10 injections;

C. On the second day after the fifth injection of oncolytic virus, the in situ tumor was removed;

D. On the seventh day after the removal of the orthotopic tumor, the mice were killed, the lungs were removed, and the number of lung metastases was observed and recorded under a microscope;

(3) Experimental results:

The results in Figure 1 show that the combination of oHSV and Olaparib can inhibit the number of lung metastases in breast cancer in mice.

Example 3

Experiment to test the combined use of oncolytic virus oHSV, Olaparib and PD-1 antibody.

(1) Experimental materials:

6-week-old BALB/C mice, mouse breast cancer cell line 4T1, RPMI1640 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) Experimental methods:

E. 5×10 ⁵ 4T1 cells were injected orthotopically into the right fat pad of mice;

F. When the tumor size reached ¹⁰⁰ mm3, treatment was started, in which oncolytic virus was injected in situ every other day, with a dosage of 5× ¹⁰⁷ PFU each time, for a total of 5 injections, and a total dosage of 25× ¹⁰⁷ PFU of oncolytic virus; Olaparib was intraperitoneally injected daily, with a dosage of 50 mg/kg each time, for a total of 10 injections, and a total dosage of 10 mg of Olaparib; anti-mouse PD-1 antibody was intraperitoneally injected at the third and fifth injections of oncolytic virus and on the second day after the removal of the in situ tumor, with a dosage of 200 μg each time, for a total of 3 injections;

G. On the second day after the fifth injection of oncolytic virus, the in situ tumor was removed and digested with DNAse, collagenase, and hyaluronidase, and then incubated with anti-mouse PD-1 antibody, anti-mouse CD45 antibody, anti-mouse CD4 antibody, and anti-mouse CD8 antibody, and finally the relevant indicators were detected by flow cytometry;

H. The treatment of short-term experiments was as follows: on the seventh day after removal of the orthotopic tumor, the mice were sacrificed, the lungs were removed, and the number of lung metastases was observed and recorded under a microscope;

The survival experiment was conducted as follows: after the orthotopic tumor was removed, no treatment was given to the mice, only the survival time of the mice was recorded and a survival curve was drawn.

(3) Experimental results

As shown in Figure 2, the combination of oncolytic virus, Olaparib and PD-1 antibody significantly inhibited the size of in situ breast cancer tumors in mice;

As shown in Figure 3, the combination of oncolytic virus, Olaparib and PD-1 antibody significantly reduced the number of lung metastases of breast cancer in mice;

As shown in Figure 4, the combination of oncolytic virus, Olaparib and PD-1 antibody significantly increased the number of CD45+ cells in mice;

As shown in Figure 5, the combination of oncolytic virus, Olaparib and PD-1 antibody significantly increased the expression of PD-1 in mouse CD4+T cells;

As shown in Figure 6, the combination of oncolytic virus, Olaparib and PD-1 antibody significantly increased the expression of PD-1 in mouse CD8+T cells;

As shown in Figure 7, the combined use of oncolytic virus, Olaparib and PD-1 antibody significantly prolonged the survival of mice.

In summary, the inventors used oncolytic virus oHSV and Olaparib in combination and found that the combination of the two can increase the PD-1 expression of CD4+T cells and CD8+T cells, thereby introducing immune checkpoint inhibitors, namely PD-1 antibodies, to further improve the efficacy of the treatment in breast cancer. The combination of oHSV and Olaparib reduced the number of lung metastases in breast cancer in mice.

The combination of oHSV, Olaparib and PD-1 antibody inhibited the size of in situ tumors and the number of lung metastases in mice with breast cancer, significantly prolonged the survival of mice, activated the body's immune system, increased the number of CD45-positive cells, and upregulated the expression level of PD-1, which enhanced the efficacy of immune checkpoint inhibitors, and ultimately prolonged the survival of tumor-bearing mice. The dosage of oHSV was 5x107PFU/mouse, the dosage of Olaparib was 50mg/kg, and the dosage of PD-1 antibody was 200μg/mouse.

The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Sequence Listing

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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

<400> 6

cggagccggc ccggcgaact 20

Claims (5)

1. An anti-tumor pharmaceutical composition, characterized in that: it comprises an oncolytic virus and a PARP inhibitor, wherein the oncolytic virus is selected from herpes simplex virus type I, the PARP inhibitor is Olaparib, and the molecular formula of Olaparib is C ₂₄ H ₂₃ FN ₄ O ₃ ; the structural formula of Olaparib is shown in Formula 1: The oncolytic virus is prepared by inserting miRT124 and hGMCSF into herpes simplex virus type I; The oncolytic virus is prepared by a method comprising the following steps: S1. Amplify the sequences at both ends of the ICP34.5 gene of HSV-1; S2. Using homologous recombination, the sequences at both ends of hGM-CSF, CMV-GFP, 4xmiRT124 and ICP34.5, as well as the plasmid pCMV-GFP, were connected to form a complete donor plasmid, which was named pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124; S3. Design sgRNA targeting the ICP34.5 region and connect it to the Lenti-CRISPR-V2 vector by enzyme digestion and ligation, recorded as Lenti-CRISPR-ICP34.5; S4. HEK293T cells were co-transfected with the donor plasmid pUL34.5-hGM-CSF-lox2272-CMV-eGFP-lox2272-4xmiRT124 and the Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5. After 24 hours, HSV-1 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, the fluorescent plaques were observed and picked; after multiple rounds of purification, HSV-1 inserted with hGMCSF, CMV-GFP, and 4xmiRT124 was obtained, which was recorded as oHSV-GFP; S5. Using the plasmid with the Cre fragment as a template, amplify the Cre fragment and connect it to the pCCL-PGK-eGFP vector by restriction digestion and ligation, which is recorded as pCCL-PGK-Cre; S6. The pCCL-PGK-Cre plasmid prepared in step S5 was transfected into HEK293T cells. After 24 hours, the oHSV-GFP prepared in step S4 was added at a dose of MOI = 1.0. After the cells were completely diseased, the culture supernatant was collected; the supernatant was added to Vero cells by a 10-fold dilution method. After 48 hours, the non-fluorescent plaques were observed and picked; after multiple rounds of purification using the above method, HSV-1 inserted with hGMCSF and 4xmiRT124 was obtained, which was recorded as oHSV. 2. The anti-tumor pharmaceutical composition according to claim 1, characterized in that the dosage of the oncolytic virus is 10×10 ⁷ -100×10 ⁷ PFU, and the dosage of Olaparib is 100-1000 mg/kg. 3. The anti-tumor pharmaceutical composition according to claim 1, characterized in that: the composition also includes PD-1 antibody. 4. The anti-tumor pharmaceutical composition according to claim 3, characterized in that the dosage of the PD-1 antibody is 450µg-750µg. 5. Use of the anti-tumor drug composition according to any one of claims 1 to 4 in the preparation of anti-tumor drugs or preparations; the tumor is breast cancer.