CN114010666A - 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 PDFInfo
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Abstract
The invention provides an application of an oncolytic virus, a PARP inhibitor and a PD-1 antibody in preparing an anti-tumor medicament, wherein miRT124 and hGMCSF are inserted into a type I herpes simplex virus to obtain an oncolytic virus oHSV, so that the safety of the oncolytic virus is improved, and the immune response of an organism is enhanced; through the combined use of the oncolytic virus oHSV and the Olaparib, the PD-1 expression quantity of the CD4+ T cell and the CD8+ T cell can be increased by combining the oncolytic virus oHSV and the Olaparib, 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 size of an in-situ tumor of the breast cancer of a mouse is further inhibited by combining the oHSV, the Olaparib and the PD-1 antibody, on the basis of the combined use of the oHSV, the Olaparib and the PD-1 antibody, the number of lung metastasis foci is further reduced, and the survival period of the mouse is remarkably prolonged.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of oncolytic virus, PARP (para-amyloid peptide) inhibitor and PD-1 antibody in preparation of antitumor drugs
Background
Oncolytic viruses with unique oncolytic mechanisms offer a new option for the treatment of tumors, especially for some tumors that do not respond to conventional treatment, wherein the replication of the oncolytic virus in tumor cells can kill the tumor cells by lysis, and furthermore, the oncolytic virus and tumor-associated antigens released by lysis can induce immune response to further kill the tumor.
Due to tumor heterogeneity and complexity of tumor microenvironment and the influence of the immune system of the body, it is difficult to achieve good therapeutic effect in some tumors by using oncolytic virus alone, for example, the first FDA approved oncolytic virus T-VEC cannot improve the overall survival rate of melanoma patients, and only 16.3% of patients receiving treatment have tumor volume that continuously shrinks within 6 months. This greatly affects the indications for oncolytic viruses.
The oncolytic virus can be combined with other medicines to realize advantage complementation, thereby improving the tumor treatment effect. For example, Amgen company and Michelia nobilis cooperate to carry out clinical results of the combination treatment of the T-VEC and the CTLA-4 antibody on the advanced melanoma stage I, which shows that the overall response rate of the combination treatment is remarkably higher than that of single drug treatment. In addition, the literature reports that histone deacetylase inhibitors, anti-tumor angiogenesis inhibitors and the like can synergistically enhance the anti-tumor treatment effect of herpes simplex virus oncolytic viruses.
Chinese patent 201711478207.X discloses the combined use of M1 oncolytic virus of alphavirus and PARP inhibitor, and the function of killing tumor cells is improved at a cellular level. The patent discloses that oncolytic virus and PARP inhibitors kill tumor cells in vitro, in-vivo experiments are not carried out, namely, the tumor cells are verified in an animal model which can best reflect the actual clinical situation, the using time of the using number and the using time of the using number of the oncolytic virus and the using time of the using number and the using time of the PARP inhibitors are not researched, in addition, the change of the body immunity situation is not researched after the oncolytic virus and the PARP inhibitors are used, and the problems are solved in the scheme. And the patent document does not mention the technical scheme of the combined use with the PD-1 antibody.
Disclosure of Invention
An object of the present invention is to provide an anti-tumor pharmaceutical composition, which comprises an oncolytic virus selected from the group consisting of herpes simplex virus type I and a PARP inhibitor.
As one embodiment of the present invention, the PARP inhibitor is Olaparib, which has the formula C24H23FN4O3(ii) a The structural formula of Olaparib is shown as formula I:
the PARP inhibitor adopted by the invention is Olaparib which can simultaneously inhibit the activities of PARP1 and PARP2, and the Olaparib is an approved medicament for clinical treatment, and the safety and reliability of the invention 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 and hGMCSF into type I herpes simplex virus. The oncolytic virus is prepared by a method comprising the following steps:
s1, amplifying sequences at two ends of HSV-1 and ICP34.5 genes respectively;
s2, connecting sequences at two ends of hGM-CSF, CMV-GFP, 4xmIRT124 and ICP34.5 and plasmid pCMV-GFP by using a homologous recombination method to form a complete donor plasmid which is marked as pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT 124;
s3, designing sgRNA aiming at an ICP34.5 region, connecting the sgRNA to a Lenti-CRISPR-V2 vector in an enzyme digestion connection mode, and marking as Lenti-CRISPR-ICP 34.5;
s4, co-transfecting HEK293T cells with donor plasmid pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT124 and 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 fluorescent plaques after several hours, and picking the fluorescent plaques; obtaining HSV-1 inserted with hGMCSF, CMV-GFP and 4xmIRT124, and marking as oHSV-GFP;
s5, amplifying a Cre fragment by using a plasmid with the Cre fragment as a template, and connecting the Cre fragment to a pCCL-PGK-eGFP vector by using an enzyme digestion connection method, wherein the pCCL-PGK-eGFP vector is marked as pCCL-PGK-eGFP;
s6, transfecting the pCCL-PGK-Cre plasmid prepared in the step S5 to a HEK293T cell, adding the oHSV-GFP prepared in the step S4 in a dosage of 1.0 MOI, and collecting a culture supernatant after the cell is completely diseased; adding the supernatant into Vero cells, observing non-fluorescent plaques, and selecting the non-fluorescent plaques; HSV-1 with hGMCSF and 4xmIRT124 inserted was obtained and designated oHSV.
Preferably, step S4 specifically includes the following steps:
co-transfecting HEK293T cells with a donor plasmid pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT124 and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, adding HSV-1 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 a 10-fold dilution method, observing fluorescent plaques after 48 hours, and selecting the fluorescent plaques; the HSV-1 inserted with hGMCSF, CMV-GFP and 4xmIRT124 is obtained by multiple rounds of purification by the method and is marked as oHSV-GFP.
Preferably, step S5 specifically includes the following steps:
transfecting HEK293T cells with the pCCL-PGK-Cre plasmid prepared in the step S5, adding the oHSV-GFP prepared in the step S4 in a dosage of MOI (1.0) after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells, observing no fluorescent plaque after 48 hours, and selecting the non-fluorescent plaque; after multiple rounds of purification by the method, HSV-1 inserted with hGMCSF and 4xmIRT124 is obtained and is marked as oHSV.
Further, the dose of the oncolytic virus is 10 × 107-100×107The dosage of PFU and the Olaparib is 100-1000 mg/kg.
Further, the dosage of the PD-1 antibody is 450 to 750 mu g.
The application of the anti-tumor medicine composition in preparing anti-tumor medicines or preparations also belongs to the protection scope of the invention.
The tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostatic cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer or gastric cancer.
It is another object of the present invention to provide an anti-tumor 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 one embodiment of the present invention, the PARP inhibitor is Olaparib, which has the formula C24H23FN4O3(ii) a The structural formula of Olaparib is shown as formula I:
the preparation method of the oncolytic virus comprises the following steps:
s1, amplifying sequences at two ends of HSV-1 and ICP34.5 genes respectively;
s2, connecting sequences at two ends of hGM-CSF, CMV-GFP, 4xmIRT124 and ICP34.5 and plasmid pCMV-GFP by using a homologous recombination method to form a complete donor plasmid which is marked as pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT 124;
s3, designing sgRNA aiming at an ICP34.5 region, connecting the sgRNA to a Lenti-CRISPR-V2 vector in an enzyme digestion connection mode, and marking as Lenti-CRISPR-ICP 34.5;
s4, co-transfecting a donor plasmid pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT124 and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5 into HEK293T cells, adding HSV-1 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 a 10-fold dilution method, observing fluorescent plaques after 48 hours, and selecting the fluorescent plaques; obtaining HSV-1 inserted with hGMCSF, CMV-GFP and 4xmIRT124 through multi-round purification by the method, and marking as oHSV-GFP;
s5, amplifying a Cre fragment by using a plasmid with the Cre fragment as a template, and connecting the Cre fragment to a pCCL-PGK-eGFP vector by using an enzyme digestion connection method, wherein the pCCL-PGK-eGFP vector is marked as pCCL-PGK-eGFP;
s6, transfecting the pCCL-PGK-Cre plasmid prepared in the step S5 into HEK293T cells, adding the oHSV-GFP prepared in the step S4 in a dosage of MOI (equal to 1.0) after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells, observing no fluorescent plaque after 48 hours, and selecting the non-fluorescent plaque; after multiple rounds of purification by the method, HSV-1 inserted with hGMCSF and 4xmIRT124 is obtained and is marked as oHSV.
The total dose of the oncolytic virus is 10 multiplied by 107-100×107The total dosage of PFU and the Olaparib is 100-1000 mg/kg.
Preferably, the total dose of the oncolytic virus is 10 x107-100×107The total dosage of PFU and the Olaparib is 100-1000 mg/kg.
The anti-tumor drug system also comprises a PD-1 antibody, and the dosage of the PD-1 antibody is 450-750 mu g.
Preferably, the total amount of the PD-1 antibody is 450. mu.g and 750. mu.g, 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 medicine system in the preparation of anti-tumor medicines 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 and hGMCSF, which improves the safety of the oncolytic virus and enhances the immune response of an organism;
2) the inventor proves that PARP inhibitors such as Olaparib can enhance the replication of the oncolytic virus through a series of experiments, thereby enhancing the curative effect of the oncolytic virus;
3) the inventor finds that the expression quantity of PD-1 of CD4+ T cells and CD8+ T cells can be increased when the oncolytic virus oHSV and Olaparib are used in combination, 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, and the combination of oHSV and Olaparib reduces the number of lung metastases of the breast cancer of a mouse;
4) the oHSV, the Olaparib and the PD-1 antibody are used in a combined manner, the size of the breast cancer in situ of the mouse is inhibited, and on the basis of the combined use of the oHSV and the Olaparib, the number of lung metastasis foci is further reduced, so that the survival period of the mouse is remarkably prolonged;
5) the OHSV, the Olaparib and the PD-1 antibody are used together to activate the immune system of the body, so that the number of CD45 positive cells is increased; the up-regulation of PD-1 expression levels without altering the number of CD4+ and CD8+ T cells is also responsible for the enhanced therapeutic efficacy of the combination with immune checkpoint inhibitors.
Drawings
FIG. 1 is a graph showing the statistical analysis of the number of lung metastases of breast cancer in mice when oncolytic virus and Olaparib are used in combination;
FIG. 2 is a graph showing the analysis of the size results of mouse breast cancer in situ tumors when oncolytic virus, Olaparib and PD-1 antibody are used in combination;
FIG. 3 is a graph showing the quantitative results of lung metastases of mouse breast cancer when oncolytic virus, Olaparib and PD-1 antibody are used in combination;
FIG. 4: graphs for the number of mouse CD45+ cells when oncolytic virus, Olaparib, and PD-1 antibodies were used in combination;
FIG. 5: results of analysis of expression levels of mouse CD4+ T cell PD-1 when oncolytic virus, Olaparib and PD-1 antibody were combined;
FIG. 6: results of analysis of expression levels of mouse CD8+ T cell PD-1 when oncolytic virus, Olaparib and PD-1 antibody were combined;
FIG. 7: results analysis of the significantly prolonged survival of mice using oncolytic virus, Olaparib in combination with PD-1 antibody.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
In the following examples, PARP inhibitors refer to the compound Olaparib (Chinese name: Olaparib) and related compounds capable of inhibiting PARP binding activity, catalytic activity; the molecular formula of Olaparib is C24H23FN4O3(ii) a The structural formula is as follows:
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 Ourhees, Denmark.
The technical solution and the technical effect of the present invention will be further described in detail with reference to the following examples.
Example 1
Preparation of oncolytic Virus oHSV
The oncolytic virus oHSV is obtained by modifying a herpes simplex virus I (HSV-1), and the preparation method comprises the following specific steps:
1) amplifying sequences at two ends of HSV-1 and ICP34.5 genes respectively by using a PCR method; among them, 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) then, the sequences at the two ends of hGM-CSF, CMV-GFP, 4xmIRT124 and ICP34.5 and the plasmid pCMV-GFP are connected by using a homologous recombination method to form a complete donor plasmid which is marked as pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT 124; wherein the sequence of hGM-CSF is shown as SEQ ID NO.3 in the sequence table; the sequence of the CMV-GFP is shown as SEQ ID NO.4 in the sequence table;
3) designing sgRNA (shown as SEQ ID NO.6 in a sequence table) aiming at an ICP34.5 region (shown as SEQ ID NO.5 in the sequence table), carrying out enzyme digestion through Esp3I, and connecting T4DNA ligase to a Lenti-CRISPR-V2 vector, wherein the vector is marked as Lenti-CRISPR-ICP 34.5;
4) co-transfecting HEK293T cells with a donor plasmid pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT124 and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, adding HSV-1 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 a 10-fold dilution method, observing fluorescent plaques after 48 hours, and selecting the fluorescent plaques; obtaining HSV-1 inserted with hGMCSF, CMV-GFP and 4xmIRT124 through multi-round purification by the method, and marking as oHSV-GFP;
5) using a plasmid with a Cre fragment as a template, amplifying the Cre fragment, and connecting the Cre fragment to a pCCL-PGK-eGFP vector by a BamHI and XhoI double enzyme digestion and T4DNA ligase connection method, and marking the pCCL-PGK-eGFP vector as pCCL-PGK-eGFP;
6) transfecting HEK293T cells with pCCL-PGK-Cre plasmids, adding oHSV-GFP in a dose of MOI (equal to 1.0) after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells, observing no fluorescent plaque after 48 hours, and selecting the non-fluorescent plaque; after multiple rounds of purification by the method, HSV-1 inserted with hGMCSF and 4xmIRT124 is obtained and is marked as oHSV.
Example 2
Test experiment for combined use of oncolytic virus oHSV and Olaparib
(1) Experimental materials:
6-week-old BALB/C mice, a mouse breast cancer cell line 4T1, RPMI1640 medium, oncolytic virus oHSV, Olaparib.
(2) The experimental method comprises the following steps:
A. will be 5X105The 4T1 cells were injected in situ into the right fat pad of the mouse;
B. when the tumor size reaches 100mm3At the time of treatment, therapy was initiated, with oncolytic virus injected every other day in situ at a dose of 5X107PFU, 5 total injections; the Olaparib is injected intraperitoneally every day, the using amount of each time is 50mg/kg, and the total injection is 10 times;
C. on the second day after the fifth oncolytic virus injection, the in situ tumor is removed;
D. on the seventh day after the removal of the orthotopic tumor, the mice were sacrificed, the lungs were taken out, and the number of lung metastases was observed and recorded under a microscope;
(3) the experimental results are as follows:
the results in FIG. 1 show that OHSV combined with Olaparib can inhibit the number of metastasis in the lung of breast cancer in mice.
Example 3
Oncolytic viruses oHSV, Olaparib were used in combination with PD-1 antibodies in the test experiment.
(1) Experimental materials:
6-week-old BALB/C mice, a mouse breast cancer cell line 4T1, an RPMI1640 medium, an oncolytic virus, Olaparib, DNase, collagenase, hyaluronidase, an anti-mouse PD-1 antibody, an anti-mouse CD45 antibody, an anti-mouse CD4 antibody, and an anti-mouse CD8 antibody.
(2) The experimental method comprises the following steps:
E. will be 5X105The 4T1 cells were injected in situ into the right fat pad of the mouse;
F. when the tumor size reaches 100mm3At the time of treatment, therapy was initiated, with oncolytic virus injected every other day in situ at a dose of 5X107PFU, 5 injections in total, total oncolytic virus dose 25X 107PFU,; injecting the Olaparib intraperitoneally every day, wherein the dosage of each injection is 50mg/kg, the total injection is 10 times, and the total dosage of the Olaparib is 10 mg; the anti-mouse PD-1 antibody is injected into the abdominal cavity of the second day after the third injection of the oncolytic virus and the removal of the in-situ tumor for the fifth time respectively, the using amount is 200 mug each time, and the total injection is 3 times;
G. on the second day after the fifth injection of the oncolytic virus, removing the in situ tumor, digesting the in situ tumor by using DNase, collagenase and hyaluronidase, then incubating by using anti-mouse PD-1 antibody, anti-mouse CD45 antibody, anti-mouse CD4 antibody and anti-mouse CD8 antibody, and finally detecting related indexes by using a flow cytometer;
H. the short-term experiment was conducted as follows: on the seventh day after the removal of the orthotopic tumor, the mice were sacrificed, the lungs were taken out, and the number of lung metastases was observed and recorded under a microscope;
survival experiments were conducted as follows: after removal of the orthotopic tumor, the mice were not treated at all, and only the survival time of the mice was recorded and a survival curve was plotted.
(3) Results of the experiment
As shown in fig. 2, the oncolytic virus, Olaparib, and PD-1 antibody used in combination significantly inhibited the size of the mouse breast cancer orthotopic tumor;
as shown in fig. 3, the use of oncolytic virus, Olaparib in combination with PD-1 antibody significantly reduced the number of lung metastases of breast cancer in mice;
as shown in fig. 4, the use of oncolytic virus, Olaparib in combination with PD-1 antibody significantly increased the number of mouse CD45+ cells;
as shown in FIG. 5, the use of oncolytic virus, Olaparib in combination with PD-1 antibody significantly increased the expression level of mouse CD4+ T cell PD-1;
as shown in FIG. 6, the use of oncolytic virus, Olaparib in combination with PD-1 antibody significantly increased the expression level of PD-1 from mouse CD8+ T cells;
as shown in fig. 7, the use of oncolytic virus, Olaparib in combination with PD-1 antibody significantly prolonged the survival of mice.
In conclusion, the inventor finds that the oncolytic virus oHSV and Olaparib can increase the expression level of PD-1 of CD4+ T cells and CD8+ T cells when used together, 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, and the oHSV and Olaparib are used together to reduce the number of lung metastasis foci of the breast cancer in mice;
the oHSV, the Olaparib and the PD-1 antibody are used together, the size of the breast cancer in situ of the mouse and the number of lung metastasis foci are inhibited, and the life cycle of the mouse is obviously prolonged; simultaneously activating the immune system of the organism to increase the number of CD45 positive cells; and up-regulates the expression level of PD-1, play a role in enhancing the curative effect of the immune checkpoint inhibitor; finally, the survival time of the tumor-bearing mice is prolonged. Wherein the using dose of the oHSV is 5x107 PFU/mouse, and the using dose of the Olaparib is 50 mg/kg; the PD-1 antibody was used at a dose of 200. mu.g/mouse.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.
Sequence listing
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Claims (10)
1. An antitumor pharmaceutical composition characterized by: comprises an oncolytic virus and a PARP inhibitor, wherein the oncolytic virus is selected from I type herpes simplex virus, the PARP inhibitor is Olaparib, and the molecular formula of the Olaparib is C24H23FN4O3(ii) a The structural formula of Olaparib is shown as formula I:
2. the antitumor pharmaceutical composition as claimed in claim 1, wherein: the oncolytic virus is prepared by inserting miRT124 and hGMCSF into type I herpes simplex virus.
3. The antitumor pharmaceutical composition as claimed in claim 1, wherein: the dosage of the oncolytic virus is 10 multiplied by 107-100×107The dosage of PFU and the Olaparib is 100-1000 mg/kg.
4. The antitumor pharmaceutical composition as claimed in claim 1, wherein: the composition further comprises a PD-1 antibody.
5. The antitumor pharmaceutical composition as claimed in claim 4, wherein: the dosage of the PD-1 antibody is 450 mu g-750 mu g.
6. The antitumor pharmaceutical composition as claimed in claim 1, wherein: the oncolytic virus is prepared by a method comprising the following steps:
s1, amplifying sequences at two ends of HSV-1 and ICP34.5 genes respectively;
s2, connecting sequences at two ends of hGM-CSF, CMV-GFP, 4xmIRT124 and ICP34.5 and plasmid pCMV-GFP by using a homologous recombination method to form a complete donor plasmid which is marked as pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT 124;
s3, designing sgRNA aiming at an ICP34.5 region, connecting the sgRNA to a Lenti-CRISPR-V2 vector in an enzyme digestion connection mode, and marking as Lenti-CRISPR-ICP 34.5;
s4, co-transfecting HEK293T cells with donor plasmid pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT124 and 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 fluorescent plaques after several hours, and picking the fluorescent plaques; obtaining HSV-1 inserted with hGMCSF, CMV-GFP and 4xmIRT124, and marking as oHSV-GFP;
s5, amplifying a Cre fragment by using a plasmid with the Cre fragment as a template, and connecting the Cre fragment to a pCCL-PGK-eGFP vector by using an enzyme digestion connection method, wherein the pCCL-PGK-eGFP vector is marked as pCCL-PGK-eGFP;
s6, transfecting the pCCL-PGK-Cre plasmid prepared in the step S5 to a HEK293T cell, adding the oHSV-GFP prepared in the step S4 in a dosage of 1.0 MOI, and collecting a culture supernatant after the cell is completely diseased; adding the supernatant into Vero cells, observing non-fluorescent plaques, and selecting the non-fluorescent plaques; HSV-1 with hGMCSF and 4xmIRT124 inserted was obtained and designated oHSV.
7. The antitumor pharmaceutical composition as claimed in claim 6,
step S4 includes the following steps:
co-transfecting HEK293T cells with a donor plasmid pULL34.5-hGM-CSF-lox 2272-CMV-eGFP-lox2272-4xmIRT124 and a Lenti-CRISPR-ICP34.5 plasmid targeting ICP34.5, adding HSV-1 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 a 10-fold dilution method, observing fluorescent plaques after 48 hours, and selecting the fluorescent plaques; after multiple rounds of purification, HSV-1 inserted with hGMCSF, CMV-GFP and 4xmIRT124 is obtained and is marked as oHSV-GFP.
Step S6 includes the steps of:
transfecting HEK293T cells with the pCCL-PGK-Cre plasmid prepared in the step S5, adding the oHSV-GFP prepared in the step S4 in a dosage of MOI (1.0) after 24 hours, and collecting culture supernatant after the cells are completely diseased; adding the supernatant into Vero cells, observing no fluorescent plaque after 48 hours, and selecting the non-fluorescent plaque; after multiple rounds of purification by the method, HSV-1 inserted with hGMCSF and 4xmIRT124 is obtained and is marked as oHSV.
8. An anti-tumor medication system, which is characterized in that: the system comprises the anti-neoplastic pharmaceutical composition of any one of claims 1-7.
9. Use of an anti-tumor pharmaceutical composition as defined in any one of claims 1 to 8 in the preparation of an anti-tumor medicament or formulation.
10. Use according to claim 9, characterized in that: the tumor is liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostatic cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer or gastric cancer.
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| US20160250267A1 (en) * | 2013-10-28 | 2016-09-01 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Oncolytic hsv vector |
| US20210322545A1 (en) * | 2016-02-24 | 2021-10-21 | 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 |
| US20200206285A1 (en) * | 2017-07-26 | 2020-07-02 | Oncorus, Inc. | Oncolytic viral vectors and uses thereof |
| 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 |
| US20210138008A1 (en) * | 2018-04-13 | 2021-05-13 | Beijing Wellgene Company Ltd. | Recombinant oncolytic virus composition and use thereof |
| 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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