CN110982794B

CN110982794B - Modified herpes simplex virus

Info

Publication number: CN110982794B
Application number: CN202010145491.4A
Authority: CN
Inventors: 田超; 赵婧姝; 刘家家; 孙春阳; 李小鹏; 周华; 闻可心
Original assignee: Beijing Weiyuan Likang Biotechnology Co ltd
Current assignee: Beijing Weiyuan Likang Biotechnology Co ltd
Priority date: 2020-03-05
Filing date: 2020-03-05
Publication date: 2020-06-16
Anticipated expiration: 2040-03-05
Also published as: CN110982794A

Abstract

The invention relates to a modified herpes simplex virus, wherein the herpes simplex virus lacks a functional ICP34.5 coding gene and a functional ICP47 gene and is derived from a gene with a preservation number of CCTCC NO: strain of V201810. The invention also relates to compositions comprising the modified herpes simplex virus and host cells infected by the herpes simplex virus.

Description

Modified herpes simplex virus

Technical Field

The present invention relates to a modified herpes simplex virus, or recombinant herpes simplex virus, and its use as an oncolytic virus. The modified herpes simplex viruses described herein are derived from non-laboratory strains of herpes viruses. Recombinant viruses which are not laboratory virus strains according to the invention have an enhanced oncolytic capacity and/or gene transfer capacity compared to laboratory virus strains.

Background

Herpes Simplex Virus (HSV) belongs to the family of herpesviridae, and is an enveloped, double-stranded DNA Virus, including two subtypes, type I (HSV-1) and type II (HSV-2).

The herpes simplex virus consists of four parts, namely a core, a capsid, a tegument protein and a cyst membrane. The viral core is composed of dense double stranded DNA wound into a fiber spool. The capsid surrounded by the DNA is in an icosahedral structure, has the diameter of 100-110 nm and consists of 162 capsomeres. The capsid is externally covered by irregularly arranged interstratified proteins. The outermost layer of the virus is a lipid bilayer envelope with a short bulge, and the diameter of the virus wrapping the envelope is 150-200 nm. The viral envelope is composed of 11 glycoproteins and at least two non-glycosylated proteins, including viral glycoprotein b (gb), viral glycoprotein c (gc), viral glycoprotein d (gd), viral glycoprotein g (gg), and viral glycoprotein m (gm). The three-dimensional structure of the envelope glycoprotein of a virus is involved in the cell-infecting ability of the virus, which determines whether the virus can enter a host cell and the amount of the virus entering the host cell.

At present, HSV-1 virus is used for gene therapy of major diseases such as tumor, nervous system degenerative disease, hereditary disease and immune system disease, and has a plurality of advantages compared with other gene therapy virus vectors.

First, the genome of HSV-1 is large and can carry large or multiple foreign genes. The HSV-1 genome is up to 152kb, whereas the adenovirus genome is only 35 kb. Among more than 80 known genes of the HSV-1 genome, about half of the genes which are non-essential genes in vitro culture can be replaced by a plurality of exogenous therapeutic genes, and the maximum exogenous gene insertion amount can reach 30-40 kb. This is particularly important for the treatment of many diseases, especially diseases associated with multiple genes.

The HSV-1 has strong infectivity, wide oncolysis spectrum and high oncolysis efficiency. HSV-1 is an enveloped virus with high infectivity, and the capability of infecting cells is obviously stronger than that of non-enveloped adenovirus. HSV-1 has a broad tumor lysis spectrum and can infect a wide variety of tumor cells. HSV-1 has short replication cycle and high oncolytic speed, and usually completes one replication cycle within 12 hours. After lysis of HSV-1 transfected tumour cells, thousands of progeny virions can be released and these viruses can continue to transfect other tumour cells. Determination of the oncolytic Activity of HSV-1 versus adenovirus by comparison with the Vero cell line revealed that the oncolytic Activity of HSV-1 was approximately 10-fold higher than that of adenovirus [ KasuyaH, Takeda S, Nomoto S, et al]. Cancer Gene Therapy, 2005, 12(9):725-36]. The clinical human dosage of the adenovirus vector is 10⁹-10¹²pfu, HSV-1 vector clinical dose is 10⁵-10⁸pfu, 1000 times less than human dose of adenovirus. HSV-1, in addition to expanding through the intercellular space, can also expand directly from one cell to another through cellular ligation. This allows the oncolytic virus to penetrate efficiently within solid tumors while being hardly accompanied by systemic transmission [ Shen Y, Nemunaitis J. hepes simplex virus 1 (HSV-1) for cancer treatment [ J.]. CancerGene Therapy, 2006, 13(11):975-992]。

HSV-1 is not integrated with cell DNA, replication can be controlled, and safety is high. HSV-1 produces little life-threatening disease in immunocompetent adults. Many nonessential genes of HSV-1 have been implicated in their neurotoxicity. Deletion of certain non-essential genes of HSV-1, such as the ICP34.5 gene, allows HSV-1 to replicate selectively in tumor cells only, and normal tissue cells are not affected by it [ Chou J, Kern E, Whitley R, et al, mapping of tumor simplex virus-1 neurovirus to gamma 34.5, gene nonessential for growth in culture [ J ] Science, 1990, 250(4985): 1262-.

Based on the above advantages, HSV-1 has become one of the most widely used viral vectors for gene therapy. In gene therapy research, HSV-1 is usually transformed into Oncolytic type I herpes simplex virus type 1 (OHSV-1) by means of gene transformation, and the main methods comprise deletion of certain genes, insertion of genes related to tumor therapy and the like. At present, a plurality of oHSV-1 are researched by preclinical and clinical tests, and a large number of results show that the product has good safety and effectiveness.

The general strategy of gene-deleted oHSV-1 construction is to mutate or delete single or multiple genes of the virus to achieve its specific killing effect on tumors, such as genes ICP34.5, ICP6, ICP0, TK, and UNG. The ICP34.5 gene in HSV-1 has been deleted by Talimogene laherparepvec, HSV1716, NV1020, G207, G47 Δ, etc. which are currently marketed and are being introduced into clinical trials. Oncolytic virus HSV1716 deleted only two copies of the ICP34.5 gene, showing tumor regression in clinical trials for refractory or relapsed high grade brain glioma treatment and no significant toxicity or side effects were found. G207 mutates the ICP6 gene while deleting the double copy ICP34.5 gene,clinical trials of treatment of malignant gliomas for recurrence and progression have shown that intratumoral injection of 10⁹No serious adverse reaction occurred after pfu G207, and good safety [ Markert JM, Liechty PG, Wang W, et al Phase Ib tertiary of mutant legacy virus G207 encapsulated pre-and post-plasmid-vector restriction for recovery GBM [ J]. Molecular Therapy, 2009, 17(1):199-207]. Talimogen laheredepvec is the first oncolytic virus approved to be marketed in the United states, eliminates ICP34.5 and ICP47 genes of HSV-1, and phase III clinical trial data show that the objective remission Rate on malignant melanoma treatment is 26.4%, and the Talimogen Laheredepepvec Impro Dual soluble Response Rate in Patients With remarkable treatment effect and good safety [ Andtbucka R H I, Kaufman H L, Colichio F, et al]. Journal of Clinical Oncology, 2015, 33(25):2780-2788]. These clinical data indicate that HSV-1 has good safety and great potential in gene therapy of tumors.

Due to the good replication capacity and safety of herpes simplex virus, it is widely regarded as important in tumor therapy. However, most of the herpes simplex viruses currently used for gene delivery/therapy or for oncolytic therapy of tumors are laboratory virus strains that have been passaged in tissue culture cells for many years. Although these viruses can enter cells to deliver genes, improvements in the characteristics required for therapy, such as oncolytic capacity and gene delivery efficiency, are desired.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a new HSV isolate, compared with a continuously-passaged standard strain HSV-1 virus 17 strain (referred to as a 17 virus strain or a 17 strain) which is used all the time before, the HSV-1 virus strain has greatly improved capability of infecting human cells in vivo and obviously enhanced capability of replicating/cracking in cells, and is suitable for serving as an oncolytic virus or serving as a carrier to destroy tumor cells through oncolytic action.

Compared with a standard strain HSV-1 virus 17 strain, the new HSV-1 isolate (strain HL-1, HL-1 or HL-1 for short) provided by the invention enhances the replication in a human tumor cell line. In the absence of ICP34.5 and/or ICP47, strain HL-1 has significantly enhanced replication, killing of tumor cells in human tumor cell lines compared to HSV-1 virus strain 17 in which ICP34.5 and/or ICP47 is also absent.

The modified HL-1 virus of the invention was subsequently used to deliver genes with anti-tumor activity, demonstrating that anti-tumor immune response activity and oncolytic effect could be further enhanced.

The invention thus provides.

1. A modified herpes simplex virus, wherein the modification is deletion of one or more of a functional ICP34.5 encoding gene, a functional ICP6 encoding gene, a functional ICP0 gene, a functional ICP47 gene, a functional US3 gene, a functional UL56 gene, a functional VP16 gene, a functional VHS gene, a functional UNG gene, a functional glycoprotein H encoding gene, a functional thymidine kinase encoding gene from the herpes simplex virus with a CCTCC accession number of V201810.

2. The modified herpes simplex virus of item 1, wherein the modification is a deletion of a functional ICP34.5 encoding gene and/or a functional ICP47 gene of the herpes simplex virus.

3. The modified herpes simplex virus of

item

1 or 2, wherein the modification further comprises introducing one or more heterologous genes into the virus.

4. The modified herpes simplex virus of item 3, wherein the one or more heterologous genes are inserted at one or more gene deletion sites selected from the group consisting of: functional ICP34.5 encoding gene, functional ICP6 encoding gene, functional ICP0 gene, functional ICP47 gene, functional US3 gene, functional UL56 gene, functional VP16 gene, functional VHS gene, functional UNG gene, functional glycoprotein H encoding gene, functional thymidine kinase encoding gene.

5. The modified herpes simplex virus of item 4, wherein the heterologous gene is selected from one or more of the following: a gene encoding a cytokine which promotes immune response, a gene encoding a tumor antigen, a gene encoding a monoclonal antibody having an effect of preventing and/or treating tumor, a gene encoding a prodrug-converting enzyme, a tumor suppressor gene, antisense RNA or small RNA.

6. The herpes simplex virus of item 5, wherein the cytokine is selected from one or more of GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IFN- α, IFN- γ, TGF- β, and TNF- α.

7. The herpes simplex virus of item 5, wherein the monoclonal antibody having tumor prevention and/or treatment effects is selected from one or more of the following: PD1 monoclonal antibody, PD-L1 monoclonal antibody, PD-L2 monoclonal antibody, CTLA-4 monoclonal antibody, CD80 monoclonal antibody, CD28 monoclonal antibody, CD137L monoclonal antibody, OX40 monoclonal antibody, OX40L monoclonal antibody, CD27 monoclonal antibody, CD70 monoclonal antibody, CD40 monoclonal antibody, CD40L monoclonal antibody, LAG-3 monoclonal antibody and TIM-3 monoclonal antibody.

8. The herpes simplex virus of item 5, wherein the tumor antigen is a tumor-derived specific antigen selected from one or more of the following: PSA, MUC1, MAGE-1, MAGE-2, MAGE-3, MAGE-12, BAGE, GAGE and LAGE.

9. The herpes simplex virus of item 5, wherein the prodrug-converting enzyme is cytosine deaminase or herpes simplex virus thymidine kinase.

10. The herpes simplex virus of item 5, wherein the tumor suppressor is P53 or PTEN.

11. The herpes simplex virus of item 5, wherein the antisense RNA or the small RNA is an RNA fragment of a protooncogene or a metabolic gene that blocks or down-regulates tumor overexpression.

12. The modified herpes simplex virus of item 5, wherein the heterologous gene encodes a cytokine polypeptide that stimulates an immune response and/or an antibody polypeptide that promotes an immune response.

13. The modified herpes simplex virus of item 12, wherein the cytokine polypeptide that stimulates an immune response is Granulocyte Macrophage Colony Stimulating Factor (GMCSF), IL-2, IL12, IFN- γ, and/or TNFa.

14. The modified herpes simplex virus of item 12, wherein the antibody polypeptide capable of promoting an immune response is an immune checkpoint antibody polypeptide.

15. The modified herpes simplex virus of item 14, wherein the immune checkpoint antibody is a PD1 antibody or a PD-L1 antibody.

16. A modified herpes simplex virus of any of claims 1-4, wherein the modification further comprises the loss of replication by preventing or reducing expression of one or more immediate early genes by genetic mutation.

17. The modified herpes simplex virus of item 16, wherein the one or more immediate early genes are one, two, three or four selected from the group consisting of ICP0, ICP4, ICP22 and ICP27 encoding genes.

18. A composition comprising the modified herpes simplex virus of any one of claims 1-17.

19. The composition of claim 18, wherein the modified herpes simplex virus has one, two, three or four foreign genes inserted at the same site or at different sites.

20. The composition of claim 18 or 19, wherein the modified herpes simplex virus is a mixture of two, three, four or more modified herpes simplex viruses, wherein each modified herpes simplex virus has a different foreign gene introduced.

21. The composition of any one of items 18-20, which is a viral culture.

22. A host cell infected with the modified herpes simplex virus of any of claims 1-17 or the composition of any of claims 18-21.

23. A composition comprising the host cell of item 22.

24. The composition of item 23, which is a cell culture.

Drawings

FIG. 1: 25000 times of electron microscope image of HL-1 strain virus.

FIG. 2: different MOIs of HL-1 and HSV (17) transfected HepG2 cells.

FIG. 3: HL-1 and HSV (17) different MOI transfected A375 cells.

FIG. 4: HL-1 and HSV (17) different MOI transfected A549 cells.

FIG. 5: different MOIs of HL-1 and HSV (17) transfected MCF-7 cells.

FIG. 6: different MOIs of HL-1 and HSV (17) transfected Hela cells.

FIG. 7: u251 cells were transfected with different MOIs of HL-1 and HSV (17).

FIG. 8: different MOIs of HL-1 and HSV (17) transfected Vero cells.

FIG. 9: IC50 comparisons of different cells transfected with HL-1 and HSV (17), respectively.

FIG. 10: CCTCC of knock-out ICP34.5 and ICP47 genes: IC50 assay results for different cells transfected with V201810 and control virus 17 strains.

FIG. 11: CCTCC expressing IL-12 exogenous gene: IC50 assay results for tumor cells transfected with V201810 and control virus 17 strains.

FIG. 12: and (3) IC50 measurement results of the HL-1 virus strain recombinant virus and the 17 virus strain recombinant virus transfected tumor cells after the IL-12 gene and the PD1 monoclonal antibody gene are inserted.

Detailed Description

The present application relates to an oncolytic virus with improved performance compared to laboratory model virus strains.

The term "oncolytic virus" as used herein refers to a class of viruses that infect and kill cancer cells, and destroy the infected cancer cells by oncolytic action, while releasing new infectious viral particles or virions that destroy the remaining cancer cells. Such viruses are known for their oncolytic effect.

"Herpes Simplex Virus (HSV)" was one of the first viruses to be selected for selective attack on cancer cells (oncolytic viruses) because of its profound research foundation and its relative harmlessness in its native state. When the virus of the invention is a herpes simplex virus, the virus may be derived, for example, from the HSV-1 strain or the HSV-2 strain or a derivative strain thereof, preferably HSV-1.

Some embodiments of the present application relate to a CCTCC collection number of: v201810 (CCTCC: V201810). In this application, the virus strain is referred to as the HL-1 strain of the HSV-1 virus, or simply the HL-1 strain, HL-1 or HL 1.

Some embodiments of the present application relate to derived strains of HSV-1 strains, for example, with a CCTCC collection number of: v201810 (CCTCC: V201810) is a strain in which the sequence homology of the genome of the strain is at least 70%, more preferably at least 80%, 85%, 90%, 95%, 98% of the sequence homology.

"herpes simplex virus vector" refers to a herpes simplex virus carrying a foreign gene.

Plaque forming units (pfu), abbreviated pfu, refer to the number of viruses that form plaques on animal cells cultured in monolayers.

An "immune checkpoint blocking (inhibitory) antibody" or "immune checkpoint blocking (inhibitory) monoclonal antibody" refers to a monoclonal antibody that inhibits or blocks an inhibitory immune checkpoint molecule. An "immune checkpoint stimulatory (agonistic) antibody" or "immune checkpoint stimulatory (agonistic) monoclonal antibody" refers to a monoclonal antibody that stimulates or agonizes a stimulatory immune checkpoint molecule. Immune checkpoints are regulators of the immune system and their role is manifested in enhancing the capacity of the immune system to clear heterosis or preventing the immune system from indiscriminately attacking cells, and thus being crucial for immune regulation. Immune checkpoints are divided into inhibitory and stimulatory checkpoint molecules. Inhibitory checkpoint molecules include, but are not limited to: a2AR, B7-H3 (CD 276), B7-H4, BTLA (CD 272), CTLA-4 (CD 152), IDO, KIR, LAG3, NOX2, PD1, TIM3, VISTA, CD 47; stimulatory checkpoint molecules include, but are not limited to: CD27, CD40, OX40, GITR, CD137, CD28, ICOS. Inhibitory and stimulatory checkpoint molecules are targets for cancer immunotherapy as they may be used in various types of cancer.

Herpes simplex virus wild strain HL-1 screened by the application and modified strain thereof

The invention relates to a herpes simplex virus in a first aspect, wherein the herpes simplex virus is a herpes simplex virus with a preservation number of CCTCC NO: herpes simplex virus type I (HSV-1) of V201810. In some embodiments, the application relates to a nucleic acid having a collection number of CCTCC NO: an attenuated variant of herpes simplex virus type I of V201810.

In some embodiments, the present application relates to a herpes simplex virus type I (HSV-1) having one or more characteristics selected from the group consisting of:

(a) compared with a reference laboratory strain, has enhanced ability of infecting tumor cells,

(b) compared with a reference laboratory strain, has enhanced replication capacity in tumor cells,

(c) compared with a reference laboratory strain, the strain has enhanced tumor cell killing capacity;

(d) the herpes simplex virus, when modified, has an enhanced ability to infect tumor cells compared to a reference laboratory strain having equivalent modifications,

(e) the herpes simplex virus is modified to have enhanced replication capacity in tumor cells as compared to a reference laboratory strain having equivalent modifications, and

(f) the herpes simplex virus, when modified, has enhanced ability to kill tumor cells compared to a reference laboratory strain having equivalent modifications.

The virus strains of the invention are "non-laboratory" strains and may be referred to as "clinical" strains. One skilled in the art can readily distinguish between laboratory strains and non-laboratory or clinical strains. A key difference between laboratory and non-laboratory strains is that the laboratory strains commonly used today have been maintained in culture for a considerable time. For virus growth and maintenance, appropriate cells are infected with the virus, the virus replicates within the cells, and the virus is then harvested; fresh cells were then reinfected. This process constitutes one cycle of serial passage. In the case of HSV, each such cycle may take, for example, several days, and such serial passages may result in a change in the characteristics of the virus strain, for example, a selection of characteristics which favour growth in culture (for example rapid replication) rather than selection and characteristics which favour practical use.

The virus strains of the invention are non-laboratory strains in that they are derived from strains newly isolated from infected individuals. The strains of the invention are modified to eliminate toxicity but to substantially retain the desirable characteristics of the original clinical isolate from which they were derived.

The viruses of the present invention are capable of efficiently infecting human target cells. Such viruses have just been isolated from infected individuals and then screened for the desired ability to enhance replication in tumor cells and/or other cells in vitro and/or in vivo as compared to standard laboratory strains. Such viruses with improved properties compared to laboratory virus strains are viruses of the invention. Identified viruses with such desired improved properties can then be engineered by mutation of the appropriate gene so that they can selectively kill tumor cells, or mutated so that they can deliver the gene to the target tissue in non-oncolytic applications without toxic effects. These modified viruses are also viruses of the present invention.

Compared with a reference laboratory strain with equivalent modifications, the virus strain of the invention has stronger ability to infect or replicate in any tumor cell, kill tumor cells or spread among cells in tissues than the reference laboratory strain with equivalent modifications. Preferably, this greater ability is a statistically significant greater ability. For example, according to the invention, the capacity of the virus strain of the invention may be at most 1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50-fold or 100-fold the capacity of the reference strain with respect to the property to be tested.

The viruses of the present invention have substantial ability to (i.e., retain) their unmodified clinical precursor strains. For example, in the case of an oncolytic virus intended for the treatment of a tumor, the virus strain of the invention preferably has substantially the ability of its unmodified clinical precursor strain to infect or replicate in, kill, or spread between cells in a tissue, any tumor cell.

The present application encompasses modified viruses in which the altered viral region can either be eliminated (in whole or in part) or rendered non-functional, or replaced by other sequences, especially by heterologous gene sequences. One or more genes may be made non-functional and one or more heterologous genes may be inserted.

In some embodiments, the virus of the present application is a modified non-laboratory oncolytic virus. These viruses are useful for oncolytic therapy for cancer. Such viruses infect and replicate within tumor cells, subsequently killing the tumor cells. Thus, such viruses are replication-competent viruses. Preferably, they are selectively replication competent in tumor cells. This means that they replicate in tumor cells but not in non-tumor cells, or they replicate more efficiently in tumor cells than in non-tumor cells. The determination of the selective replication capacity can be performed by the assays described herein for determining replication capacity and tumor cell killing capacity.

Preferably, the oncolytic viruses of the invention are more potent (preferably statistically significant) than a reference laboratory strain with the same modifications in infecting or replicating within tumor cells, killing tumor cells, or spreading between cells in a tissue.

The ability of a virus to infect tumor cells can be quantified by measuring the dose of virus required to infect a certain percentage of cells (e.g., 50% or 80% of cells). The ability to replicate in tumor cells can be determined by measuring the growth of the virus in the cells, for example by measuring the virus growth in the cells over a period of time of 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours or more. The ability of a virus to kill tumor cells can be quantified by counting the number of viable cells remaining at a given time point and moi (multiplicity of infection) for a given cell type over a specified time period. The cells or combination of cells in the examples of the present application can be used, as can other tumor cell types. To count the number of viable cells remaining at a given time point, the number of cells that exclude trypan blue (i.e., viable cells) can be counted. Quantitative determination may also be performed by flow cytometry (FACS), MTT, CCK-8 or CTG method analysis. Tumor cell killing ability can also be determined in vivo, for example by measuring the reduction in tumor volume due to a particular virus.

To determine the characteristics of the viruses of the invention, it is generally preferred to use standard laboratory reference strains for comparison. Any suitable standard laboratory reference strain may be used. As for HSV, it is preferable to use one or more of HSV-1 strain 17, HSV-1 strain F, or HSV-1 strain KOS. The reference strain typically has equivalent modifications, such as gene deletions and/or heterologous gene insertions, to the test strain of the invention. For example, with respect to HSV strains, if ICP34.5, ICP6 and/or Thymidine Kinase (TK) encoding genes have been rendered non-functional in the viruses of the invention, they are rendered non-functional in a reference strain as well.

In the embodiment of the application, the reference strain is laboratory standard model strain HSV-1 virus 17 strain, or HSV-1 virus 17 strain, HSV (17), 17 virus strain, 17 strain.

The application relates to a human herpes virus type I HL-1 strain for patent program preservation with the preservation number of CCTCC NO: and V201810.

In the present application, herpes virus type I (type 1), herpes simplex virus type I (type 1), and HSV-1 have the same meanings and may be used interchangeably. The human herpesvirus type I HL-1 strain may be abbreviated herein as HL-1 strain, HL1 strain, HL-1 or HL 1.

Herpes Simplex Virus (HSV) is used both as a gene delivery vector in the nervous system and other systems, and for oncolytic therapy for cancer. However, in both applications, the virus must be made defective so that it is no longer pathogenic, but so that it can still enter the cell and perform the desired function.

The HSV-1 genome has many genes that can be deleted or mutated to confer safety and/or tumor targeting specificity. For example, one of the common features that distinguish cancer cells from most normal cells (post-mitosis) is the sustained proliferation of cancer cells and thus the maintenance of sufficient nucleotides for DNA replication. HSV-1 contains the relevant genes involved in nucleotide metabolism (thymidine kinase, ribonucleotide reductase and uracil DNA glycosylase, etc.) allowing the virus to replicate in non-dividing cells that lack sufficient nucleotides. Deletion or mutation of these genes can confer specificity to dividing cells (tumor cells) and also often attenuate the pathogenicity of the virus. In addition, there are various mechanisms in the cell to detect and inactivate invading viruses, and part of the genes of the viruses (ICP 34.5, ICP0, UL56, etc.) can escape or block the antiviral mechanism of the cell, and deletions or mutations of the part of the genes can also confer specificity to the virus for dividing cells (tumor cells).

In some embodiments, an HSV strain of the present invention is modified to lack one or more of a functional ICP34.5 encoding gene, a functional ICP6 encoding gene, a functional glycoprotein H encoding gene, a functional thymidine kinase encoding gene. In some embodiments, the virus lacks a functional ICP34.5 encoding gene. In some embodiments, the virus may be further modified to delete a functional ICP47 gene. In some embodiments, one or more of the ICP34.5, ICP47, Us3, ICP0, UL56 genes are mutated to produce Oncolytic type I herpes simplex virus type 1, oHSV-1.

For the oncolytic treatment of cancer (which may also include delivery of genes that enhance the effect of the treatment), a number of mutations in HSV have been identified (pets C. design here Viruses as Oncolytics. molecular Therapy Oncolytics, 2015.) which still allow the virus to replicate in culture or in actively dividing cells in vivo (e.g. intratumoral), but prevent its efficient replication in normal tissues. Such mutations include disruption of genes encoding ICP34.5, ICP6 and thymidine kinase. To date, among these mutant viruses, viruses having the ICP34.5 mutation or the ICP34.5 mutation and, for example, the ICP6 mutation have shown the most favorable safety. Viruses that only lack ICP34.5 have been shown to replicate in many tumor cell types in vitro and selectively replicate in mouse brain tumors without harming surrounding tissues.

Any other gene-deleted/mutated virus that provides oncolytic properties (i.e., selectively replicates in tumors compared to surrounding tissues) is also contemplated by the present application.

Heterologous genes can be inserted into such viruses of the invention, e.g., viruses having a CCTCC accession number of V201810 (CCTCC: V201810), using techniques known in the art and/or techniques described herein. In oncolytic viruses, the heterologous gene is typically a gene that enhances the ability of the virus to fight tumors. Any gene conferring anti-tumor properties on the virus may therefore be inserted. In particular, the heterologous gene may be a gene, in particular an immunostimulatory polypeptide, which is capable of improving the immune response against tumor cells in a beneficial manner.

Pharmaceutical compositions, articles of manufacture, and uses of the HL-1 Virus

The oncolytic virus of the invention can be used for preparing medicines for treating cancers. In particular, the oncolytic viral formulations of the present invention may be used in the treatment of cancer, for example by direct intratumoral injection. The oncolytic virus formulations of the present invention can be used to treat any solid tumor in a mammal, preferably a human. For example, the viruses of the present invention can be administered to a subject suffering from: liver cancer, melanoma, brain glioma, sarcoma, lung cancer, colorectal cancer, head and neck tumor, breast cancer, renal cell carcinoma, ovarian cancer, cervical cancer, prostate cancer, stomach cancer, lymphoma, pancreatic cancer, and bladder cancer.

In some embodiments, the present application relates to a recombinant oncolytic virus, or designated modified oncolytic virus, characterized in that the recombinant oncolytic virus or modified oncolytic virus is derived from a CCTCC with a accession number of: v201810 (CCTCC NO: V201810), wherein for the CCTCC: the V201810 virus is genetically modified, so that one or more of a functional ICP34.5 encoding gene, a functional ICP6 encoding gene, a functional ICP0 gene, a functional ICP47 gene, a functional US3 gene, a functional UL56 gene, a functional VP16 gene, a functional VHS gene, a functional UNG gene, a functional glycoprotein H encoding gene and a functional thymidine kinase encoding gene are deleted, a foreign gene is inserted at the position of the deletion of one or more genes, for example, a cytokine polypeptide for stimulating an immune response or an antibody polypeptide for promoting an immune response is encoded, for example, the cytokine polypeptide for stimulating an immune response is Granulocyte Macrophage Colony Stimulating Factor (GMCSF), IL-2, IL12, IFN-gamma or TNFa, and the antibody polypeptide for promoting an immune response is an immune check point antibody.

The recombinant oncolytic viruses of the invention, or so-called modified oncolytic viruses, can be used for the preparation of a medicament for the treatment of cancer. In particular, the oncolytic viral formulations of the present invention may be used in the treatment of cancer, for example by direct intratumoral injection. The oncolytic virus formulations of the present invention can be used to treat any solid tumor in a mammal, preferably a human. For example, the viruses of the present invention can be administered to a subject suffering from: liver cancer, melanoma, brain glioma, sarcoma, lung cancer, colorectal cancer, head and neck tumor, breast cancer, renal cell carcinoma, ovarian cancer, cervical cancer, prostate cancer, stomach cancer, lymphoma, pancreatic cancer, and bladder cancer.

Some embodiments of the present application relate to pharmaceutical compositions comprising a virus of the present application and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents are known in the art and include, but are not limited to, phosphate buffered saline solutions.

In some embodiments, the present application relates to an article of manufacture or a kit comprising a vial containing the aforementioned pharmaceutical composition and a package insert comprising instructions for use of the article of manufacture or kit for treating a disease.

The pharmaceutical compositions of the present application can be injected directly into a target tissue, such as a cancer tissue, for oncolytic therapy and/or gene delivery into cells for therapeutic purposes. The pharmaceutical compositions comprising the virus or recombinant virus of the present application are administered in a viral dose in the range of 10^4 to 10^12pfu/ml, preferably 10^5 to 10^8pfu/ml, more preferably 10^6 to 10^8 pfu/ml. For oncolytic or non-oncolytic therapy, the pharmaceutical composition consisting essentially of the virus and a pharmaceutically acceptable suitable carrier or diluent is usually injected in an amount of up to 10ml, usually 1-5ml, preferably 1-3ml, when injected. However, for some oncolytic therapeutic applications, larger volumes of more than 10ml may also be used, depending on the tumor and the inoculation site.

The optimal route of administration and dosage can be readily determined by one skilled in the art based on the subject and the disease condition. The dosage can be determined according to various parameters, in particular according to the age, weight and condition of the patient to be treated, the severity of the disease or condition and the route of administration. The preferred route of administration for cancer patients is direct injection into the tumor. The virus may also be administered systemically, e.g., intravenously, or by injection into the blood vessels that supply the tumor. The optimal route of administration will depend on the location and size of the tumor. The dosage can be determined according to various parameters, in particular according to the location of the tumor, the size of the tumor, the age, weight and condition of the patient to be treated and the route of administration.

Examples

Example 1 obtaining of HL-1 Strain Virus having oncolytic Effect

a) Collection of viruses

63 healthy volunteers with recurrent cold sores were recruited and the herpes fluid was collected aseptically.

b) Screening for viruses

The purpose of this section is to test and screen primary clinical isolates of HSV-1 and select the most oncolytic strains.

Experiments were performed simultaneously using 5 to 8 virus strains each time. Using HSV-1 virus strain 17 as a control, the virus strains were infected at the same MOI into cells in a six-well plate, tested in parallel, and observed for the phenomenon of cpe (cytopathic effect) caused by the virus 48 hours after infection and evaluated for the number of plaques after crystal violet staining. CPE should be the typical HSV-1 infection causes, cell rounding and from the center of infection to gradually shed and form plaque. Viral strains showing significant CPE and high numbers of plaques after staining were evaluated.

The virus strain having the best oncolytic effect is evaluated and screened as HL-1 strain by the method.

c) Biological characterization of HL-1 Virus strains

1) Observation by electron microscope

After the HL-1 strain virus infects cells, cell debris is removed and observed under a 25000-fold electron microscope. The virus is shown in a typical HSV-1 appearance, with four components being observed for genomic DNA, capsid, tegument protein and envelope (see FIG. 1).

2) Analysis of genomic DNA sequencing results for HL-1 Virus strains

The genome DNA of the type I herpes simplex virus (HSV-1) HL-1 strain is handed over to Shanghai Europe Yiyi organism for third generation sequencing analysis, the virus genome sequences of the HL-1 strain and a standard model strain HSV-1 virus 17 strain (NC _ 001806.2) are compared, the results show that the genome sequences of the HL-1 strain and the standard model strain HSV-1 virus 17 strain have the difference of 1700 bases, the amino acid sequences of a plurality of important coding genes including membrane protein, a promoter gene and the like have great difference, and the comparison results are shown in the following table 1 and the table 2.

TABLE 1 comparison of the amino acid sequences of the membrane proteins of HL-1 and 17 strains

TABLE 2 alignment of amino acid sequences of important genes of HL-1 and 17 strains of viruses

The difference of the amino acid sequences of gD, gG, gI, gJ, gL, gM and gN in the envelope proteins of the HL-1 strain and the 17 strain is more than 1%, especially the difference of the amino acid sequences of gG and gI is more than 3%, wherein the gG reaches 5.46%. The membrane proteins of the herpes simplex virus envelope are involved in virus entry, release, and direct intercellular transmission, suggesting that the infectivity of the HL-1 strain differs from that of the 17 strain.

Comparing the amino acid sequences of the early genes ICP0, ICP4, ICP22, ICP27 and ICP47 and the important genes ICP6 and ICP34.5, the sequence differences of the early genes ICP4, ICP22 and ICP47 of the HL-1 strain and 17 strain are large, and the amino acid sequence differences are more than 1%. ICP4 and ICP22 are used as the immediate early genes of HSV-1, can stimulate the expression and DNA synthesis of early genes of viruses and induce the expression of late genes, and are key factors for triggering infection by the expression of HSV-1 genes. ICP34.5 is also a gene with larger difference between genes of HL-1 strain and 17 strain, and the amino acid difference reaches 6.25%. ICP34.5 is an important neurotoxic factor of HSV-1 virus, and plays a key role in the replication and pathogenicity of the virus.

d) Collection of HL-1 Virus strains

The HL-1 virus strain is preserved in China center for type culture Collection, named as human herpesvirus type I HL-1 strain, with the preservation number of CCTCC NO: and V201810.

Example 2 comparison of oncolytic Effect of HL-1 wild-type and 17 strains

This example provides comparative experiments and results of the replication of HL-1 and 17 strains on different cells and their ability to transfect tumor cells. The experimental cells include: hep G2 human liver cancer cell, A375 human skin cancer cell, A549 human lung cancer cell, MCF7 human breast cancer cell, HeLa human cervical cancer cell, U251 human glioma cell, Vero monkey kidney cell.

The experimental method is as follows:

1, microscopic examination observation after different cells are transfected by HL-1 and HSV (17) respectively

And (3) taking the cells in the logarithmic growth phase, digesting, and then, subculturing the cells in a six-well plate. After 24 hours the old medium was discarded and 2ml of medium was added to each well. And (3) transfecting viruses with different MOI quantities, adding the viruses, putting the viruses into a carbon dioxide incubator at 37 ℃ for culturing for 48 hours, taking out the viruses, observing the viruses under a mirror, and photographing and recording the viruses.

IC50 assay of different cells transfected with HL-1 and HSV (17)

Cells in logarithmic growth phase were taken and passaged to 96-well plates after digestion. After 24 hours the old medium was discarded and 9 dilutions of HL-1 and HSV (17) virus were added, 100. mu.l/well, giving MOI of 20, 5, 1.25, 0.3125, 0.0781, 0.0195, 0.0049, 0.0012 and 0.0003, with blanks. After 3 days of incubation at 37 ℃ all the liquid in the wells was discarded, 10% CCK-8 reagent (Cell Counting Kit-8, Dojindo) was added to 100. mu.l/well, incubated at 37 ℃ for 1 h, the OD of the sample was measured at 450 nm, and IC50 was calculated from the fitted curve.

The experimental results are as follows:

1.1 results of microscopic examination of HepG2 cells transfected by HL-1 and HSV (17)

Microscopic observations 48 hours after transfection of HepG2 cells with HL-1 and HSV (17) showed that the transfection efficiency of HL-1 was higher than that of HSV (17) under each of the same MOI conditions (see FIG. 2).

1.2 results of A375 cell microscopic examination of HL-1 and HSV (17) transfection

Microscopic observations 48 hours after transfection of A375 cells with HL-1 and HSV (17) showed that the transfection efficiency of HL-1 was significantly higher than that of HSV (17) under each of the same MOI conditions. It was even observed under the mirror that the morphology of HL-1 transfected cells had been completely rounded under the condition of transfection of the virus MOI0.1, while normal cells were still visible in HSV (17) -transfected cells (see FIG. 3).

1.3 microscopic examination of A549 cells transfected by HL-1 and HSV (17)

Microscopic observation of HL-1 and HSV (17) transfected A549 cells for 48 hours showed that the transfection efficiency of HL-1 was significantly higher than that of HSV (17) under the same MOI conditions. The number of HL-1 transfected cells was observed under the mirror to be significantly greater than that of HSV (17) transfected cells, almost no normal cells were observed at an MOI of 0.1, while normal cells were still visible in the HSV (17) transfected cells and the number was increased as the MOI was decreased (see FIG. 4).

1.4 results of microscopic examination of MCF-7 cells transfected with HL-1 and HSV (17)

According to the experience of comparing the effect of virus transfection cells in the above experiments, only two virus concentrations of MOI0.1 and MOI 0.01 are selected for comparison in the following experiments.

Results of microscopic observation 48 hours after transfection of MCF-7 cells with HL-1 and HSV (17) showed that the transfection efficiency of HL-1 was significantly higher than that of HSV (17). HL-1 transfected MCF-7 cells were fully rounded in cell morphology at MOI0.1, while HSV (17) transfected MCF-7 cells remained partially normal (see FIG. 5).

1.5 results of HeLa cell microscopic examination of HL-1 and HSV (17) transfection

Results of microscopic observation after transfection of Hela cells with HL-1 and HSV (17) for 48 hours show that the transfection efficiency of HL-1 is obviously higher than that of HSV (17) under the condition of MOI 0.1. The cell morphology of the HL-1 transfected Hela cells became mostly round at the MOI of 0.1, while the HSV (17) transfected Hela cells still had a certain number of normal cells (see FIG. 6).

1.6 results of microscopic examination of U251 cells transfected with HL-1 and HSV (17)

Results of microscopic observation 48 hours after transfection of U251 cells with HL-1 and HSV (17) showed that the transfection efficiency of HL-1 was significantly higher than that of HSV (17). HL-1 transfected U251 cells were mostly rounded in cell morphology at MOI 0.01, whereas HSV (17) transfected U251 cells were rarely morphologically rounded by viral transfection (see FIG. 7).

1.7 HL-1 and HSV (17) transfection Vero cell microscopic examination result

Vero cell non-tumor cell is HSV virus sensitive cell line, is one of the commonly used production cells, so that comparison of HL-1 and HSV (17) infection on Vero cell is of great significance for production capacity.

Results of microscopic observation 48 hours after transfection of Vero cells with HL-1 and HSV (17) show that the transfection efficiency of HL-1 is obviously higher than that of HSV (17) under the conditions of MOI0.1 and 0.01. The cell morphology of HL-1 transfected Vero cells was fully rounded at a virus MOI of 0.1, while HSV (17) transfected Vero cells still remained partially normal at an MOI of 0.1 (see FIG. 8).

2. IC50 determination of HL-1 and HSV (17) transfection of different cells A comparison of IC50 of HL-1 and HSV (17) transfection of different cells is shown in FIG. 9. The results in the figure show that HL-1 has less IC50 (MOI) than HSV (17), indicating that HL-1 has stronger killing ability to cells than HSV (17).

This example is intended to compare the difference in the ability of the two virus strains HL-1 and HSV (17) to transfect tumor cells. HL-1 and HSV (17) were transfected into different tumor cells (HepG 2, A375, A549, MCF-7, Hela and U251) and Vero cells which are normal passage cells, and the results of microscopic observation after 48 hours of transfection of the cells show that the transfection efficiency of HL-1 is obviously higher than that of HSV (17) under the same MOI, although the sensitivity of each cell to viruses is different. IC50 values were further determined for HL-1 and HSV (17) transfected cells by the CCK-8 method, and the results showed that both HL-1 transfected cells had smaller IC50 values than HSV (17). In conclusion, compared with the HSV (17) virus strain, the HL-1 virus strain has better infection efficiency and replication capacity in cells, strong oncolytic effect and better application prospect when being used as an oncolytic virus.

Example 3 comparison of the oncolytic Effect of HSV (HL-1) recombinant Virus and HSV (17) recombinant Virus

The ICP34.5 gene and ICP47 gene of the virus strain with the CCTCC preservation number of V201810 (CCTCC: 201810) and the virus strain of the control virus 17 are knocked out by a genetic engineering method, the difference of the capacities of the recombinant viruses HL1- △ 34.5.5- △ 47 and 17- △ 34.5.5- △ 47 after gene knockout are compared, and the experimental cells comprise a Hep G2 human liver cancer cell, an A375 human skin cancer cell, an A549 human lung cancer cell, an MCF7 human breast cancer cell, a HeLa human cervical cancer cell, a U251 human glioma cell and a Vero monkey kidney cell.

Cells in logarithmic growth phase were taken and passaged to 96-well plates after digestion. After 24 hours the old medium was discarded and 9 dilutions of different viruses were added, 100. mu.l/well, to give MOI of 20, 5, 1.25, 0.3125, 0.0781, 0.0195, 0.0049, 0.0012 and 0.0003, respectively, with blanks. After 3 days of incubation at 37 ℃ all fluid in the wells was discarded, 10% CCK-8 reagent was added, 100. mu.l/well was incubated at 37 ℃ for 1-2 h, OD was measured at 450 nm and IC50 values were calculated from the fitted curve.

A comparison of the results of IC50 from the transfection of two recombinant viruses into different cells is shown in FIG. 10.

Experimental results show that the IC50 values of HL1- △ 34.5.5- △ 47 transfected cells are all smaller than those of 17- △ 34.5.5- △ 47, and the results show that the HL-1 virus strain has better infection efficiency and replication capacity in the cells and strong oncolytic effect compared with HSV (17) virus strain recombinant viruses after gene recombination and reconstruction, and has better application prospect when being used as an oncolytic virus.

Example 4 oncolytic Effect of HL-1 Virus Strain as a vector carrying foreign genes on tumor cells

This example is intended to compare the oncolytic effects of HL-1 recombinant virus and 17 strain recombinant virus carrying foreign genes. The experimental cells comprise A375 human skin cancer cells, A549 human lung cancer cells, AGS human gastric cancer cells, AsPC-1 human pancreatic cancer cells, HeLa human cervical cancer cells, Hep G2 human liver cancer cells, HT-29 human large intestine colorectal cancer cells, MCF7 human breast cancer cells, PC-3 human prostate cancer cells, T24 human bladder cancer cells, U-2 OS human bone cancer cells and U-87 human brain glioma cells.

Comparison of oncolytic Effect of oncolytic Virus expressing IL-12 foreign Gene

The ICP34.5 Gene and ICP47 Gene of the virus strain with the CCTCC preservation number of V201810 (CCTCC: V201810) and the virus strain of the contrast virus 17 are knocked out by a genetic engineering method, and an artificially and chemically synthesized foreign Gene is inserted into the position where the ICP34.5 Gene is knocked out, wherein the foreign Gene sequentially comprises an EF1 α promoter, a Gene (Gene ID: 16159,16160) for encoding IL-12 and TK PolyA from the 5 'end to the 3' end, and the encoding Gene is verified to be correctly inserted into a herpes simplex virus vector by sequencing in New industries of Beijing Ongchou, a successfully constructed recombinant virus vector and the cell strain are subjected to 5% CO at 37 ℃ to obtain the recombinant virus vector₂And (4) proliferating under the condition.

The results of the IC50 assay for different virus-transfected tumor cells are shown in FIG. 11. Experimental results show that the difference of the capacity of the HL-1 recombinant virus (HL 1-IL 12) and 17 strains of recombinant virus (17-IL 12) for transfecting the tumor cells after the IL-12 gene is inserted is different, and the HL-1 recombinant virus inserted with the IL-12 gene has smaller IC50 value than the 17 strains of recombinant virus inserted with the IL-12 gene for transfecting the tumor cells, so that the HL-1 virus strain recombinant virus inserted with the IL-12 gene has better infection efficiency and replication capacity in the tumor cells, has strong oncolytic effect and better application prospect as an oncolytic virus.

Oncolytic virus introduced with exogenous gene IL-12 for melanoma mouse

HL1-IL12 and HL1-mock (HL1- △ 34.5.5- △ 47) were constructed according to the methods of examples 3 and 4, and the oncolytic effect of oncolytic virus on melanoma mice was evaluated C57BL/6J mice were subcutaneously inoculated with B16F10 tumor cells to establish an allograft melanoma model control groups were phosphate solution, treatment groups were oxaliplatin (10mg/kg), tested oncolytic virus HL1-mock (10 ^7 pfu), HL1-IL12 (10 ^7 pfu), HL1-IL12 (10 ^6 pfu) and HL1-IL12 (10 ^5 pfu), respectively, 10 mice per group, control group and test oncolytic virus-administered group were administered by intra-injection, and oxaliplatin-administered group was administered by intraperitoneal injection.

Relative tumor inhibition ratio TGI (%): TGI% = (1-T/C) × 100%. T/C% is the relative tumor proliferation rate, i.e., the percentage value of the relative tumor volume of the treatment group and the control group at a particular time point. T and C are the Relative Tumor Volumes (RTV) at specific time points in the treated and control groups, respectively. RTV = tumor volume of animals after treatment/tumor volume of control group. The evaluation of the therapeutic effect was carried out on the basis of the tumor inhibition ratio (TGI).

The research results are as follows: HL1-IL12 (10) by mean tumor volume analysis in mice on day 10 post-dose⁷PFU) tumor inhibition rate of 76%, HL1-IL12 (10)⁶PFU) tumor inhibition rate of 47% and HL1-mock (10)⁷PFU) tumor inhibition rate is 52%, and the three have statistically significant difference (P) in anti-tumor effect compared with the control group<0.05); while HL1-IL12 (10)⁵PFU) and oxaliplatin (10mg/kg) alone had no statistically significant difference (P) in antitumor effect compared with the control group>0.05), the tumor inhibition rate is 6% and 20%, respectively. Control group died entirely on day 14, HL1-IL12 (10)⁷PFU) administration group 3 mice had lost tumor and continued to survive. Except for the above 3 mice, all the other mice in the group were dead on day 21.

The remainder was HL1-IL12 (10)⁷PFU) group, survival continued to 91 days, re-challenged with B16F10 tumor cells inoculated contralaterally to mice, and control group with 5C 57BL/6J mice inoculated subcutaneously with B16F10 tumor cells to observe tumor growth.

After the mice are inoculated, tumors grow and grow continuously on the 10 th day in the control group, tumors do not grow in the challenge group, and the tumors do not grow after continuous observation, so that the mice have tumor immunity after being treated by HL1-IL 12.

Oncolytic effect of oncolytic virus expressing GM-CSF and IL-2 exogenous genes

The ICP34.5 Gene and the ICP47 Gene of the virus strain with the CCTCC preservation number of V201810 (CCTCC: V201810) and the virus strain of the control virus 17 are knocked out by a Gene engineering method, and an artificially and chemically synthesized foreign Gene is inserted into the position where the ICP34.5 Gene is knocked out, wherein the foreign Gene sequentially comprises a CMV promoter, a Gene (Gene ID: 12981) for encoding GM-CSF, BGH PolyA, a EF1 α promoter, a Gene (Gene ID: 16183) for encoding IL-2 and TKPolyA from the 5 'end to the 3' end, and the encoding Gene is sequenced and identified to be correctly inserted into a herpes simplex virus vector by Beijing Mianbo company.

An animal model is constructed by inoculating a murine melanoma B16F10 cell line subcutaneously into a C57BL/6 mouse. Selecting mice successfully modeled, and setting 9 groups of 5 mice each, wherein the HL-1 recombinant virus treatment group comprises: administering HL-1 recombinant virus introduced with exogenous genes GM-CSF and IL-2; 17 virus strain recombinant virus treatment groups: administering a recombinant virus of 17-strain introduced with foreign genes GM-CSF and IL-2; HSV-mock group: the oncolytic virus is an oncolytic virus which does not insert exogenous genes and knocks out ICP34.5 genes and ICP47 genes; negative control: PBS was administered. The total pfu of virus administration for each group was 10⁶pfu. The relative tumor suppression rate 14 days after administration was used as a criterion.

Relative tumor inhibition ratio TGI (%): TGI% = (1-T/C) × 100%. T/C% is the relative tumor proliferation rate, i.e., the percentage value of the relative tumor volume of the treatment group and the control group at a particular time point. T and C are the Relative Tumor Volumes (RTV) at specific time points in the treated and control groups, respectively. RTV = tumor volume of animals after treatment/tumor volume of control group.

The experimental results are as follows: the relative tumor inhibition rate of the HL-1 recombinant virus treatment group is 79 percent; the relative tumor inhibition rate of the 17 virus strain recombinant virus treatment group is 58%; the relative tumor inhibition rate of the HSV-mock group is 39%.

Oncolytic effect of oncolytic virus expressing anti-PD 1 monoclonal antibody in vitro-derived gene

An oncolytic virus inserted with an anti-PD 1 antibody foreign gene comprising, in order from the 5 'end to the 3' end: CMV promoter, gene encoding anti-PD 1 antibody (J43, BioXCell), BGHPolyA sequence. C57BL/6J mice were inoculated with B16F10 tumor cells subcutaneously to establish an allograft melanoma model. The control group was phosphate solution, and the treatment groups were PD1 monoclonal antibody (5 mg/kg) and test oncolytic virus HL1-mock (10)⁷pfu），HL1-PD1（10⁷pfu），HL1-PD1（10⁶pfu) and HL1-PD1 (10)⁵pfu), 10 mice per group; the control group and the test group administered with oncolytic virus were administered by intratumoral injection, and the group administered with PD1 monoclonal antibody was administered by intraperitoneal injection, and the relative tumor inhibition rates were calculated as described above.

The research results are as follows: by analyzing the mean tumor volume of the mice on day 13 after administration, the group administered with the oncolytic virus of each dose group of HL1-PD1 has a statistically significant difference in antitumor effect compared with the control group (P < 0.05); the HL1-PD1 high, medium and low dose groups have obvious dose effects, the tumor inhibition rates are respectively 86%, 65% and 51%, and the tumor inhibition rates of the HL1-mock group and the PD1 monoclonal antibody group are respectively 34% and 42%.

Oncolytic effect of oncolytic virus expressing IL-12 and PD1 monoclonal antibody exogenous gene

The HL-1 strain and 17 HSV virus with ICP34.5 and ICP47 genes deleted as above were inserted with IL-12 gene and PD1 monoclonal antibody gene at ICP34.5 and ICP47 genes, respectively, and IC50 of HL1-IL12+ PD1 (HL-1 is inserted with IL-12 gene and PD1 monoclonal antibody gene), 17-IL12+ PD1 (17 is inserted with IL-12 gene and PD1 monoclonal antibody gene) transfected into different cells was determined. The experimental cells comprise A375 human skin cancer cells, A549 human lung cancer cells, AGS human gastric cancer cells, AsPC-1 human pancreatic cancer cells, HeLa human cervical cancer cells, Hep G2 human liver cancer cells, HT-29 human large intestine colorectal cancer cells, MCF7 human breast cancer cells, PC-3 human prostate cancer cells, T24 human bladder cancer cells, U-2 OS human bone cancer cells and U-87 human brain glioma cells.

This example is intended to compare the difference in the ability of the recombinant virus of HL-1 strain and the recombinant virus of 17 strain to transfect tumor cells after insertion of IL-12 gene and PD1 monoclonal antibody gene. The IC50 values of virus-transfected tumor cells were determined by the CCK-8 method, and the results of the IC50 assay of different virus-transfected tumor cells are shown in FIG. 12. The result shows that the IC50 values of the HL-1 recombinant viruses inserted with the IL-12 gene and the PD1 monoclonal antibody gene for transfecting tumor cells are smaller than those of 17 strains of recombinant viruses inserted with the IL-12 gene and the PD1 monoclonal antibody gene, and the HL-1 virus strain recombinant viruses inserted with the IL-12 gene and the PD1 monoclonal antibody gene have better infection efficiency and replication capacity in the tumor cells and strong oncolytic effect.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A modified herpes simplex virus lacking a functional ICP34.5 gene and a functional ICP47 gene, wherein the herpes simplex virus is derived from a virus having a accession number of CCTCC NO: strain of V201810.

2. The herpes simplex virus of claim 1, wherein the herpes simplex virus has one or more heterologous genes introduced.

3. The herpes simplex virus of claim 2, wherein the heterologous gene is selected from the group consisting of a gene encoding a cytokine that promotes an immune response and a gene encoding a monoclonal antibody having a tumor preventing and/or treating effect.

4. A herpes simplex virus of claim 3, wherein the cytokine is selected from one or more of the following: IL-2, IL-12 and GM-CSF.

5. The herpes simplex virus of claim 3, wherein the monoclonal antibody having an effect of preventing and/or treating a tumor is an immune checkpoint antibody.

6. The herpes simplex virus of claim 5, wherein the monoclonal antibody having an effect of preventing and/or treating a tumor is PD1 monoclonal antibody.

7. A composition comprising the herpes simplex virus of any one of claims 1-6.

8. The composition of claim 7, wherein the modified herpes simplex virus has one, two or three foreign genes inserted at the same site or at different sites.

9. The composition of claim 8, which is a mixture of two, three or four herpes simplex viruses, wherein different herpes simplex viruses have different foreign genes introduced.

10. The composition of any one of claims 7-9, which is a viral culture.

11. A host cell infected with a herpes simplex virus of any one of claims 1-6 or a composition of any one of claims 7-9.

12. A composition comprising the host cell of claim 11.

13. The composition of claim 12, which is a cell culture.