Genetically engineered bovine herpesvirus type 1 (BHV-1) for use in cancer therapy

Abstract

Recombinant BHV-1 oncolytic viruses are provided, including BHV-1 mutant strains that have enhanced cancer selectivity and / or enhanced immunostimulatory activity compared to wild-type BHV-1. The BHV-1 mutant strains are genetically engineered to express one or more immunomodulatory molecules that induce anti-tumor immune responses. Methods for producing the recombinant BHV-1 oncolytic viruses are also provided.

Description

[Technical Field]

[0001] The present invention relates generally to oncolytic viruses, and more particularly to a genetically engineered oncolytic bovine herpesvirus type 1 (BHV-1) that targets tumor cells and induces an anti-tumor immune response. [Background technology]

[0002] Oncolytic viruses are being actively researched as novel cancer treatments. Oncolytic viruses preferentially target and kill cancer cells. Some oncolytic viruses have a natural tumor tropism, while others are engineered to replicate in cancer cells. To be successful as cancer treatments, oncolytic viruses must be safe and effective. Some of the first oncolytic viruses tested in clinical trials were derivatives of the human herpesvirus herpes simplex virus type 1 (HSV-1). Researchers deleted specific HSV-1 genes to promote viral replication in actively dividing cells (i.e., cancer cells) rather than normal cells. The first and only FDA-approved oncolytic virus is an HSV-1-based vector called T-Vec. Because of pre-existing general population immunity to T-Vec (50–90% of the general population is latently infected with HSV-1, resulting in the production of neutralizing antibodies against the virus), its use is limited to accessible tumors.

[0003] Bovine herpesvirus type 1 (BHV-1), a close relative of HSV-1, possesses promising properties for clinical development of oncolytic viruses. BHV-1 has the ability to kill immortalized and transformed cells, suggesting its potential for activity across a range of tumor types, from preneoplastic to overt cancer. It targets tumor bulk and tumor-initiating cells, regardless of tumor status or subtype. Furthermore, BHV-1 does not cause disease in humans; therefore, pre-existing immunity to this virus does not exist in the human population. When oncolytic HSV-1 is mixed with commercially available human serum, the virus is neutralized, preventing it from infecting susceptible human tumor cells. However, the lack of neutralization with BHV-1 suggests the absence of neutralizing antibodies against BHV-1 in human serum.

[0004] BHV-1 glycoprotein I and glycoprotein E (gI and gE) are nonessential proteins that form noncovalent heterodimers within infected cells and the virion envelope. Mutations in gE and gI do not affect the kinetics of BHV-1 cell entry or cell exit in vitro, but they significantly reduce the size of BHV-1 plaques. BHV-1 gE deletion mutants and gI deletion mutants fail to form plaques in the presence of anti-BHV-1 antibodies, indicating that both glycoproteins gE and gI are involved in the cell-to-cell spread mechanism. Although the genes encoding BHV-1 glycoproteins gE and gI are nonessential for viral replication in cell culture, BHV-1 with single mutations in gI or gE or with double mutations in gI and gE exhibits significantly reduced pathogenicity in cattle.

[0005] BHV-1 possesses all the characteristics required for a successful oncolytic virus, including safety (it cannot replicate in normal human cells), efficacy (it can replicate in a wide range of cancer cell types), and broad applicability (it can be administered systemically because there is no pre-existing immunity), so it would be desirable to provide a BHV-1 that is engineered to have effective effects in vivo. Summary of the Invention [Means for solving the problem]

[0006] We have now constructed a recombinant BHV-1 vector that has high targeting selectivity for tumor cells and secretes molecules that enhance the host's anti-tumor immune response.

[0007] Thus, in one aspect of the present invention, a recombinant BHV-1 oncolytic virus is provided, comprising a BHV-1 mutant genetically engineered to express one or more immunomodulatory molecules that induce an anti-tumor immune response, wherein the BHV-1 mutant exhibits enhanced cancer selectivity and / or enhanced immunostimulatory activity compared to wild-type BHV-1 due to at least partial deletions or alterations introduced into one or more endogenous viral target genes.

[0008] In another embodiment, there is provided a method for producing a recombinant BHV-1 oncolytic virus, comprising: i) nucleofecting a cell line with a vector capable of expressibly incorporating a gene encoding an immunomodulatory molecule that induces an anti-tumor immune response into a target gene of BHV-1; and ii) A method for producing a recombinant BHV-1 oncolytic virus is provided, which comprises the step of infecting the cell line with wild-type BHV-1 under appropriate conditions to obtain a recombinant BHV-1 oncolytic virus that expresses the immunomodulatory molecule and has at least a partial deletion or modification introduced into the target gene of BHV-1, wherein the recombinant BHV-1 oncolytic virus exhibits higher cancer selectivity and / or higher immunostimulatory activity compared to wild-type BHV-1.

[0009] In another aspect, a method is provided for treating an individual with cancer, comprising administering to the individual a BHV-1 mutant strain, wherein the BHV-1 mutant strain exhibits increased cancer selectivity and / or increased immunostimulatory activity compared to wild-type BHV-1 due to partial or complete deletion or alteration in the expression of BHV-1 target genes.

[0010] These and other aspects of the present invention will be described with reference to the following drawings. [Brief explanation of the drawings]

[0011] Embodiments of the present invention are shown in more detail in the following specific description of the following drawings.

[0012] [Figure 1] Schematic diagram of the donor plasmid used to generate recombinant BHV-1 ΔgE mutant strains expressing immunomodulatory molecules (genes of interest: GOI).

[0013] [Figure 2] This is a graph showing the production amount of the protein encoded by the target gene (human GMCSF) in the supernatant of CRIB cells infected with BHV-1 wild-type (wt) or BHV-1 ΔgE-EGFP-huGMCSF isolate 3E, or in the supernatant of CRIB cells nucleofected with the donor plasmid of Figure 1.

[0014] [Figure 3] The effects of infection of normal human lung fibroblasts (HEL) and human lung cancer cells (A549) with BHV-1 wild-type (wt), BHV-1 ΔgI mutant, or BHV-1 ΔgE mutant at various multiplicities of infection (MOI, plaque-forming units per cell [pfu]) are shown by the disappearance of the cell monolayer after Giemsa staining.

[0015] [Figure 4]Graphs showing the results of preclinical testing of BHV-1 ΔgI-GFP in a syngeneic mouse model of melanoma. A) Graph showing individual tumor progression in C10 tumor-bearing mice treated with different combinations of mitomycin c (Mito), BHV-1 ΔgI-GFP, and checkpoint inhibitors (CPs). B) Graph showing survival curves. C) Results of an immunogenic cell death (ICD) assay. D) Graph showing in vitro viral replication in C10 cells in the presence or absence of Mito. E) Graph showing the mean tumor progression and corresponding survival curves for C10 tumor-bearing mice treated with PBS, C10 tumor-bearing mice treated with intratumoral injection of mitomycin c + BHV-1 ΔgI-GFP + checkpoint inhibitors (Triple comb IT), and C10 tumor-bearing mice treated with intravenous injection of mitomycin c + BHV-1 ΔgI-GFP + checkpoint inhibitors (Triple comb IV).

[0016] [Figure 5] Figure 1 shows DNA (A) and protein (B) sequence alignments of the UL49.5 gene of BHV-1 wild-type (WT), BHV-1-UL49.5Δ30-32ΔCT isolate 5C1, and the donor plasmid used to generate 5C1.

[0017] [Figure 6] Western blot analysis of TAP-1 production in A549 cells not treated with IFNγ (−IFNγ), A549 cells treated with IFNγ (+IFNγ), A549 cells treated with IFNγ and infected with wild-type BHV-1 (+IFNγ +wt), and A549 cells treated with IFNγ and infected with BHV-1-UL49.5Δ30-32ΔCT isolate 5C1 (+IFNγ +5C1).

[0018] [Figure 7]1 is a graph showing the viral replication levels of BHV-1 wild-type (wt) and UL49.5Δ30-32ΔCT isolate 5C1 in human normal cells (HEL) and human tumor cells (A549).

[0019] [Figure 8] Figure 10 is a graph showing tumor regression and increased survival in mice bearing C10 tumors treated with a combination of low-dose mitomycin c, anti-PD-L1 and anti-CTLA-4 antibodies, and wild-type (wt) BHV-1 (Comb. with wt), or a combination of low-dose mitomycin c, anti-PD-L1 and anti-CTLA-4 antibodies, and BHV-1 isolate 5C1 (Comb. with 5C1).

[0020] [Figure 9] This graph shows the amount of protein encoded by the target gene (human GMCSF) produced in the supernatant of multiple cell types infected with a recombinant BHV-1 overlap mutant (mutated UL49.5 and gE) prepared by co-infection of BHV-1 UL49.5Δ30-32ΔCT-RFP isolate 5C1 and BHV-1 ΔgE-EGF-huGMCSF isolate 3E.

[0021] [Figure 10] The effects of infection of normal human lung fibroblasts (HEL) and mouse tumor cells (C10) with BHV-1 wild-type (wt), BHV-1 ΔgI mutant, BHV-1 ΔgE mutant, or BHV-1 ΔgIΔgE mutant at various MOIs are shown by the disappearance of the cell monolayer after Giemsa staining (A) and cell viability quantified with AlamarBlue (B).

[0022] [Figure 11]Graphs showing the results of preclinical testing of BHV-1 ΔgIΔgE in a syngeneic mouse model of melanoma. A) Survival curves for mice bearing C10 tumors treated with three different combinations of mitomycin c, oncolytic viruses (BHV-1 wt, ΔgI, ΔgE, or ΔgIΔgE), and checkpoint inhibitors. B) Tumor progression in individual mice. DETAILED DESCRIPTION OF THE INVENTION

[0023] A recombinant BHV-1 oncolytic virus is provided, which comprises a BHV-1 mutant strain that has higher cancer selectivity and / or higher immunostimulatory activity compared to wild-type BHV-1, characterized in that the BHV-1 mutant strain has been genetically modified to express one or more molecules that induce an anti-tumor immune response.

[0024] Bovine herpesvirus type 1 (BHV-1 or BoHv-1) is a virus of the Herpesviridae family, subfamily Alphaherpesvirinae, known to cause diseases such as rhinotracheitis, vaginitis, balanoposthitis, abortion, growth retardation, conjunctivitis, and enteritis in cattle worldwide. The genome of the BHV-1 strain contains approximately 135 kb of double-stranded DNA with approximately 72 coding regions.

[0025] As used herein, the term "BHV-1 mutant" refers to a mutant virus prepared from wild-type BHV-1 virus that has been mutated to exhibit high cancer selectivity and / or high immunostimulatory activity. The term "high cancer selectivity" is used herein to describe a BHV-1 mutant that has low normal cell killing ability, e.g., at least 10% less than the killing ability of wild-type BHV-1 against normal cells such as HEL fibroblasts, or little or no killing ability, and / or a BHV-1 mutant that has high cancer cell killing ability, e.g., at least 10% more than the killing ability of wild-type BHV-1 against cancer cells such as A549 cells. Viral killing ability can be visually confirmed by Giemsa staining after viral infection and measuring the area of ​​the cell monolayer that has disappeared. Viral killing ability can also be confirmed by measuring the degree of decrease in cell viability after a certain period of time has passed since viral infection. Cell viability is measured using commercially available cell viability assays such as, but not limited to, AlamarBlue, MTS test or CellTiter-Glo.

[0026] As used herein, the term "high immunostimulatory activity" refers to a BHV-1 mutant strain that stimulates host immune defenses by losing the function of the viral protein UL49.5, which activates the degradation of viral proteins involved in evading host immune defenses, such as the transporter associated with antigen processing (TAP). The loss of function of such viral proteins in a BHV-1 mutant strain can be confirmed based on the expression of cellular genes or proteins involved in immune surveillance, such as TAP expression. For example, a BHV-1 mutant strain is considered to have high immunostimulatory activity if the expression of viral proteins (e.g., UL49.5 or other proteins involved in evading host immune defenses) is reduced or absent or has little or no effect on the immune defenses of host cells upon infection, i.e., the host immune defenses are essentially equivalent to those of uninfected host cells. Therefore, host cells infected with a BHV-1 UL49.5 mutant strain exhibit TAP expression levels comparable to those of uninfected host cells. Cellular gene or protein expression is measured using methods such as, but not limited to, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting.

[0027] Generally, in the production of BHV-1 mutants, deletions or modifications can be introduced into portions of one or more target genes in the viral nucleic acid backbone to obtain BHV-1 mutants with enhanced cancer selectivity and / or enhanced immunostimulatory activity compared to wild-type BHV-1. Specifically, BHV-1 with enhanced cancer selectivity can be obtained by completely or at least partially inhibiting or otherwise altering the expression of BHV-1 genes, such as genes associated with BHV-1 pathogenicity, through deletion of the entire target gene or deletion or modification of one or more portions of the target gene. For example, genes that affect viral replication; genes that affect host defense mechanisms; genes that affect viral tropism, spread, or infectivity; and genes that encode or produce products that are directly toxic to the host are target genes that can be mutated to obtain BHV-1 mutants with enhanced cancer selectivity and / or enhanced immunostimulatory activity. Examples of BHV-1 genes that can be deleted or modified to enhance cancer selectivity include, but are not limited to, the glycoprotein E gene (gE) and the glycoprotein I gene (gI). Additionally, an example of a BHV-1 gene into which modifications can be introduced to enhance immunostimulatory activity is the UL49.5 gene.

[0028] In one embodiment, a BHV-1 mutant strain is provided that contains a mutation that inhibits expression of one or more glycoproteins involved in viral cell-to-cell spread. For example, the BHV-1 mutant strain contains a genetic mutation that eliminates expression of one or more of the glycoproteins gI and gE. Those skilled in the art will appreciate that various mutations can be incorporated into the nucleic acid backbone of BHV-1 to obtain such mutant strains, for example, mutant strains with alterations in targeted glycoprotein genes or mutant strains with the entire glycoprotein gene deleted.

[0029] In another embodiment, a BHV-1 mutant strain is provided that contains a mutation that stimulates the host immune response by promoting antigen presentation. One example is a mutant strain that expresses a mutant UL49.5 that does not degrade the host immune defense proteins TAP1 or TAP2. The UL49.5 protein interferes with peptide transport by inhibiting the transporter associated with antigen processing (TAP). Examples of UL49.5 mutant strains include, but are not limited to, mutant strains that lack amino acids 30-32 (RRE) and the C-terminal tail (by introducing a stop codon).

[0030] In a further embodiment, a BHV-1 mutant strain is provided that contains at least two mutations, for example, mutations in different BHV-1 target genes. Thus, a BHV-1 mutant strain is provided that inhibits a glycoprotein involved in viral propagation and reduces viral replication in healthy cells by inhibiting or inactivating a specific protein, or a BHV-1 mutant strain is provided that inhibits a glycoprotein involved in viral propagation and has a mutation that enhances the host immune response. As an example, a mutant strain is provided in which the expression of at least one of gI and gE is inhibited and UL49.5 is inactivated. As another example, a mutant strain is provided in which the expression of both gI and gE is inhibited and, optionally, UL49.5 is inactivated.

[0031] Selected BHV-1 mutant viruses with high cancer selectivity and / or high immunostimulatory activity may be further genetically modified to express one or more immunomodulatory molecules that enhance the virus-mediated anti-tumor immune response.

[0032] This genetic modification can be achieved by inserting a construct into the viral genome that is designed to express a gene of interest, such as a gene encoding an immunomodulatory molecule of interest. Examples of immunomodulatory molecules that induce anti-tumor responses include, but are not limited to, ectocalreticulin (ecto-CRT), high-mobility group box 1 protein (HMGB1), and chemokines and cytokines that enhance tumor immunogenicity (e.g., interferons such as interferon-alpha (INF-α), interleukins such as interleukin-2 (IL-2), erythropoietin, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and hematopoietic growth factors such as granulocyte-colony-stimulating factor (G-CSF)).

[0033] Generally, the construct contains a gene of interest and includes restriction enzyme sites at both ends. This configuration allows the gene of interest to be inserted adjacent to and downstream of a promoter that drives expression of the gene of interest, located between flanking regions homologous to viral sequences in a platform vector (e.g., a plasmid). In one embodiment, the homologous viral sequence in the platform vector comprises the gE locus of BHV-1 into which the construct is designed to be inserted. The gE protein of BHV-1 is a non-essential protein and is therefore not required for viral replication, but it functions to promote viral neuronal spread. In another embodiment, the homologous viral region in the platform vector comprises the gI locus of BHV-1 into which the construct is designed to be inserted.

[0034] The promoter in the construct may be any promoter suitable for driving expression of a gene of interest. Commonly used promoters include the CMV promoter, PGK1 promoter, EF1a promoter, SV40 promoter, and CAG promoter. The platform vector may also contain a reporter gene that is not naturally expressed in cells, for use in confirming vector integration into BHV-1. Commonly used reporter genes include those that produce visually identifiable results, such as genes expressing fluorescent or luminescent proteins, such as green fluorescent protein (GFP or EGFP), red fluorescent protein (RFP), or blue fluorescent protein (BFP), and genes expressing enzymes that catalyze luminescent / colorigenic reactions, such as luciferase, an enzyme that catalyzes a reaction with luciferin to produce light.

[0035] Recombinant BHV-1 mutant strains are generated by introducing a platform vector encoding a gene of interest into the wild-type BHV-1 genome. Generally, recombinant BHV-1 mutant strains are generated by co-transfecting wild-type BHV-1 genomic DNA and the platform vector into a cell line capable of BHV-1 replication and efficient transfection of the platform vector and BHV-1. Transfection can be achieved by chemical methods, such as calcium phosphate, cationic polymers, or liposomes; physical methods, such as electroporation, sonoporation, or optical transfection; or particle methods, such as particle bombardment or gene gun delivery.

[0036] In a preferred embodiment, transfection of the platform vector into a selected cell line is performed using a modified electroporation method known as "nucleofection," which combines electrical parameters generated by a device called a Nucleofector with cell-type-specific reagents. The vector is introduced into the cell nucleus and cytoplasm. Optimal nucleofection conditions depend on the selected cell type, not the vector being transfected. Many nucleofection programs and reagent kits are available. In one embodiment, the Nucleofector kit R and program X-100 are used to introduce the platform vector into selected cells, such as CRIB cells (bovine cells resistant to BVDV infection), and then this vector is integrated into BHV-1 to generate a BHV-1 mutant. Other nucleofection kits and programs are also available.

[0037] After nucleofection, the cells are infected with wild-type BHV-1 using methods established in the art.

[0038] The generation of the desired recombinant BHV-1 mutant can be confirmed based on the expression of a reporter gene or a gene of interest in the recombinant BHV-1 mutant. To facilitate detection and isolation of the recombinant BHV-1 mutant, an enrichment step may be performed to isolate cells expressing the reporter gene. For example, fluorescence-activated cell sorting (FACS) may be used.

[0039] BHV-1 mutant strains, whether or not genetically engineered to express one or more immunomodulatory molecules, can be administered to individuals to treat cancers, including, but not limited to, lung cancer, colon cancer, breast cancer, prostate cancer, kidney cancer, ovarian cancer, and CNS cancer. Generally, the BHV-1 mutant strains of the present invention may be combined with a pharmaceutically acceptable carrier to form a formulation suitable for administration. Here, a pharmaceutically acceptable carrier refers to a carrier that does not adversely affect the viability or activity of the BHV-1 mutant strain and does not exhibit unacceptable toxicity or other undesirable properties upon administration to an individual. Those skilled in the art will appreciate that the carrier selected will depend on the mode of administration of the formulation. In one embodiment, the BHV-1 mutant strain is formulated into a formulation suitable for administration by infusion or injection, for example, subcutaneous, intraperitoneal, intramuscular, intravenous, or intratumoral administration. Therefore, the BHV-1 mutant strain is prepared as a suspension in a physiologically acceptable medical-grade carrier, such as a pyrogen-free sterile aqueous solution (optionally buffered or isotonic). The carrier may be distilled water, a sterile sugar-containing solution (e.g., sucrose or dextrose), or sterile saline containing sodium chloride, which may be buffered. Suitable sterile saline solutions include those containing various concentrations of sodium chloride. Physiological saline may optionally contain additional components, such as sugars such as dextrose or sucrose. Examples of saline solutions containing additional components include Ringer's solution, e.g., lactated Ringer's solution or acetate Ringer's solution, phosphate-buffered saline (PBS), TRIS ((hydroxymethyl)aminomethane)-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard citrate saline (SSC), HEPES-buffered saline (HBS), and Gey's balanced salt solution (GBSS). The formulation may also contain a cryoprotectant.

[0040] The BHV-1 mutant strain can be administered to an individual in a therapeutically effective amount for the treatment of cancer. As used herein, the term "individual" refers to humans and non-human mammals (e.g., excluding cattle), preferably humans. The term "therapeutically effective amount" refers to an amount of the BHV-1 mutant strain sufficient to treat cancer, but not exceeding an amount that may cause significant side effects. Those skilled in the art will appreciate that the therapeutically effective dose of the BHV-1 mutant strain will vary depending on various factors, such as the nature of the condition being treated, the individual being treated, and the mode of administration. The appropriate dose can be determined by appropriate controlled clinical trials. Generally, the dose is about 10 6 ~10 11 Doses in the range of plaque-forming units (pfu) are preferred, e.g., 10 7 pfu, 10 8 pfu, 10 9 pfu or 10 10 It may be pfus.

[0041] The recombinant BHV-1 mutant viruses of the present invention may be used in combination with other anti-cancer therapies, including, but not limited to, chemotherapeutic agents such as mitomycin c, dacarbazine, 5-fluorouracil, epirubicin, cyclophosphamide, or 5-azacytidine; immune checkpoint inhibitors such as pembrolizumab or nivolumab; immunogenic antibodies; or immune cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR) T-cell therapy.

[0042] The oncolytic virus provided by the recombinant BHV-1 mutant strain of the present invention is advantageous because it exhibits higher killing ability against a broader range of cancer cell types than against normal human cells, and / or exhibits high immunostimulatory activity, and further expresses immunomodulatory molecules that enhance antitumor effects. Furthermore, the BHV-1 of the present invention is equally effective when administered intravenously or intratumorally. Also provided is a method for producing the recombinant BHV-1 mutant strain of the present invention, which overcomes the challenges involved in producing the recombinant BHV-1 mutant strain of the present invention. [Example]

[0043] The following specific examples illustrate embodiments of the present invention, but should not be construed as limiting the invention.

[0044] Example 1: Generation of BHV recombinant mutant strains A recombinant BHV mutant strain useful as an oncolytic virus was prepared and genetically engineered to express a target gene. The methods and materials used are as follows.

[0045] The following cells and viruses were used: 1) CRIB cells: Obtained from Dr. Clinton Jones (University of Oklahoma). These cells are a derivative of MDBK cells that are resistant to BVDV infection. When BHV-1 was propagated in CRIB cells, 2.6 × 10 8 The titers of pfu / mL were shown, demonstrating that CRIB was equivalent to MDBK in terms of BHV-1 growth. 2) U2OS: Obtained from ATCC. 3) HEK293: Obtained from Dr. Frank Graham. 4) BHV-1 Cooper strain: Wild-type BHV-1 was obtained from ATCC (catalog number VR-864, lot number 61236466).

[0046] Co-transfection of viral DNA and donor plasmid: We attempted co-transfection of viral DNA (BHV-1 wild-type) and donor plasmid (Figure 1), but this was unsuccessful due to the difficulty in finding cell lines capable of both transfection and viral replication. Specifically, we attempted recombination using the co-transfection method in HEK-293 cells and CRIB cells, but were unable to recover virus.

[0047] Transfection-infection method: Because virus recovery was not achieved by co-transfection of viral DNA, an alternative method was employed. The transfection-infection method involves transfecting various cell lines with a donor plasmid (Figure 1) using a lipid-based reagent, followed by infection of the transfected cells with virus (BHV-1 wild-type). This method yielded minimal success in various cell lines, as shown below (Table 1). [Table 1]

[0048] To obtain recombinant viruses, a cell line that can transfect donor plasmids efficiently and maintain high levels of BHV-1 replication is required. Unfortunately, we were unable to isolate recombinant viruses using this method.

[0049] Nucleofection-infection method: It has been reported that transfection efficiency using lipid-based transfection reagents is low in MDBK cells (the parent cells of CRIB cells). Because efficient viral replication is maintained in CRIB cells, we investigated alternative methods for delivering plasmid DNA into these cells, such as nucleofection. Nucleofection (e.g., Amaxa's Nucleofector® technology) uses a combination of cell-specific reagents and electrical parameters to form pores in both the cytoplasmic and nuclear membranes of cells. This technique has been reported as a method for DNA delivery into difficult-to-transfect cell lines (Hamm et al. Tissue Eng. 2002; 8(2):235-45; Maasho et al. J Immunol Methods. 2004; 284(1-2):133-40). An additional protocol advantage of this technique is that it allows DNA to be delivered directly into the nucleus, where viral replication occurs.

[0050] CRIB cells (1 x 10 6Nucleofection was performed using an Amaxa Nucleofector® II device, Nucleofector® kit R, and program X-001 in combination with 1.5 μg of pMAX-GFP plasmid DNA (nucleofection control plasmid). Images were taken with an inverted fluorescence microscope 5 hours after nucleofection. GFP fluorescence was confirmed in approximately 75% of the cells, indicating that the plasmid DNA had been incorporated. Because of this improved DNA delivery capacity into CRIB cells, recombinant virus production was performed using co-nucleofection (viral DNA + donor plasmid) or nucleofection-infection protocols, as described below.

[0051] Construction of BHV-1 ΔgE-EGFP-huGMCSF: A platform plasmid (clone ID: X31749) containing the pCAG promoter (CMV enhancer / chicken β-actin promoter) was ordered from Genscript. A construct containing a gene of interest to be inserted into the BHV-1 gE locus within the platform plasmid was prepared. This construct contains green fluorescent protein (EGFP) and the gene of interest (GOI) (human GMCSF), separated by a P2A self-cleavage (ribosomal skip) site. In preparation for homologous recombination, this construct was cloned downstream of the promoter between the flanking sequences of BHV-1 gE within the platform plasmid, as shown in Figure 1. Recombinants were screened for the presence or absence of EGFP. The EGFP protein is cleaved from the protein expressed by the gene of interest via the P2A site. The huGMCSF gene is contained in the T-VEC genomic sequence and can be measured by ELISA using cell supernatants. After transfection of CRIB cells, expression of GMCSF in the cell culture medium was confirmed by ELISA. Following the nucleofection-infection protocol, recombinant viruses were screened by fluorescence microscopy. Eight 6-well plates were examined, and one fluorescent plaque was observed.

[0052] An additional enrichment step was performed to facilitate detection and isolation of the recombinant virus. Fluorescence-activated cell sorting (FACS) was used to isolate CRIB cells expressing EGFP (approximately 0.05% of the cell population) after viral infection. After enrichment by FACS, the EGFP-positive cells were plated onto a monolayer of uninfected CRIB cells and examined for the presence of EGFP-expressing viral plaques. A total of eight GFP-positive plaques were obtained in two 6-well plates, and several of these were purified. The sequence of isolate 3E was confirmed by sequencing, and expression of human GMCSF was also confirmed by ELISA (Figure 2). GMCSF was not detected when cells were infected with wild-type virus (BHV-1 wt). Furthermore, GMCSF production was significantly lower when cells were nucleofected with the donor plasmid (Figure 1) than when infected with BHV-1 isolate 3E.

[0053] Validation of BHV-1 recombination / purification method: A plasmid containing the EGFP-P2A-muGMCSF sequence (clone ID: M17993) was obtained from Genscript and cloned into the ΔgE platform vector. The BHV-1-ΔgE-EGFP-murine GMCSF virus (virus isolate A5) was generated using the nucleofection-infection method and FACS purification. The sequence of this virus was confirmed by sequence analysis. Expression of murine GMCSF in cell culture medium was confirmed by ELISA. protocol BHV-1 Recombination Schedule Day 1: Design the sequence. Day 24: Receive plasmid DNA. Day 24: Nucleofection / infection with CRIB is performed. Day 26: Virus is harvested and titered. Day 29: Stain titer plates. Day 31: Divide CRIB into T150 for fractionation. Day 32: Infect with T150 CRIB. - Prepare cells for sorting 12-18 hours after infection. - The sorted cells are seeded at a density of 0.3 cells / well into a 96-well plate containing uninfected CRIB (approximately 60% confluent). Day 34: GFP+ cells in 96-well plates are screened using Typhoon and confirmed under a fluorescent microscope. Day 36: Harvest virus from GFP+ cells. Day 39: The virus is diluted and used to infect CRIB cells seeded in a 96-well plate at 0.3 pfu / well to obtain isolated viral particles. Day 43: Serially dilute and infect CRIB cells seeded in 96-well plates at 0.3 pfu / well. Day 46: Titer the virus to ensure that all plaques express GFP. If there are any GFP-negative plaques, further purification is required. Day 48: If the virus forms pure plaques, infect one T150 for seed stock generation. Day 51: Harvest the preparation and titer it. Day 54: Infect 20 T150s to obtain a sufficient preparation. Day 56: Recovery Day 57: Purification Day 58: Titer measurement Day 61: Ready for characterization Nucleofection / infection protocol for BHV-1 homologous recombination Nucleofection: 1. Remove the Nucleofector Reagent (Kit R) from 4°C and allow it to warm to room temperature. 2. Add 2 mL of DMEM + 5% HS to each well of a 6-well plate and equilibrate in a 37°C incubator. 3. Trypsinize CRIB cells (1x T150). 4. 1×10 6 Transfer 10 cells (per reaction) to a 15 mL conical tube. 5. Centrifuge (650 rpm, 10 minutes, room temperature). 6. Suspend the cell pellet in 100 μL (per reaction) of Nucleofector solution R (82 μL Nucleofector solution + 18 μL supplement). 7. Prepare plasmid DNA (1-2 μg) in a sterile Eppendorf tube. 8. Transfer 100 μL of cells (suspended in Nucleofector solution) to the Eppendorf tube containing the DNA. 9. Transfer cells / DNA to a cuvette. 10. Perform nucleofection using program X-001. 11. Add 500 μL of equilibrated medium to the cuvette. 12. Transfer the cells to one well of a 6-well plate containing 1.5 mL of equilibrated medium. 13. Return the plate to the incubator. infection 1. Check for fluorescence under a microscope. 2. Five hours after nucleofection, cells are infected with BHV (MOI=0.05). -Wash the cells with PBS. - Add 400 μL to each well for infection and leave at 37°C for 1 hour with shaking every 10 minutes. -Add DMEM + 1% FBS. Virus recovery 1. Collection will be made once 100% CPE is confirmed (usually the next day). 2. Detach the cells by pipetting and resuspend them in the medium. 3. Transfer to a 15 mL conical tube. 4. Freeze (-80℃) / thaw (37℃) three times. 5. Sonicate (1 x 30 seconds). 6. Centrifuge (1500 rpm, 10 minutes, 4°C). 7. Transfer the supernatant to a 2 mL tube. 8. Take a 25 μL aliquot for titration and store the remaining virus frozen at -80°C. Flow sorting for recombinant BHV enrichment 1. Seed 1x T150 for each recombination reaction to be screened (also 1x T150 for the -ve control). 2. The next day, infect at an MOI of 0.1 (nucleofection / infection) or MOI of 0.01 (co-infection). 3. At the time of infection, prepare a flow sorting tube (4 mL, polypropylene) containing harvest buffer (medium + 1% L-glutamine + 30% FBS). 4. 12 hours after infection, prepare samples as follows: 5. Wash the cells with PBS. 6. Add 2 mL of TrypLE. 7. Place back in the 37°C incubator and wait until the cells begin to detach. 8. Add 10 mL DMEM + 5% FBS to inactivate TrypLE. 9. Spin down (650 x g, 10 minutes, 4°C). 10. Resuspend the pellet in sorting buffer (PBS, 1% BSA, 5mM EDTA). 11. Count the cells using a hemocytometer. 12. 2.5×10 6 Dilute to 1000 / mL. 13. Pass through a filter cap just before separation. Cells are sorted. This is an enrichment step, not a purification step. Sorted cells are added at 0.3 cells / well to uninfected CRIB monolayers in 96-well plates. After sorting 1. Count the total number of cells sorted into the GFP+ve population. 2. Add the sorted cells at 0.3 cells / well to a 96-well plate containing a CRIB monolayer (approximately 60% confluent). 3. Place the plate in an incubator overnight. 4. Check for GFP expression. 5. Once plaques are observed, select GFP+ve plaques under a fluorescence microscope. 6. Harvest all cells and virus from GFP+ve wells. 7. Repeat the dilution and isolation in 96-well plates until pure virus is obtained.

[0054] Example 2: BHV-1 ΔgE mutant and BHV-1 ΔgI mutant The BHV-1 ΔgE mutant was generated as described in Example 1. The BHV-1 ΔgE-EGFP-muGMCSF mutant was compared with the BHV-1 ΔgI-GFP mutant obtained from Dr. Gunther Keil (Friedrich-Loeffler-Institut, Germany). For in vitro safety characterization, human lung fibroblasts (HEL) and human lung adenocarcinoma cells (A549) were infected with BHV-1 wild-type (wt), BHV-1 gE deletion mutant (ΔgE), and BHV-1 gI deletion mutant (ΔgI) at various MOIs. All BHV-1 deletion mutants exhibited lower killing potency against normal human fibroblasts than the wild-type at the highest MOI tested, with the ΔgE mutant exhibiting the least cytopathic effect (Figure 3). On the other hand, the wild-type and ΔgE mutant strains showed comparable killing ability against A549 cancer cells. Both mutants also showed comparable killing ability against C10 mouse tumor cells. These results indicate that both mutants have higher cancer selectivity than the wild-type strain, suggesting that deletion of both gI and gE may result in a virus that retains its killing ability against tumor cells while being safer than the wild-type.

[0055] We also conducted preclinical studies of BHV-1 ΔgI-GFP. Testing of BHV-1 ΔgI-GFP was performed using the C10 tumor model in C57Bl / 6 mice (Miller et al. Mol Ther. 2001;3(2):160-8). The C10 model is the first syngeneic mouse cancer model susceptible to BHV-1 entry. Because mouse cells lack the receptor essential for BHV-1 entry, they are not susceptible to BHV-1. C10 is a B16 mouse melanoma cell clone expressing human nectin-1. C10 cells can reproducibly form tumors in C57Bl / 6 mice, and human nectin-1 expression does not induce detectable tumor immunogenicity in vivo. Preliminary in vitro results confirmed that nectin-1 expression significantly enhanced BHV-1 entry into non-susceptible B16 cells.

[0056] The treatment regimen used was as follows: Once tumors reached a treatable size, a single 100 μg dose of mitomycin C was administered intratumorally (day 1), followed by 2×10 7 Plaque-forming units (pfu) of BHV-1 ΔgI-GFP were administered intratumorally or intravenously three times (on days 2, 3, and 4), and anti-CTLA-4 checkpoint inhibitory antibodies and anti-PD-L1 checkpoint inhibitory antibodies (200 μg each) were administered intraperitoneally once every three days for a total of 10 times.

[0057] Even though BHV-1 ΔgI-GFP showed low in vitro infection proliferation and complete inhibition of viral replication by chemotherapy (Figure 4C), BHV-1ΔgI-GFP demonstrated improved tumor control (Figure 4A / 4E) and animal survival (Figure 4B / 4E) compared to control groups. Furthermore, infection with the slow-growing BHV-1 ΔgI-GFP was sufficient to induce a host immune response against C10 tumors (Figure 4D). The initiation or enhancement of a systemic antitumor immune response can be explained in part by the ability of therapeutics such as oncolytic viruses to induce immunogenic cell death (ICD) of cancer cells. A standard assay for assessing ICD uses dying tumor cells as a vaccine to determine whether the type of cell death is sufficient to induce an immune response that suppresses or controls subsequent tumor formation (Kepp et al. Oncoimmunology. 2014:3(9):e955691). This assay revealed that vaccination with BHV-1 ΔgI-GFP-infected dying cells was more effective in suppressing tumor growth than mitomycin c treatment alone and was as effective as mitomycin c and BHV-1 ΔgI-GFP treatment (Figure 4D). Thus, BHV-1 ΔgI-GFP was sufficient to induce ICD in C10 cells, even when de novo virus production was very low and even when de novo virus production was inhibited by mitomycin c. Therefore, in vivo antitumor activity did not correlate with viral replication. While previous findings have shown that the in vivo efficacy of oncolytic viruses correlates with their ability to activate antitumor immune responses, our findings do not show the same trend, highlighting the specificity of BHV-1's mechanism of action.

[0058] These experiments demonstrated the high cancer selectivity of the recombinant BHV-1 ΔgE and ΔgI mutants, and the in vivo efficacy of the recombinant BHV-1 ΔgI mutants in combination with low-dose chemotherapy and checkpoint immunotherapy. Furthermore, these experiments demonstrated that intravenous administration was as effective as intratumoral administration in inhibiting tumor progression and survival.

[0059] Example 3: BHV-1 UL49.5 mutant strain The BHV-1 UL49.5 gene product, glycoprotein N, normally inhibits antigen presentation in virus-infected cells, "hiding" the virus from the immune system and maximizing viral replication and spread. However, the most important goal of oncolytic viruses is thought to be to stimulate and stimulate the immune system against cancer, presenting both viral and tumor-specific proteins. Therefore, introducing mutations into the UL49.5 gene may enhance antigen presentation and promote a stronger anti-cancer immune response.

[0060] Therefore, we constructed a BHV-1 mutant strain, the BHV-1-UL49.5Δ30-32ΔCT mutant, that retains the expression and function of the host antigen processing-associated transporter 1 (TAP-1). Specifically, two mutations were introduced into the UL49.5 gene of BHV-1: a deletion of amino acids 30–32 (RRE) and a deletion of the C-terminal tail (by introducing a stop codon). To confirm these two mutations, we plaque-purified the constructed recombinant virus and sequenced the UL49.5 gene. The nucleic acid (Figure 5A) and protein (Figure 5B) sequence alignments of the UL49.5 gene of the wild-type (wt), recombinant virus (clone 5C1), and donor plasmid (delta3032deltaC) are shown in Figure 5. The consensus (cons) sequence confirmed that there were no additional mutations in the UL49.5 gene and the corresponding protein.

[0061] Plasmid DNA: The plasmid (pUC57simple) used to generate the BHV-1 UL49.5 mutant strain contains the desired UL49.5 gene sequence, i.e., deletion of amino acids 30–32 and the cytoplasmic tail. This plasmid was also modified to express mCherry (red fluorescent protein; RFP) and to contain a P2A cleavage site immediately upstream of the UL49.5 initiation codon.

[0062] Generation of UL49.5Δ30-32ΔCT-RFP virus: The above plasmid DNA was nucleofected into CRIB cells using the Nucleofector kit R and program X-100. Five hours after nucleofection, the cells were infected with wild-type BHV-1 (MOI = 0.05). Plaque screening in a 6-well plate yielded six RFP-positive plaques. The resulting isolate (5C1) was further plaque-purified twice to confirm its purity. The introduction of the mutation was confirmed by PCR amplification and sequence analysis (Figure 5).

[0063] Characterization of BHV-1-UL49.5Δ30-32ΔCT: We confirmed that the BHV-1-UL49.5Δ30-32ΔCT isolate 5C1 does not possess the ability to degrade TAP-1. Figure 6 shows that TAP-1 is produced when induced with IFNγ (+IFNγ), but further infection with BHV-1 wild-type (+IFNγ +wt) results in TAP-1 degradation. However, infection with the BHV-1 mutant strain 5C1, but not the wild-type, did not result in TAP-1 degradation (+IFNγ +5C1).

[0064] We also confirmed that the BHV-1-UL49.5Δ30-32ΔCT mutant retained its therapeutic index in human cancer cells. Figure 7 shows that BHV-1-UL49.5Δ30-32ΔCT grows to titers similar to wild-type BHV-1 (wt) in human lung adenocarcinoma (A549 cells) but not in normal human lung fibroblasts (HEL) (Figure 7). Furthermore, BHV-1-UL49.5Δ30-32ΔCT has the same cytopathic effect (cell killing ability) as the wild-type strain across a wide range of multiplicities of infection. Therefore, recombinant BHV-1-UL49.5Δ30-32ΔCT mutants engineered to express a selected GOI are also suitable for use in the present invention.

[0065] BHV-1-UL49.5Δ30-32ΔCT isolate 5C1 was tested in the C10 tumor model as in Example 2, using the same treatment regimen (a single intratumoral injection of 100 μg mitomycin C (day 1), 2×10 BHV-1 wild-type or 5C1). 7 The mice were treated with three intratumoral injections of 100 μg pfu each (days 2, 3, and 4), and anti-CTLA-4 and anti-PD-L1 checkpoint inhibitor antibodies (200 μg each) were administered intraperitoneally once every three days (10 times total). There were no significant differences in tumor growth or survival between BHV-1 wild-type (WT) and BHV-1 5C1 (Figure 8). All three combination treatments using WT and 5C1 more than doubled median survival, demonstrating significant improvement over the PBS control group.

[0066] This experiment demonstrated that the BHV-1-UL49.5Δ30-32ΔCT mutant strain possesses high immunostimulatory activity and retains wild-type in vivo efficacy.

[0067] Example 4: BHV-1 with UL49.5 mutations and gE deletions and expressing human GMCSF Using the BHV-1-UL49.5Δ30-32ΔCT vector as a foundation, further engineered recombinant vectors can be constructed, for example, by incorporating the gE plasmid construct (Figure 1) designed to express a GOI, such as an immunostimulatory molecule, that enhances immunogenicity and induces a stronger anti-tumor immune response. Thus, a mutant strain that combines these two modifications, i.e., a viral UL49.5 mutant with a gE (or gI) deletion engineered to express a GOI of choice, can induce a stronger anti-tumor immune response than wild-type BHV-1. An additional advantage of introducing a molecule of interest into the gE or gI locus is that the gE or gI deletion enhances the safety of the vector by reducing its ability to kill normal, healthy cells while still allowing viral replication in permissive cancer cells.

[0068] Therefore, we combined the co-infection method with FACS enrichment (described in Example 1) to generate the overlapping mutant virus Q5A from the two single-mutant viruses described above (BHV-1-UL49.5Δ30-32ΔCT isolate 5C1 and BHV-1 ΔgE-EGFP-huGMCSF isolate 3E). This virus contains the UL49.5 mutation (described in Example 3), a gE deletion, and expresses human GMCSF (described in Example 1). Expression of GMCSF by this virus was confirmed in several different cell types, even in the absence of viral replication (Figure 9).

[0069] This experiment demonstrated that it is possible to generate BHV-1 overlapping mutants, for example, BHV-1 mutants with UL49.5 mutations and gE / gI deletions that express immunomodulatory molecules such as GMCSF.

[0070] Example 5: BHV-1 ΔgIΔgE mutant strain The BHV-1 ΔgIΔgE mutant was generated as described in Example 1. The BHV-1 ΔgIΔgE mutant was compared with the BHV-1 wild-type (wt) and the single mutants BHV-1 ΔgE and BHV-1 ΔgI. For in vitro safety characterization, human lung fibroblasts (HEL) were infected with the BHV-1 wild-type and each mutant at various MOIs. All mutants exhibited lower killing activity against normal human fibroblasts than the wild-type at the highest MOI tested (Figures 10A and 10B). The wild-type and mutant strains exhibited comparable killing activity against C10 mouse tumor cells (Figures 10A and 10B). These results indicate that the BHV-1 ΔgIΔgE mutant, like the single mutants, has higher cancer selectivity than the wild-type strain, and suggest that deletion of either or both gI and gE may result in a virus that is safer than the wild-type strain while retaining its killing ability against tumor cells.

[0071] Furthermore, a preclinical study of the BHV-1 ΔgIΔgE double mutant was performed using a C10 tumor model in a similar manner to that described in Example 2, and compared with the wild-type and single mutant strains BHV-1 ΔgI and BHV-1 ΔgE. The same treatment regimen as in Example 2 (a single intratumoral administration of 100 μg of mitomycin C (day 1) and 2 × 10 BHV-1 wild-type or mutant strains) was used. 7 pfu three times intratumoral injection (days 2, 3, and 4), and anti-CTLA-4 checkpoint inhibitor antibody and anti-PD-L1 checkpoint inhibitor antibody (200 μg each) were administered intraperitoneally once every three days for a total of 10 injections. The three combination treatments with wild-type and mutant strains more than doubled median survival time, demonstrating significant improvement in survival compared to the PBS group (Figure 11A). Furthermore, combination treatment with the overlapping mutant BHV-1 ΔgIΔgE strain demonstrated improved tumor control compared to the wild-type group (Figure 11B).

Claims

1. A formulation for human administration containing a combination of a recombinant bovine herpesvirus type 1 (BHV-1) mutant virus and a pharmaceutically acceptable carrier, the virus is an oncolytic virus, and is characterized in that it exhibits higher cancer selectivity and / or higher immunostimulatory activity compared to wild-type BHV-1 due to mutations introduced into one or more target genes of BHV-1; the target gene is a gene selected from the group consisting of a gene expressing glycoprotein I (gI), a gene expressing glycoprotein E (gE), and a gene expressing UL49.5; The preparation, wherein the mutation inhibits expression of the target gene.

2. The formulation described in claim 1, wherein the virus has been genetically modified to express a gene encoding an immunomodulatory molecule.

3. 2. The formulation of claim 1, wherein the target genes comprise two genes selected from the group consisting of a gene expressing gI, a gene expressing gE, and a gene expressing UL49.

5.

4. The formulation of claim 1, wherein the virus exhibits a lower killing ability than wild-type BHV-1 against normal human cells and / or a higher killing ability than wild-type BHV-1 against human tumor cells.

5. The formulation of claim 1, wherein the BHV-1 target genes include a gene encoding gI and a gene encoding gE, or a gene encoding gE and a gene encoding UL49.

5.

6. The formulation of claim 2, wherein the immunomodulatory molecule is a chemokine or cytokine.

7. 3. The formulation of claim 2, wherein the immunomodulatory molecule is selected from the group consisting of ectocalreticulin (ecto-CRT), high-mobility group box 1 protein (HMGB1), interferon, interleukin, hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-colony-stimulating factor (G-CSF).

8. The formulation of claim 2, wherein the immunomodulatory molecule is GM-CSF.

9. 1. A method for producing a recombinant bovine herpesvirus type 1 (BHV-1) oncolytic virus for human administration, comprising: i) nucleofecting a cell line with a vector capable of expressibly incorporating a gene encoding an immunomodulatory molecule that induces an anti-tumor immune response into a target gene of BHV-1; and ii) Infecting the cell line with wild-type BHV-1 under appropriate conditions to obtain a recombinant BHV-1 oncolytic virus that expresses the immunomodulatory molecule and has at least a partial deletion or alteration introduced into the target gene of BHV-1. Including, the target gene is a gene selected from a gene encoding gI and a gene encoding gE, A method for producing the recombinant BHV-1 oncolytic virus, characterized in that the virus exhibits higher cancer selectivity and / or higher immunostimulatory activity compared to wild-type BHV-1.

10. the immunomodulatory molecule is a chemokine or cytokine; or 10. The method of claim 9, wherein the immunomodulatory molecule is selected from the group consisting of ectocalreticulin (ecto-CRT), high-mobility group box 1 protein (HMGB1), interferon, interleukin, hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-colony-stimulating factor (G-CSF).

11. The method of claim 9, wherein the immunomodulatory molecule is GM-CSF.

12. A formulation for treating an individual with cancer, comprising a combination of a bovine herpesvirus type 1 (BHV-1) mutant strain and a pharmaceutically acceptable carrier, comprising: the BHV-1 mutant strain is characterized by exhibiting enhanced cancer selectivity and / or enhanced immunostimulatory activity compared to wild-type BHV-1 due to partial or complete deletions or alterations introduced into one or more target genes of BHV-1; the target gene is selected from the group consisting of a gene expressing gI, a gene expressing gE, and a gene expressing UL49.5; The formulation, wherein the alteration inhibits expression of the target gene.

13. 13. The formulation of claim 12, wherein the individual is a human.

14. 13. The formulation of claim 12, used in combination with at least one of a chemotherapeutic agent, an immune checkpoint inhibitor, or an immunogenic antibody.

15. 13. The formulation of claim 12, used in combination with immune cell therapy.

16. The formulation described in claim 12, wherein the BHV-1 mutant strain exhibits low killing ability against normal human cells and / or high killing ability against human tumor cells.

17. The formulation of claim 12, wherein the BHV-1 mutant strain is genetically modified to express a gene encoding an immunomodulatory molecule that induces an anti-tumor immune response.

18. 18. The formulation of claim 17, wherein the gene encoding the immunomodulatory molecule is inserted into a target gene of BHV-1.

19. 18. The formulation of claim 17, wherein the immunomodulatory molecule is a chemokine or cytokine.

20. 20. The formulation of claim 19, wherein the immunomodulatory molecule is selected from the group consisting of ectocalreticulin (ecto-CRT), high-mobility group box 1 protein (HMGB1), interferon, interleukin, hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-colony-stimulating factor (G-CSF).

21. The formulation of claim 12, wherein the BHV-1 mutant strain contains mutations in two target genes selected from the gI gene, the gE gene, and the UL49.5 gene.

22. The formulation according to claim 12, characterized in that it is used for intratumoral administration.

23. 13. The preparation according to claim 12, characterized in that it is used for intravenous administration.

24. The BHV-1 mutant strain is 10 6 ~10 11 13. The formulation according to claim 12, characterized in that it is administered in an amount of plaque-forming units.

25. 18. The formulation of claim 17, wherein the immunomodulatory molecule is GM-CSF.

Images