Tumor treatment using a combination of oncolytic viruses and immune checkpoint inhibitors

Abstract

This application relates to a drug for tumor treatment. A novel attenuated oncolytic virus strain is provided by site-specific mutagenesis of the matrix protein M of wild-type vesicular stomatitis virus. An oncolytic virus vaccine is provided by inserting a foreign gene into the attenuated oncolytic virus strain. The combination of the oncolytic virus vaccine with an immune checkpoint inhibitor provides a drug that can efficiently treat multiple types of tumors.

Description

[Technical field]

[0001] The present application relates to the field of biopharmaceuticals, and in particular to drug combinations for anti-tumor or cancer treatment made using an attenuated strain of an oncolytic virus and an immune checkpoint inhibitor. [Background technology]

[0002] At present, malignant tumors (cancer) have become one of the major public health issues that pose a serious threat to the health of China and even the world. Traditional cancer treatments mainly include surgery, chemotherapy, radiation therapy, molecular targeted therapy, etc., but traditional treatments have no clear effect on tumor recurrence and metastasis.

[0003] Previous studies have shown that vesicular stomatitis virus (VSV) can be used as a novel tumor vaccine vector to promote immune responses in tumor therapy. VSV is a prototype non-segmented negative-strand RNA virus, whose genome, 11 kb in size, encodes five proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase protein (L). Compared with other oncolytic virus platforms currently under development, VSV has a smaller genome, is easier to handle, has a shorter replication time, has an independent cell cycle, can be rapidly expanded in various cell lines, has high titers, can be produced on a large scale, and replicates in the cytoplasm of host cells without the risk of transformation. Such oncolytic viruses do not integrate into DNA, and are attenuated to avoid the inflammation of the nervous system caused by wild-type viruses. Therefore, VSV has great potential as a tumor immunotherapy.

[0004] However, when VSV is used alone as a tumor immunotherapy, the therapeutic response rate is poor. Therefore, more and more research is being conducted on the combination of VSV with other therapies. A study on a mouse model of papilloma found that the combination of VSV and a tumor vaccine significantly improved the antitumor effect. Manish R. Patel et al. from the University of Minnesota Medical School reported the treatment of lung cancer with a combination of a JAK / STAT inhibitor (Ruxolitinib) and VSV-IFNβ, and the results showed that the combination of Ruxolitinib and VSV-IFNβ achieved good tumor lytic therapeutic effects. Oncolytic viruses carrying various cytokines have also been combined with CAR-T cell therapy, and in xenograft tumor models, this combination method has enhanced antitumor activity.

[0005] Currently, great strides have been made in immunotherapy by using some immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1 (programmed death ligand 1) for tumor treatment. Unfortunately, most patients are not sensitive to treatment with PD-1 or PD-L1 antibodies, and most sensitive patients do not achieve complete remission after treatment.

[0006] PD-1 or PD-L1 antibody therapy has the greatest antitumor efficacy when used in tumors with high amounts of tumor-infiltrating lymphocytes (TILs), high mutational burden, and increased expression of PD-L1. Such responsive tumors are immunologically called "hot tumors," in contrast to poorly responsive "cold tumors." Studies have shown that the inability of "cold tumors" to be treated with PD-1 or PD-L1 antibodies is primarily due to lack of expression and / or presentation of tumor-associated antigens (TAAs), low density of TILs, inhibited infiltration of immune cell subsets (e.g., neutrophils, macrophages, regulatory T cells, myeloid-derived suppressor cells, natural killer cells), and lack of expression of immunosuppressants (e.g., IL-10, indoleamine-2,3-dioxygenase, CD73, PD-L1, and prostaglandin E2). From the above studies, turning "cold tumors" into "hot tumors" has become a hot spot for improving anti-PD-1 or PD-L1 antibody therapy, and oncolytic viruses are an ideal therapeutic tool to enhance the response of patients and certain types of tumors to PD-1 or PD-L1 antibody therapy.

[0007] As can be seen from the above, previous research has made it possible to treat tumors by combining VSV with PD-1 or PD-L1 antibodies. However, there are still at least the following problems when performing tumor immunotherapy by combining VSV with PD-1 or PD-L1 antibodies. (1) When directly combining wild-type or attenuated VSV with PD-1 or PD-L1 antibodies, the cure rate is not high, and the improvement in efficacy is not clear compared to the use of one therapy alone. (2) Wild-type VSV still has some safety risks, and it has been found to have strong neurotoxicity in rodents so far. For clinical use, it is necessary to modify its genes to further reduce its pathogenicity and improve the efficacy of treatment. (3) Random gene modification will not produce good tumor lysis effect or successful packaging, much less recombinant virus production.

[0008] Therefore, providing a VSV recombinant virus with good safety and high cure rate and combining it with PD-1 or PD-L1 antibodies or other immune checkpoint inhibitors (e.g., antibodies to CTLA-4, LAG-3, TIM-3, etc.) as a drug would be of great research value and practical significance in the field of tumor immunotherapy. Summary of the Invention [Problem to be solved by the invention]

[0009] The objective of the present invention is to provide a tumor therapeutic drug, specifically, a tumor therapeutic drug that combines an oncolytic virus vaccine with an immune checkpoint inhibitor (e.g., an antibody against PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, SIGLEC15, etc.). [Means for solving the problem]

[0010] In order to achieve the above object of the invention, the technical solution adopted by the present invention is as follows: An antitumor or cancer therapeutic drug, the drug simultaneously comprises an attenuated oncolytic virus strain and an immune checkpoint inhibitor, the method for producing the attenuated oncolytic virus strain is to modify the matrix protein M of the oncolytic virus, and the gene sequence of the modified matrix protein M is as shown in SEQ ID NO:3.

[0011] Preferably, the attenuated oncolytic virus strain is obtained by site-specific mutagenesis of the VSV MuddSummer subtype strain, including at least a mutation of methionine at position 51 to arginine, a knockout of the base encoding leucine at position 111, a mutation of valine at position 221 to phenylalanine, and a mutation of serine at position 226 to arginine.

[0012] Preferably, an oncolytic virus vaccine is produced from the attenuated oncolytic virus strain, and a drug is further produced.

[0013] Preferably, said oncolytic virus vaccine is produced by inserting an antigen into said attenuated strain of oncolytic virus.

[0014] Preferably, the antigen is a specific tumor antigen.

[0015] Preferably, the antigen is any one of NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX / Y, HPV, alpha-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopachrome tautomerase, BAGE protein, GAGE ​​protein, survivin, tyrosinase, SSX2, cyclin A1, KIF20A, MUC5AC, Meloe, Lengsin, kallikrein 4, IGF2B3, and glypican 3.

[0016] Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, or an anti-SIGLEC15 antibody.

[0017] Preferably, the immune checkpoint inhibitor is released in the body prior to the oncolytic viral vaccine.

[0018] Preferably, the tumor or cancer is any of head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, and synovial sarcoma.

[0019] The present invention has the following beneficial effects: The present invention provides a tumor treatment drug by combining an oncolytic virus vaccine with an immune checkpoint inhibitor (e.g., antibodies against PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, SIGLEC15, etc.), which can effectively treat multiple types of tumors, and surprisingly, in a mouse lung cancer model, the cure rate reached 87.5%, and it has a good therapeutic effect on large tumors.

[0020] In one aspect of the present application, a drug combination is provided that includes an attenuated oncolytic virus strain and an immune checkpoint inhibitor, wherein the attenuated oncolytic virus strain has a modified matrix protein M compared to a VSV MuddSummer subtype strain, the modification including knockout of the base encoding leucine at amino acid position 111.

[0021] In a particular embodiment, the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111.

[0022] In a particular embodiment, the modification of the matrix protein M of the attenuated strain of oncolytic virus further comprises a mutation of the methionine at amino acid position 51 to arginine.

[0023] In a particular embodiment, the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111 and a mutation of methionine at position 51 to arginine.

[0024] In a particular embodiment, the modification of the matrix protein M of the attenuated oncolytic virus strain further comprises a mutation of valine at position 221 to phenylalanine.

[0025] In a particular embodiment, the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at position 111 and a mutation of valine at position 221 to phenylalanine.

[0026] In certain embodiments, the modification of the matrix protein M of the attenuated strain of oncolytic virus further comprises an amino acid mutation at amino acid position 226, serine to arginine.

[0027] In a particular embodiment, the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111 and a mutation of serine at position 226 to arginine.

[0028] In a specific embodiment, the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111, a mutation of valine at position 221 to phenylalanine, and a mutation of serine at position 226 to arginine.

[0029] In a specific embodiment, the modifications in the matrix protein M of the attenuated oncolytic virus strain are a mutation of methionine at position 51 to arginine, a knockout of the base encoding leucine at position 111, a mutation of valine at position 221 to phenylalanine, and a mutation of serine at position 226 to arginine.

[0030] In a specific embodiment, the matrix protein M of the attenuated oncolytic virus strain has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0031] In another aspect of the present application, there is further provided a drug combination comprising an oncolytic virus vaccine and an immune checkpoint inhibitor, wherein the oncolytic virus vaccine is produced using an attenuated strain of the oncolytic virus.

[0032] In a particular embodiment, the oncolytic virus vaccine is produced by inserting an antigen into an attenuated strain of the oncolytic virus.

[0033] In a particular embodiment, the antigen is a specific tumor antigen.

[0034] In a particular embodiment, the antigen is selected from the group consisting of NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX / Y, HPV, alpha-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopachrome tautomerase, BAGE proteins, GAGE ​​proteins, survivin, tyrosinase, SSX2, cyclin A1, KIF20A, MUC5AC, Meloe, Lengsin, kallikrein 4, IGF2B3, and glypican 3.

[0035] In certain embodiments, the immune checkpoint inhibitor of the drug combination described herein is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, and an anti-SIGLEC15 antibody.

[0036] In certain embodiments, the immune checkpoint inhibitor is released in the body prior to the oncolytic viral vaccine.

[0037] In certain embodiments, the drug combination is capable of anti-tumor or cancer treatment.

[0038] In certain embodiments, the tumor or cancer is selected from the group consisting of head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.

[0039] In another aspect of the present application, there is further provided the use of said drug combination in the manufacture of an anti-tumor or tumor treatment medicament.

[0040] Other aspects and advantages of the present application can be readily ascertained by those skilled in the art from the following detailed description. The following detailed description shows and describes exemplary embodiments of the present application only. As those skilled in the art will appreciate, the present application will enable those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the present invention. For this reason, the drawings and description of the present application are illustrative only, rather than limiting. [Brief description of the drawings]

[0041] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments in the following detailed description and the drawings, the brief description of which is as follows: [Figure 1A] FIG. 1A is a schematic diagram showing the replication ability of each attenuated strain in LLC cells in vitro. [Figure 1B] FIG. 1B is a schematic diagram showing the replication ability of each attenuated strain in MEF cells in vitro. [Figure 2A] FIG. 2A is a schematic diagram showing the killing ability of each attenuated strain against LLC cells in vitro. [Figure 2B] FIG. 2B is a schematic diagram showing the in vitro killing ability of each attenuated strain against HeLa cells. [Diagram 3] FIG. 3 is a schematic diagram showing the killing ability of each attenuated strain against MEF cells in vitro. [Figure 4A] FIG. 4A is a schematic diagram showing the effect of each attenuated strain on the expression level of IFN-β in LLC cells in vitro. [Figure 4B] FIG. 4B is a schematic diagram showing the effect of each attenuated strain on the expression level of IFN-β in MEF cells in vitro. [Diagram 5] FIG. 5 is a schematic diagram of the construction of an oncolytic viral vaccine. [Figure 6]FIG. 6 is a schematic diagram showing the effect of each attenuated strain on the volume of non-small cell lung cancer (transplanted tumor) in mice. [Figure 7] FIG. 7 is a schematic diagram showing the effect of each vaccine on the volume of non-small cell lung cancer (transplanted tumor) in mice. [Figure 8] FIG. 8 is a schematic diagram showing the volume of non-small cell lung cancer (transplanted tumor) in mice treated with each attenuated strain and vaccine at the end of the experiment. [Figure 9] FIG. 9 is a schematic diagram showing the effects of each attenuated strain and vaccine on the metastasis status of non-small cell lung cancer cells in mice. [Figure 10] FIG. 10 is a schematic diagram showing the effect of each vaccine on the volume of fibrosarcoma (transplanted tumor) in the mouse body. [Figure 11] FIG. 11 is a schematic diagram showing the volume of fibrosarcoma (transplanted tumor) in mice treated with each vaccine at the end of the experiment. [Figure 12] FIG. 12 is a schematic diagram showing the effect of each vaccine on the volume of melanoma (transplanted tumor) in the mouse body. [Figure 13] FIG. 13 is a schematic diagram showing the volume of melanoma (transplanted tumor) in mice treated with each vaccine at the end of the experiment. [Figure 14] FIG. 14 is a schematic diagram showing the effect of different doses of JBS004 on the volume of non-small cell lung cancer (transplanted tumor) in mice. [Figure 15] FIG. 15 is a schematic diagram of the volume of non-small cell lung cancer (transplanted tumor) in mice treated with different doses of JBS004 at the end of the experiment. [Figure 16] FIG. 16 is a schematic diagram of the effects of different doses of JBS004 on the metastatic status of non-small cell lung cancer cells in mice. [Figure 17] FIG. 17 is a schematic diagram of the effect of different doses of JBS004 on the body weight of lung cancer mice. [Figure 18] FIG. 18 is a schematic diagram of the effects of different doses of JBS004 on the body temperature of lung cancer mice. [Figure 19] FIG. 19 is a quantitative standard curve for the PCR detection method. [Figure 20] FIG. 20 is a schematic diagram showing the nucleic acid copy number of JBS004 in tumors at different times in an LLC transplant tumor model. [Figure 21] FIG. 21 is a schematic diagram of the effect of different doses of JBS004 at different times on the body temperature of female mice. [Figure 22] FIG. 22 is a schematic diagram of the effect of different doses of JBS004 at different times on body temperature in male mice. [Diagram 23] FIG. 23 is a schematic diagram of the effect of different doses of JBS004 at different times on the body weight of female mice. [Figure 24] FIG. 24 is a schematic diagram of the effect of different doses of JBS004 at different times on the body weight of male mice. [Diagram 25] Figure 25 is a schematic diagram showing the effect of JBS004 treatment alone or in combination with a PD-1 antibody on the volume of lung cancer (transplanted tumor). [Figure 26A] Figure 26A is a schematic diagram showing the volume of lung cancer (transplanted tumor) at the end of the experiment when the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application was administered alone or in combination with a PD-1 antibody. [Figure 26B] Figure 26B is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Figure 26C] Figure 26C is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Figure 26D] Figure 26D is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Figure 26E]Figure 26E is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Figure 26F] Figure 26F is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Figure 26G] Figure 26G is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Fig. 26H] Figure 26H is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment, when treated with the attenuated oncolytic virus strain / oncolytic virus vaccine described in the present application alone or in combination with a PD-1 antibody. [Figure 27] Figure 27 is a schematic diagram showing the effects of JBS004 treatment alone or in combination with a PD-1 antibody on the metastatic status of lung cancer cells. [Figure 28] Figure 28 is a schematic diagram showing the effect of combined treatment with JBS004 and different doses of PD-1 antibody on the volume of lung cancer (transplanted tumor). [Figure 29] Figure 29 is a schematic diagram of the volume of lung cancer (transplanted tumor) at the end of the experiment when JBS004 was combined with different doses of PD-1 antibody. [Diagram 30] Figure 30 is a schematic diagram showing the effect of combined treatment of JBS004 with different doses of PD-1 antibody on the metastatic status of lung cancer cells. [Diagram 31] Figure 31 is a schematic diagram showing the effect of combined treatment of JBS004 and PD-1 antibody using different administration routes on the volume of lung cancer (transplanted tumor). [Diagram 32] Figure 32 is a schematic diagram of the volume of lung cancer (transplanted tumors) at the end of the experiment after combined treatment with JBS004 and PD-1 antibody using different administration routes. [Diagram 33]Figure 33 is a schematic diagram showing the effect of combined treatment of JBS004 and PD-1 antibody administered via different routes on the metastasis status of lung cancer cells. [Diagram 34] Figure 34 is a schematic diagram showing the effect of combined treatment of JBS004 and PD-1 antibody with different administration sequences on lung cancer (transplanted tumor) volume. [Diagram 35] Figure 35 is a schematic diagram of the volume of lung cancer (transplanted tumor) upon combination treatment of JBS004 and PD-1 antibody in different administration orders at the end of the experiment. [Diagram 36] Figure 36 is a schematic diagram showing the effects of combined treatment with JBS004 and PD-1 antibody in different administration orders on the metastatic status of lung cancer cells. [Figure 37] Figure 37 is a schematic diagram showing the effect of combined treatment with JBS004 and PD-1 antibody on the volume of breast cancer (transplanted tumor). [Figure 38] Figure 38 is a schematic diagram of the volume of breast cancer (transplanted tumor) during combined treatment with JBS004 and PD-1 antibody at the end of the experiment. [Figure 39] Figure 39 is a schematic diagram showing the effect of combined treatment with JBS004 and PD-1 antibody on the volume of colon cancer (transplanted tumors, large tumors). [Diagram 40] Figure 40 is a schematic diagram of the volume of colon cancer (transplanted tumors, large tumors) during combined treatment with JBS004 and PD-1 antibody at the end of the experiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Next, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art can easily know other advantages and effects of the present invention from the contents disclosed in this specification.

[0043] "Definition of Terms" In this application, the term "modified" generally refers to the alteration of the structure and / or properties of a naturally occurring organism / molecule by artificial means. The manner of alteration may be, for example, modification, mutation, synthesis and / or insertion of an exogenous molecule into said molecule. Something "modified" is different from what is naturally occurring. For example, a cell or organism is considered "modified" if its genetic information has been altered by manipulation (e.g., by introducing new genetic material not previously present, such as by transformation, mating, somatic hybridization, transfection, transduction or other mechanisms, or by altering or removing previously present genetic material, such as by substitution or deletion mutation). For example, a modified oncolytic virus may be one in which a gene encoding a protein of the oncolytic virus has been mutated, a foreign gene has been inserted into a gene of the oncolytic virus, or an amino acid of a protein of the oncolytic virus has been mutated.

[0044] In this application, the term "drug combination" generally refers to a combination that includes at least two components. Each component of the drug combination may be used in combination. Used in combination may refer to simultaneous use or each use. Each component of the combination may be mixed or placed in a different container. For example, the drug combination includes an attenuated oncolytic virus strain and the immune checkpoint inhibitor described in this application. The attenuated oncolytic virus strain may be administered simultaneously with the immune checkpoint inhibitor or each may be administered separately. The attenuated oncolytic virus strain may be mixed with the immune checkpoint inhibitor or placed in a different container.

[0045] In this application, the term "oncolytic virus" generally refers to a virus that can replicate in and kill tumor cells. In certain embodiments, the virus is modified to increase selectivity for tumor cells. In certain embodiments, the oncolytic virus is modified to reduce its immunogenicity. In certain embodiments, the oncolytic virus is a vesicular stomatitis virus. In certain embodiments, the vesicular stomatitis virus is a MuddSummer subtype strain of vesicular stomatitis virus. In certain embodiments, site-directed mutagenesis may be performed on the gene for matrix protein M of vesicular stomatitis virus.

[0046] In this application, the term "matrix protein M" is used interchangeably with "M protein" and generally refers to the matrix protein of vesicular stomatitis virus. Matrix protein M is an important virulence factor of VSV and is known to be a protein that can interfere with the innate immune response of mice in vesicular stomatitis virus. The term "matrix protein M" further includes its homologs, orthologs, variants, functionally active fragments, and the like. In this application, the matrix protein M of wild-type vesicular stomatitis virus MuddSummer subtype Indiana strain may comprise the amino acid sequence shown in SEQ ID NO:2.

[0047] In the present application, the protein mutation site is generally represented by "amino acid + amino acid position number + (mutated amino acid)". In the present application, the mutation includes, but is not limited to, addition, substitution, deletion and / or removal of amino acids. For example, the term "M51R" generally refers to a mutation of methionine M at position 51 to arginine R. In the present application, the symbol "Δ" represents an amino acid deletion. For example, the term "ΔL111" generally refers to a deletion of leucine at position 111.

[0048] In this application, the term "antigen" generally refers to a protein having immunogenicity. The antigen is used, for example, to generate an antibody capable of binding to the antigen. In some cases, the antigen comprises a tumor antigen. In some cases, the antigen comprises a melanoma. In some cases, for example, the antigen may be NY-ESO-1.

[0049] As used herein, the term "NY-ESO-1," also known as "New York esophageal squamous cell carcinoma 1," is a cancer-testis antigen (CTA). NY-ESO-1 as described herein includes variants, fragments, and / or truncated forms thereof. For example, the sequence of human NY-ESO-1 is available in GeneBank under accession number 1485.

[0050] As used herein, the terms "PD-1" and "PD1" are used interchangeably, for example, "PD-1 antibody" and "PD1 antibody" are used interchangeably.

[0051] In the present application, the term "medicament" generally refers to a formulation in which the biological activity of the active ingredient is present in a form that is effective and does not contain other ingredients that have intolerable toxicity to the subject to which the formulation is administered. In certain embodiments, these formulations may contain the active ingredient of a drug and a pharma- ceutical acceptable carrier. In certain embodiments, the pharmaceuticals include pharmaceuticals for parenteral, transdermal, intraluminal, intraarterial, intrathecal and / or intranasal administration or for direct injection into tissue. The pharmaceuticals may be administered by different modes, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0052] In this application, the term "pharmaceutically acceptable" generally refers to an adjuvant that, in reasonable medical judgment, is suitable for contact with human or animal tissues, does not cause toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable benefit-to-risk ratio. In certain embodiments, a pharmaceutically acceptable adjuvant may refer to an adjuvant for use in animals (e.g., humans) that is approved by a regulatory agency (e.g., the U.S. Food and Drug Administration, the Chinese Food and Drug Administration, or the European Medicines Agency) or registered in a generally recognized pharmacopoeia (e.g., the United States Pharmacopoeia, the Chinese Pharmacopoeia, or the European Pharmacopoeia). In this application, a pharmaceutically acceptable adjuvant may be an aluminum-based adjuvant, an inorganic salt adjuvant, an isotonic active adjuvant, a bacterial-derived adjuvant, an emulsion adjuvant, a liposomal adjuvant, a cytokine adjuvant, a carbohydrate adjuvant, a DNA / RNA oligomer adjuvant, and others.

[0053] In this application, the term "combination" is also referred to as "co-administration", and generally refers to administration before, after, or simultaneously with another drug component. Two or more drug components used in combination may be administered in the same or different administration modes, simultaneously, or sequentially. For example, one therapeutic agent used in combination may be the oncolytic virus attenuated strain / oncolytic virus vaccine, and a second therapeutic agent may be the immune checkpoint inhibitor. The combination may also include a third or more therapeutic agents.

[0054] In this application, the term "prevention" generally refers to taking certain measures in advance to prevent the onset, onset, recurrence, and / or spread of a disease or one or more symptoms thereof. In this application, the term "treatment" generally refers to eliminating or ameliorating a disease or one or more symptoms associated with a disease. In certain embodiments, treatment generally refers to eliminating or remitigating a disease by administering one or more drugs to a patient having the disease. In certain embodiments, "treatment" may refer to administering the drug combination and / or pharmaceutical agent in the presence or absence of other drugs after a particular disease symptom has onset. For example, the drug combination and / or pharmaceutical agent described in this application is used to prevent the onset, progression, recurrence, and / or metastasis of a tumor.

[0055] In this application, the term "comprising" generally refers to the inclusion of the explicitly specified features but not to the exclusion of other elements.

[0056] In this application, the term "homology" generally refers to an amino acid sequence or a nucleic acid sequence that has a certain degree of homology with the amino acid sequence and the nucleic acid sequence to which it is compared. The term "homology" may be interchanged with the "identity" of a sequence. A homologous sequence may include an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% identical to the subject sequence. In general, a homolog contains the same active site, etc., as the subject amino acid sequence. Homology may be considered as similarity (having amino acid residues with similar chemical properties / functions) or may be expressed by sequence identity.

[0057] Detailed Description of the Invention 1. An antitumor drug or cancer therapeutic drug produced using an attenuated oncolytic virus strain, wherein the attenuated oncolytic virus strain has a modified substrate protein M of the oncolytic virus, the genetic sequence of the modified substrate protein M being as shown in SEQ ID NO: 3, and the drug simultaneously contains the attenuated oncolytic virus strain and an immune checkpoint inhibitor.

[0058] 2. An antitumor drug or cancer therapeutic agent produced using the attenuated oncolytic virus strain described in embodiment 1, characterized in that the attenuated oncolytic virus strain is obtained by introducing site-specific mutagenesis into the VSV MuddSummer subtype strain, at least comprising mutating methionine at position 51 to arginine, knocking out the base encoding leucine at position 111, mutating valine at position 221 to phenylalanine, and mutating serine at position 226 to arginine.

[0059] 3. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain described in embodiment 1 or 2, characterized in that an oncolytic virus vaccine is produced from the attenuated oncolytic virus strain, and a drug is further produced.

[0060] 4. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain described in embodiment 3, wherein the oncolytic virus vaccine is produced by inserting an antigen into the attenuated oncolytic virus strain.

[0061] 5. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain described in embodiment 4, wherein the antigen is a specific tumor antigen.

[0062] 6. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain according to embodiment 5, characterized in that the antigen is any one of NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX / Y, HPV, α-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopachrome tautomerase, BAGE protein, GAGE ​​protein, survivin, tyrosinase, SSX2, cyclin A1, KIF20A, MUC5AC, Meloe, Lengsin, kallikrein 4, IGF2B3, and glypican 3.

[0063] 7. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain described in embodiment 1 or 2, wherein the immune checkpoint inhibitor is any one of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, and an anti-SIGLEC15 antibody.

[0064] 8. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain described in embodiment 1 or 2, characterized in that the immune checkpoint inhibitor is released in the body prior to the oncolytic virus vaccine.

[0065] 9. An antitumor drug or cancer therapeutic drug produced using the attenuated oncolytic virus strain described in embodiment 1 or 2, characterized in that the tumor or cancer is any one of head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.

[0066] The present invention provides a drug that can effectively treat multiple tumors, which simultaneously comprises an oncolytic virus vaccine and an immune checkpoint inhibitor.

[0067] The method for producing the oncolytic virus vaccine is to insert the NY-ESO-1 gene or gene fragment into the attenuated oncolytic virus strain. The specific method for producing the vaccine includes the following steps: 1. Preparation of an attenuated oncolytic virus strain. Vesicular stomatitis virus (VSV) is selected, specifically, the MuddSummer subtype strain of the Indiana strain of vesicular stomatitis virus is selected, the gene sequence of its M protein is as shown in SEQ ID NO:1, and the amino acid sequence of its matrix protein M is as shown in SEQ ID NO:2.

[0068] 2. Site-specific mutagenesis of the gene encoding the matrix protein M of the vesicular stomatitis virus. The mutation sites include (1) a mutation of methionine (M) at position 51 to arginine (R), (2) a knockout of the base encoding leucine (L) at position 111, (3) a mutation of valine (V) at position 221 to phenylalanine (F), and (4) a mutation of serine (S) at position 226 to arginine (R). The number of the vesicular stomatitis virus after the mutation is JBS003 and it is named XN2-M51R-△L111-V221F-S226R. The gene sequence of the M protein is as shown in SEQ ID NO:3, and the amino acid sequence of the M protein is as shown in SEQ ID NO:4.

[0069] Compared with wild-type VSV and other attenuated VSV strains, JBS003 is safer and can be used as a vector (backbone) for substances such as antigens and cytokines, and can be combined with substances such as antigens and cytokines to be used as a vaccine or drug. In addition, JBS003 can be used directly as a drug in tumor immunotherapy without combining with other substances, and its therapeutic effect is superior to that of wild-type VSV and other attenuated VSV strains.

[0070] 3. Insertion of NY-ESO-1 gene or gene fragment. A gene capable of expressing NY-ESO-1 was inserted between the G protein and L protein genome of JBS003 to construct an oncolytic virus vaccine, designated JBS004.

[0071] NY-ESO-1 (New York esophageal squamous cell carcinoma 1) belongs to the cancer-testis antigen (CTA) family and is expressed in testis, ovary and various tumor tissues, but not in other normal tissues. It is the most immunogenic tumor-specific antigen discovered to date. The expression level of NY-ESO-1 varies depending on the tumor tissue, and the highest protein expression is in myxoid round cell liposarcoma (89-100%), neuroblastoma (82%), synovial sarcoma (90%), melanoma (46%) and ovarian cancer (43%). The NY-ESO-1 antigen is immunogenic and safe, and is the clinically predominant antigen used in immunotherapy.

[0072] At present, recurrent and metastatic head and neck squamous cell carcinoma, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, etc. cannot be effectively treated. The JBS004 oncolytic virus vaccine constructed by introducing NY-ESO-1 can efficiently induce specific anti-tumor immune responses in the peripheral lymphatic system and tumor tissues in the body. The study showed that in anti-tumor immunotherapy, especially in the treatment of the above cancers and tumors, oncolytic virus vaccines have obvious advantages in immunogenicity, efficacy, targeting, safety and tolerability.

[0073] The attenuated strain of JBS003 oncolytic virus or the JBS004 oncolytic virus vaccine can be used alone as a tumor treatment drug, and the method of use is as follows: The attenuated strain of JBS003 oncolytic virus or the JBS004 oncolytic virus vaccine is injected intratumorally or intravenously. Intratumor injection refers to injection into the corresponding tumor in the body of a cancer-bearing mammal. A method of multiple injections in small amounts is adopted.

[0074] 4. JBS004 oncolytic virus vaccine is combined with immune checkpoint inhibitors to form a tumor treatment drug. The immune checkpoint inhibitors include antibodies to PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, SIGLEC15, etc., and are preferably antibodies to PD-1 or PD-L1. For the sake of comparison and ease of handling, the following examples all use antibodies to PD-1.

[0075] The method of using the drug is as follows: A small amount of oncolytic virus or oncolytic virus vaccine is provided multiple times (intratumoral or intravenous injection). The preferred administration order is to administer the immune checkpoint inhibitor first, and then administer the oncolytic virus or oncolytic virus vaccine. The injection mode of the immune checkpoint inhibitor is preferably intraperitoneal injection or intravenous injection, and the administration mode of the oncolytic virus or oncolytic virus vaccine is preferably intratumoral injection or intravenous injection.

[0076] In one aspect of the present application, a drug combination is provided that includes an attenuated oncolytic virus strain and an immune checkpoint inhibitor, wherein the attenuated oncolytic virus strain has a modified matrix protein M compared to a VSV MuddSummer subtype strain, and the modification includes knocking out a base encoding leucine at amino acid position 111. For example, the matrix protein M of the attenuated oncolytic virus strain has a knockout of a base encoding leucine at amino acid position 111 compared to the matrix protein M of the VSV MuddSummer subtype strain. For example, the amino acid sequence of the matrix protein M of the attenuated oncolytic virus strain is as shown in SEQ ID NO: 7.

[0077] In certain embodiments, the matrix protein M of the VSV MuddSummer subtype strain comprises the nucleic acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the matrix protein M of the VSV MuddSummer subtype strain comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0078] In the present application, the mutation of the gene of the substrate protein M of the attenuated oncolytic virus strain may further include a mutation of methionine at amino acid position 51 to arginine. For example, compared with the substrate protein M of the VSV MuddSummer subtype strain, the substrate protein M of the attenuated oncolytic virus strain has a base encoding leucine at position 111 knocked out and methionine at position 51 mutated to arginine. For example, the amino acid sequence of the substrate protein M of the attenuated oncolytic virus strain is as shown in SEQ ID NO:8.

[0079] In the present application, the mutation of the gene of the substrate protein M of the attenuated oncolytic virus strain may further include a mutation of valine at position 221 to phenylalanine. For example, compared with the substrate protein M of the VSV MuddSummer subtype strain, the substrate protein M of the attenuated oncolytic virus strain has a base encoding leucine at position 111 knocked out and valine at position 221 mutated to phenylalanine. For example, the amino acid sequence of the substrate protein M of the attenuated oncolytic virus strain is as shown in SEQ ID NO:9.

[0080] In the present application, the mutation of the gene of the substrate protein M of the attenuated oncolytic virus strain further includes an amino acid mutation that is a mutation of serine at amino acid position 226 to arginine. For example, in the substrate protein M of the attenuated oncolytic virus strain, compared with the substrate protein M of the VSV MuddSummer subtype strain, the base encoding leucine at amino acid position 111 is knocked out, and serine at position 226 is mutated to arginine. For example, the amino acid sequence of the substrate protein M of the attenuated oncolytic virus strain is as shown in SEQ ID NO: 10. For example, in the substrate protein M of the attenuated oncolytic virus strain, compared with the substrate protein M of the VSV MuddSummer subtype strain, the base encoding leucine at amino acid position 111 is knocked out, valine at position 221 is mutated to phenylalanine, and serine at position 226 is mutated to arginine. For example, the amino acid sequence of the substrate protein M of the attenuated oncolytic virus strain is as shown in SEQ ID NO: 11. For example, in the substrate protein M of the attenuated oncolytic virus strain, compared with the substrate protein M of the VSV MuddSummer subtype strain, the methionine at position 51 is mutated to arginine, the base encoding the leucine at position 111 is knocked out, the valine at position 221 is mutated to phenylalanine, and the serine at position 226 is mutated to arginine. For example, the amino acid sequence of the substrate protein M of the attenuated oncolytic virus strain is as shown in SEQ ID NO:4.

[0081] In the present application, the immune checkpoint inhibitor may be any one selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody and anti-SIGLEC15 antibody.

[0082] In another aspect of the present application, there is further provided a pharmaceutical composition comprising an oncolytic virus vaccine and an immune checkpoint inhibitor, wherein the oncolytic virus vaccine is produced using an attenuated oncolytic virus strain described herein. In a particular embodiment, the oncolytic virus vaccine is produced by inserting an antigen into the attenuated oncolytic virus strain. For example, the antigen may be selected from the group consisting of NY-ESO-1, gp33, gp100, TX103, Mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, SURVIVIN, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX / Y, HPV, α-fetoprotein, carcinoembryonic antigen, CA 125, Her2, dopachrome tautomerase, BAGE protein, GAGE ​​protein, survivin, tyrosinase, SSX2, cyclin A1, KIF20A, MUC5AC, Meloe, Lengsin, kallikrein 4, IGF2B3, and glypican 3.

[0083] In a particular embodiment, said oncolytic viral vaccine is produced by inserting a tumor-associated antigen into an attenuated strain of VSV whose matrix protein M carries the ΔL111 mutation.

[0084] In a particular embodiment, the oncolytic virus vaccine is produced by inserting a tumor-associated antigen into an attenuated VSV strain whose matrix protein M carries the ΔL111 and M51R mutations.

[0085] In a particular embodiment, the oncolytic virus vaccine is produced by inserting a tumor-associated antigen into an attenuated strain of VSV, whose matrix protein M carries the ΔL111 and V221F mutations.

[0086] In a particular embodiment, the oncolytic virus vaccine is produced by inserting a tumor-associated antigen into an attenuated strain of VSV whose matrix protein M carries the ΔL111 and S226R mutations.

[0087] In a particular embodiment, the oncolytic viral vaccine is produced by inserting a tumor-associated antigen into an attenuated strain of VSV whose matrix protein M carries the ΔL111, V221F and S226R mutations.

[0088] In a particular embodiment, the oncolytic viral vaccine is produced by inserting a tumor-associated antigen into an attenuated strain of VSV whose matrix protein M has the ΔL111, M51R, V221F and S226R mutations.

[0089] In the present application, the immune checkpoint inhibitor of the pharmaceutical composition may be any one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, and an anti-SIGLEC15 antibody. In a specific embodiment, the immune checkpoint inhibitor is a PD-1 antibody (anti-PD-1-antibody). For example, the heavy chain variable region of the PD-1 antibody may comprise the amino acid sequence set forth in SEQ ID NO: 12. For example, the light chain variable region of the PD-1 antibody may comprise the amino acid sequence set forth in SEQ ID NO: 13.

[0090] In the present application, the immune checkpoint inhibitor may be released in the body prior to the oncolytic virus vaccine.

[0091] In the present application, the drug combination can be used for anti-tumor or cancer treatment, and the tumor or cancer may be selected from the group consisting of head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.

[0092] In another aspect of the present application, there is further provided a pharmaceutical product comprising said drug combination and, optionally, a pharma- ceutically acceptable carrier.

[0093] In the present application, the attenuated oncolytic virus strain may be obtained by a virus rescue process, which may include the following steps: inoculating BHK-21 cells with a poxvirus expressing RNA polymerase by infection, preparing a plasmid transfection premix using expression plasmids in which VSV N, VSV P, and VSV L genes are cloned, and a VSV M gene expression plasmid or a mutant VSV M gene expression plasmid, and then performing lipofectamine transfection to obtain a mutant strain.

[0094] In this application, the drug combination and / or each component of the pharmaceutical product may be located in the same container or in different containers.

[0095] In the present application, the drug combination and / or each component of the pharmaceutical product may be mixed or placed separately.

[0096] The present application further provides a use of the attenuated oncolytic virus strain / oncolytic virus vaccine and the immune checkpoint inhibitor together for manufacturing a drug.

[0097] The present application further provides a use of the expression vector of the attenuated oncolytic virus strain / oncolytic virus vaccine and the immune checkpoint inhibitor together for manufacturing a drug.

[0098] In certain embodiments, the attenuated oncolytic virus strain / oncolytic virus vaccine and the immune checkpoint inhibitor may be administered simultaneously. Simultaneous administration may be administration of each component mixed together or separately. They may be administered in the same manner, or in different manners, for example, in the same vein or other blood vessel, for example, intravenous administration and intratumoral administration at the same time.

[0099] In certain embodiments, the attenuated oncolytic virus / oncolytic virus vaccine and the immune checkpoint inhibitor may be administered sequentially. The order of administration may be to administer the attenuated oncolytic virus / oncolytic virus vaccine first and then the immune checkpoint inhibitor, or to administer the immune checkpoint inhibitor first and then the attenuated oncolytic virus / oncolytic virus vaccine. They may be administered in the same manner or in different manners. Each component may be administered once or in multiple doses. In certain embodiments, the sequential administration may be administered at any time interval, such as minutes, hours, days, weeks, months, or years. In certain embodiments, sequential administration refers to administration separated by a time interval of at least 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0100] In certain embodiments, the dosage of administration may be any dosage.

[0101] In certain embodiments, the dosage of administration may be a therapeutically effective amount.

[0102] In certain embodiments, the attenuated oncolytic virus / oncolytic virus vaccine may be formulated for intratumoral administration. In certain embodiments, the attenuated oncolytic virus may be formulated for intravenous administration.

[0103] In certain embodiments, the immune checkpoint inhibitor may be formulated for systemic administration. In certain embodiments, the immune checkpoint inhibitor may be formulated for local administration.

[0104] Without being limited by any theory, the following examples are intended to illustrate the technical solutions of the present invention, but are not intended to limit the scope of the present invention.

[0105] "Example" Example 1: Construction and demonstration of site-directed mutagenesis attenuated strains 1. Site-directed mutagenesis was performed on the M matrix protein of the vesicular stomatitis virus Indiana strain to obtain 7 groups of mutated attenuated strains according to the method in Table 1. The group without gene mutation was numbered JBS000 and served as the control. [Table 1] Table 1: Indication of mutation status in each group

[0106] A specific method for constructing an attenuated strain is a common technique in the art, and is outlined as follows. (1) Plasmid construction. Using the pVSV-XN2 plasmid as a template, the different mutation sites listed in Table 1 were introduced using PCR. The plasmid was subjected to PCR with primers for each mutation site, and the PCR product was subjected to 1% agarose gel electrophoresis. The gel was then excised and recovered using a gel recovery kit to obtain plasmids with different mutations in the M substrate protein.

[0107] (2) Virus rescue. BHK-21 cells were inoculated with poxvirus vTF7-3 expressing T7 RNA polymerase at MOI=5 for infection, and after 1 hour of infection, the BHK-21 cells were rinsed once using DPBS buffer. Then, a plasmid transfection premix was prepared, specifically containing pBS-N, pBS-P, and pBS-L, the mutant plasmids prepared in step (1). Here, pBS-N, pBS-P, and pBS-L refer to expression plasmids in which the genes of VSV N, VSV P, and VSV L proteins were cloned, respectively, and each expresses the N, P, and L proteins required for virus rescue. Plasmid transfection was performed according to the method described in the lipofectamine 2000 instruction manual, and after 4 hours, the medium was replaced with a new DMEM complete medium containing 10% fetal bovine serum. After 48 hours, the supernatant was aspirated and the poxvirus was removed using a 0.22 μm membrane filter. The filtrate was added to fresh BHK-21 cells, and the lesions of the cells were observed every day. When the lesions appeared on the cells, the supernatant was collected, and after the success was confirmed by RTPCR identification, the virus was purified by virus plaque experiment, and an attenuated strain was obtained.

[0108] (3) Sequencing of the M protein gene. Viral genomic RNA was extracted using a Trizol kit, reverse transcription was performed using random primers, and the cDNA obtained by reverse transcription was PCR-run using primers designed for the M protein gene sequence. The primer sequences were 5'-AAAAAAGTAACAGATATCAC-3' (sequence number 5) and 5'-ACATTTTTCCAGTTTCCTTTTTGG-3' (sequence number 6). The product was electrophoresed on a 1% agarose gel, recovered, and sent to a professional sequencing company for sequencing.

[0109] 2. This was an indication of the in vitro infectivity of different attenuated strains against cells. 200 pfu each of JBS000, JBS001, JBS002, JBS003, JBS008, JBS009, JBS010, and JBS014 were added to MEF cell (human fibroblast) culture medium and LLC cell (mouse non-small cell lung cancer cell) culture medium, and the 50% tissue culture infective dose (TCID50) of the attenuated strains in each group was detected. The specific test method was as follows. (1) Add 3 mL of cell suspension to each well of a 6-well culture plate to obtain a cell volume of 4 × 10 5 The cells were cultured at 37°C in 5% CO2 for 16 hours in six wells in total.

[0110] (2) 200 pfu of each of the viruses JBS000, JBS001, JBS002, JBS003, JBS008, JBS009, JBS010, and JBS014 were added to each well, and two wells were used as normal cell controls. After 24 hours, 100 μL of cell supernatant was obtained.

[0111] (3) Add 100 μL of the Vero cell suspension to each well of a 96-well culture plate to give a cell volume of 1 × 10 4 The cells were cultured at 37°C in 5% CO2 for 16 hours at a concentration of 1.5 cells / mL.

[0112] (4) Serially dilute the supernatant obtained in step (2) 10-fold in 1.5 mL EP tubes to obtain 10 -1 ~10 -11 The total titer was 11.

[0113] (5) The diluted supernatants were inoculated into the 96-well culture plate from step (3), with one row for each dilution (total of 8 wells), and 100 μL was inoculated into each well. Row 1 was used as a normal cell control group.

[0114] (6) After 48 hours, the fluorescence state of the cells in each well was observed, and if there was fluorescence, the well was recorded as infected.

[0115] (7) TCID50 was calculated using the Karber method.

[0116] The results are shown in Figures 1A and 1B. The replication and amplification ability of each of the attenuated oncolytic virus strains constructed in vitro in lung cancer cells (LLC) was stronger than that of each of the attenuated oncolytic virus strains in normal fibroblasts (MEF), and JBS003 in particular had a stronger replication and amplification ability in lung cancer cells (LLC) in vitro, and the number of virus particles generated 24 hours after infection was close to that of the wild-type virus. On the other hand, the replication and infection ability of each of the attenuated oncolytic virus strains in normal fibroblasts (MEF) was reduced. Therefore, the JBS003 vector has a relatively strong specific infection ability in tumor cells.

[0117] 3. Display of the in vitro killing ability of different attenuated strains against cells. The same amount of each attenuated strain was used to infect different cells in vitro, and the cell activity was detected by MTT method after 24 hours. The method was specifically as follows: (1) Add 100 μL of cell suspension to each well of a 96-well culture plate to give a cell volume of 1 × 10 4 The cells were cultured at 37°C and 5% CO2 for 16 hours. The types of cells tested were LLC, MEF, and Hela (human tumor cells).

[0118] (2) JBS000, JBS001, JBS002, JBS003, JBS008, JBS009, JBS010, and JBS014 were diluted to MOI (multiplicity of infection) of 0.001, 0.01, 0.1, and 1.0, respectively, and each dilution was inoculated into four wells, with 100 μL inoculated into each well, and cultured at 37°C and 5% CO2 for 40 hours.

[0119] (3) The supernatant was discarded from the 96-well culture plate, and fresh DMED medium was added, followed by the addition of 5 mg / mL MTT solution (20 μL / well). Culture was performed for 4 hours at 37°C and 5% CO2.

[0120] (4) The 96-well plate was centrifuged at a speed of 2500 g / min at room temperature for 5 minutes, and then the supernatant was gently sucked up using a 1 mL disposable sterile syringe.

[0121] (5) Further, DMSO was added to each well at 100 μL / well, and the plate was left to stand at 37° C. for 10 minutes.

[0122] (6) After shaking for 2 minutes, the OD value of each well was measured at a wavelength of 570 nm using a multi-mode microplate reader.

[0123] The results are shown in Figures 2A, 2B, and 3. The results showed that each attenuated oncolytic virus strain exhibited good tumor cell killing ability, and none of them had significant killing in MEF cells, except for JBS000. That is, in vitro, each attenuated strain, except for JBS000, specifically killed tumor cells, and had no significant effect on normal cells.

[0124] 4. This was a test of the difficulty of intracellular elimination of different attenuated strains. IFN-β was used as the test index. The cells were cultured according to steps (1) and (2) of step 3 above, and the attenuated strains were added. Then, the cells of each group were disrupted, and total RNA was extracted from each cell using TRIzol (Invitrogen), reverse transcribed to cDNA using the PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription kit, stained with LightCycler 480SYBR Green I Master (Roche) dye, and the Ct value of each gene was detected using a LightCycler 480 quantitative PCR device. The relative expression levels of the target genes IFN-β and VSV-G were calculated using the ΔΔCt method. The results are shown in Figure 4. In LLC cell lines, all attenuated strains except JBS000 were able to induce an increase in IFN-β expression, with the JBS003 vector showing the lowest regulatory ability, while in MEF cells, all viruses were able to upregulate IFN-β expression, with the JBS003 vector showing the highest level, three times higher than that of the wild-type virus vector (JBS000). This means that JBS003 is not easily eliminated in tumor cells, but is easily eliminated in normal cells.

[0125] Example 2: Construction and efficacy demonstration of oncolytic virus vaccine 1. Oncolytic virus vaccines were constructed by inserting the NY-ESO-1 gene into each attenuated strain and wild-type virus prepared in Example 1, and the construction diagram is shown in Figure 5. The insertion status of genes or gene fragments of each group is shown in Table 2. [Table 2] Table 2: Indication of the insertion status of genes or gene fragments in each group

[0126] The specific manufacturing methods of JBS004 to JBS007, JBS011 to JBS013, and JBS015 are common techniques in this field, and are summarized as follows: Note that the following content does not limit the manufacturing methods of JBS004 to JBS007, JBS011 to JBS013, and JBS015 to the following methods, but merely shows examples.

[0127] (1) Construction of the attenuated strain plasmid. A link sequence having the restriction enzyme cleavage sites Xho I and Mlu I was artificially synthesized and inserted into the non-coding region between the G protein and the L protein of each of the attenuated strains prepared in Example 1 using biological and recombinant gene technology to obtain each of the attenuated strain plasmids.

[0128] (2) Insertion of a foreign gene. Each attenuated strain plasmid was double-digested with XhoI and MluI, and then the NY-ESO-1 foreign gene was inserted to obtain an attenuated strain recombinant plasmid carrying NY-ESO-1.

[0129] (3) Vaccine rescue: Each oncolytic virus vaccine was constructed by rescuing the vaccine corresponding to each attenuated strain recombinant plasmid with reference to the method of "virus rescue" in Example 1.

[0130] 2. This was an indication of the therapeutic effect of LLC-NY-ESO-1 non-small cell lung cancer (transplanted tumor).

[0131] 136 C57BL / 6 mice with no significant difference were selected and each was cultured at 2 × 10 5 LLC cells (mouse lung cancer cells) were subcutaneously inoculated and cultured for 9 days under the same appropriate conditions. On the 9th day after inoculation, the volume of the transplanted tumor was 100 mm 3When the number of mice reached about 100, the mice were divided into 17 groups (n=8). The control group (PBS group) was injected with 50μL of PBS into the tumor, and the other 16 groups were treated with JBS000, JBS001, JBS002, JBS003, JBS004, JBS005, JBS006, JBS007, JBS008, JBS009, JBS010, JBS011, JBS012, JBS013, JBS014, and JBS015, respectively. The mice were administered once every two days for a total of three doses. Each dose was 10 7 pfu / animal. From the start of administration until the end of the experiment, the volume of the transplanted tumor was recorded every two days, and the volume (mm 3 )=(long diameter x short diameter 2 ) / 2. The metastasis rate of cancer cells was detected, and the detection method was as follows: LLC cells have red fluorescent protein, so they emit yellow fluorescence under a green fluorescent microscope. If cancer cells metastasize to lung tissue, the lung tissue is placed under a microscope, a fluorescent image is taken, and the grayscale value of the image is analyzed by Image J to analyze the proportion of lung cancer cells, and the metastasis rate of cancer cells is obtained.

[0132] The changes in tumor volume are shown in Figures 6 to 8. The results showed that each treatment group had a certain degree of inhibitory effect on the transplanted tumors. One animal in the JBS003 group was completely cured. The cure rate of JBS004 against transplanted tumors reached 37.5%. The metastasis of cancer cells is shown in Figure 9. Figures 6 to 9 show that there is a more or less certain correlation between the volume of transplanted tumors and lung metastasis. The treatment of lung metastases by JBS003 was superior to the JBS000 and JBS001 groups, and the ability of JBS004 to inhibit or prevent lung cancer cell metastasis was superior to the other groups.

[0133] 3. MCA-205-NY-ESO-1 was an indication of therapeutic efficacy against fibrosarcoma (transplanted tumor).

[0134] Mice were treated with the method described in "Treatment of LLC-NY-ESO-1 non-small cell lung cancer transplanted tumors" and incubated for 10 min. 6 MCA-205-NY-ESO-1 fibrosarcoma cells were inoculated subcutaneously, and the volume of the transplanted tumor was 100 mm 3Similarly, 50 μL of PBS was injected intratumorally as a control group, and JBS004, JBS005, JBS006, JBS007, JBS011, JBS012, JBS013, and JBS015 were injected intratumorally in the treatment groups. Each group consisted of six animals, and the injections were administered once every two days for a total of three injections. The injection dose was 10 8 pfu / animal. The volume of the transplanted tumor was recorded every two days from the start of administration to the end of the experiment. The results are shown in Figures 10 and 11.

[0135] The results showed that each treatment group could reduce tumor volume to some extent. After treatment with JBS004, two mice had completely eliminated the implanted tumors (rate 33.33%), and the remaining mice had effectively controlled tumor volume, which was significantly different from the other groups. The overall response rate of fibrosarcoma treatment with JBS004 was 100%.

[0136] 4. This was an indication of the therapeutic effect of B16-F10-NY-ESO-1 melanoma (transplanted tumor).

[0137] Mice were treated with the same method as in the transplanted tumor test, and 2 × 10 6 B16-F10-NY-ESO-1 melanoma cells were inoculated subcutaneously, and the volume of the transplanted tumor was 100 mm 3 Similarly, 50 μL of PBS was injected intratumorally as a control group, and JBS004, JBS005, JBS006, JBS007, JBS011, JBS012, JBS013, and JBS015 were injected intratumorally in the treatment groups. Each group consisted of six animals, and the injections were administered once every two days for a total of three injections. The injection dose was 10 8 pfu / mouse. The volume of the transplanted tumor was recorded every two days from the start of administration to the end of the experiment. The results are shown in Figures 12 and 13. The results showed that each treatment group had a certain degree of therapeutic effect on melanoma, and the effect was particularly good in the JBS004 group.

[0138] 5. The effect of different doses of JBS004 was shown.

[0139] Select C57BL / 6 mice aged 6–8 weeks and weighing approximately 18 g, and incubate them at 2 × 10 5 LLC cells (mouse lung cancer cells) were subcutaneously inoculated. On the 9th day after inoculation, the volume of the transplanted tumor was 100 mm 3 When the number of mice reached about 100, they were divided into 5 groups, each with 6 animals. The control group (PBS group) was injected with 50 μL of PBS into the tumor, and the other 4 groups were treated with JBS004 for 10 min. 6 pfu / fish, 10 7 pfu / fish, 10 8 pfu / fish, 10 9 pfu / mouse was inoculated into the tumor, and the administration was performed once every two days, for a total of three administrations. From the start of administration to the end of the experiment, the volume of the transplanted tumor was recorded once every two days, and the results are shown in Figures 14 and 15. At the end of the experiment, the euthanized mice were autopsied, and lung tissue was taken from the mice to detect the metastasis rate of the cancer cells, and the results are shown in Figure 16.

[0140] The results showed that JBS004 at different doses all had a certain therapeutic effect on lung cancer mice, and 8 At the dose of pfu / animal, the cure rate reached 33.33%, the effective control rate reached 33.33%, and the rate of no detection of lung cancer cell metastasis was 66.67%, which was significantly superior to other dose groups.

[0141] In addition, as shown in Figure 17 and Figure 18, the body temperature and weight of the mice were maintained within the normal range throughout the experiment, and no abnormal body temperature or weight occurred, indicating that different doses of JBS004 had no significant effect on the body temperature and weight of the lung cancer mice. Regarding body weight, the weight of the mice in the PBS group increased steadily, while the weight of the mice in the other treatment groups increased slowly, which may be related to the shrinkage of the transplanted tumor. At the end of the experiment, there was no significant difference in the weight of the mice in each group, proving that JBS004 has good safety.

[0142] Example 3: Results of pharmacokinetic and acute toxicity experiments of JBS004 1. Pharmacokinetics experiment. C57BL / 6 mice were selected and 2 × 10 5 LLC cells were subcutaneously inoculated, and on the 9th day after inoculation, the volume of the transplanted tumor reached 100 mm 3 When the cells grew to about 1000 cells / ml, an LLC tumor model was established. 8 After a single intratumoral injection of pfu / animal, tumor tissue samples were taken at 0 minutes (+15 minutes), 6 hours, 12 hours, 48 ​​hours, 96 hours, 120 hours, and 14 days (repeated three times), and the tissues were crushed with an automatic grinder, and total RNA was extracted from the tumor tissues with Trizol. Finally, the viral nucleic acid copy number was analyzed using quantitative PCR (fluorescent probe method). The results are shown in Figure 19 and Figure 20.

[0143] The results showed that the viral load in the tumors reached its peak 6 hours after infection, replicating approximately 500 times more than initially, and that 48 hours after infection, the viral load fell below the initial injection amount, and 14 days later, the viral nucleic acid copy number was undetectable.

[0144] Therefore, JBS004 can replicate rapidly and efficiently in the tumor, and after 14 days, JBS004 was not detectable, proving that it will not accumulate in the body for a long time and cause later damage, and has good safety profile.

[0145] 2. Acute toxicity experiment. 40 C57BL / 6 mice were selected, half of which were male and half female. They were divided into three treatment groups and a control group, and were administered a single intramuscular injection of JBS004 solution. The dose of each group was 10 3 pfu / fish, 10 6 pfu / fish, 10 9 pfu / animal. The blank control group received a single dose of the solvent control (a single intramuscular injection of PBS), and the administration volume was 100 μL in each group. The day of administration to the animals was defined as the first day of observation of the animals in that group. The animals were observed for 14 days after administration, and dissected on the 15th day after administration.

[0146] During the experiment, the body temperature and weight of the mice were recorded every two days, and the results are shown in Figures 21 to 24. The mice were observed in detail through their cages before administration and 30 minutes, 1 hour, 2 hours, 4 hours, and 10 hours after administration, and were observed through their cages at least once every day in the subsequent experiments. At the end of the experiment, peripheral blood was collected from the mice for hematology and blood biochemistry detection (blood glucose, creatinine, urea nitrogen, blood urea nitrogen / creatinine, phosphorus ion, calcium ion, total protein, albumin, globulin, etc.), and the major organs were collected by necropsy, and the tissues of the heart, liver, spleen, lung, kidney, brain, and testis / ovary were weighed, and the organ coefficients were calculated.

[0147] Due to space limitations, the test results of all hematology and blood biochemistry indices are not shown here. The test results showed that no mice died abnormally and no JBS004-related clinical symptoms appeared. Injection of different doses of JBS004 had no significant effect on the organ weight of mice, and no significant effect on the hematology and blood biochemistry indices of mice. In this experimental condition, the maximum tolerated dose (MTD) was at least 10 9 pfu / animal. Therefore, the optimal dose (10 8 pfu / animal) was within the safe dose.

[0148] (Example 4: Demonstration of the efficacy of the combination of an oncolytic virus vaccine and a PD-1 antibody (PD1 antibody) in the treatment of non-small cell lung cancer) C57BL / 6 mice aged 6 to 8 weeks and weighing approximately 18 g were selected and each mouse was inoculated with 2 × 10 5 Non-small cell lung cancer LLC-NY-ESO-1 cells were subcutaneously inoculated, and the volume of the transplanted tumor was 100 mm 3When the PD-1 antibody concentration reached about 100%, treatment was started. The treatment conditions for each group are shown in Table 3, and the mice were divided into a control group (injected with PBS), a PD-1 antibody group, a JBS004 group, a JBS004 and PD-1 antibody combination group, a JBS005 group, a JBS005 and PD-1 antibody combination group, a JBS006 group, a JBS006 and PD-1 antibody combination group, a JBS007 group, a JBS007 and PD-1 antibody combination group, a JBS011 group, a JBS011 and PD-1 antibody combination group, a JBS012 group, a JBS012 and PD-1 antibody combination group, a JBS013 group, a JBS013 and PD-1 antibody combination group, a JBS014 group, and a JBS014 and PD-1 antibody combination group, each group consisting of 8 mice. The sequence of the heavy chain variable region of the PD-1 antibody was as shown in SEQ ID NO: 12, and the sequence of the light chain variable region was as shown in SEQ ID NO: 13. JBS004 was injected intratumorally once every two days for a total of three doses. 8 pfu / mouse, and the PD-1 antibody was intraperitoneally injected once every two days for a total of three doses, with each dose being 5 mg / kg, and the administration time being the same as that of JBS004. [Table 3] Table 3: Descriptor of treatment for each group

[0149] From the start of administration to the end of the experiment, the volume of the transplanted tumor was recorded every two days. At the end of the experiment, the mice were autopsied, and the lungs were removed and placed under a fluorescent microscope to take fluorescent images of the lungs. The results of the volume change of the transplanted tumor in each group are shown in Figure 25 and Figure 26. The results of the metastasis of lung cancer cells in each group are shown in Figure 27.

[0150] As can be seen from Figure 25 and Figure 26, the tumor growth trend in the PBS group proved that the lung cancer transplantation tumor model was successfully established. The tumor inhibition effect of the combination treatment of oncolytic virus vaccine and PD-1 antibody in each group was superior to the effect of single administration. Although the single intraperitoneal injection of PD-1 antibody had an effect on the growth of transplanted tumors, the mice did not show complete cure of transplanted tumors, while the mice in the JBS004 group and the JBS004 + PD1 antibody combination treatment group all had the volume of transplanted tumors controlled to some extent. Among them, 2 mice in the JBS004 group were completely cured, the rate was 25%, and 5 mice in the JBS004 + PD1 antibody group were completely cured, the rate was 62.5%. As can be seen from Figure 27, in both the JBS004 group and the JBS004 + PD1 antibody group, the cancer cell metastasis of the lung cancer transplant tumor was inhibited to a certain extent, and after combined administration of JBS004 and PD1 antibody, the inhibitory effect on cancer cell metastasis was significantly superior to the other groups.

[0151] Example 5: Effect of different doses of PD-1 antibody when co-administering oncolytic virus vaccine and PD-1 antibody A lung cancer transplanted tumor model (LLC-NY-ESO-1, mouse) was established by the method of Example 4, and the volume of the transplanted tumor was 100 mm 3 Treatment was started when the tumor size reached approximately 100%. There was a control group (PBS injection), a JBS004 group (JBS004 intratumoral injection only), and a JBS004 + PD-1 antibody group. In the JBS004 + PD-1 antibody group, the PD-1 antibody was administered intraperitoneally at doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg, respectively, with 8 mice in each group. In each group, JBS004 was injected intratumorally at a dose of 10 mg / kg. 8 pfu / animal, administered once every two days for a total of three doses, with the administration times for JBS004 and the PD-1 antibody being the same.

[0152] The changes in tumor volume in each group are shown in Figure 28 and Figure 29, and the metastasis of cancer cells is shown in Figure 30. As can be seen from the figures, except for the 1mg / kg JBS004 + PD-1 antibody group, all other combination administration groups showed a certain degree of improvement in therapeutic efficiency. The cure rate of JBS004 was 25%, and when the PD-1 antibody was 5mg / kg, the cure rate was 62.5%, showing the most obvious inhibitory effect on cancer cell metastasis, and when the PD-1 antibody was 10mg / kg, the cure rate was 37.5%, showing a weaker inhibitory effect on cancer cell metastasis than the JBS004 alone group and the 1mg / kg PD-1 antibody group.

[0153] As can be seen above, the optimal dose of PD-1 antibody when combined with JBS004 was 5 mg / kg.

[0154] Example 6: Demonstration of the effect of different administration routes of JBS004 when co-administered with an oncolytic virus vaccine and a PD-1 antibody The administration routes of JBS004 include intranasal administration, intratumoral injection, intravenous injection, and intraperitoneal injection. From the viewpoint of clinical administration convenience, only intratumoral injection and intravenous injection were targeted in this example. A lung cancer transplanted tumor model (LLC-NY-ESO-1, mouse) was established by the method of Example 4, and the volume of the transplanted tumor was 100 mm 3 The treatment was started when the tumor size reached about 100%. There were 8 mice in each group, including a control group (PBS injection), a group receiving intratumoral injection (IT) of JBS004 alone, a group receiving intravenous injection (IV) of JBS004 alone, a group receiving intratumoral injection (IT) of JBS004 in combination with PD-1 antibody, and a group receiving intravenous injection (IV) of JBS004 in combination with PD-1 antibody. 9 The intratumoral dose was 10 pfu / animal. 8pfu / animal, and the PD-1 antibodies were all administered by intraperitoneal injection, with each dose being 5mg / kg. They were administered once every two days for a total of three doses, and the administration times for JBS004 and the PD-1 antibodies were the same. The intravenous dose was greater than the intratumoral dose because, after intravenous injection, some of the drug is lost in the body before it reaches the tumor, and the intravenous dose was increased to maximize the amount of drug that reaches the tumor being equivalent to that of the intratumoral injection group.

[0155] The changes in tumor volume in each group are shown in Figure 31 and Figure 32, and the metastasis of cancer cells is shown in Figure 33. Each figure shows that the intratumoral injection method was significantly superior to intravenous injection. After the combination treatment of JBS004 intratumoral injection and PD-1 antibody, the cure rate was 62.5%, while under the same conditions, the cure rate of JBS004 intravenous injection was only 12.5%.

[0156] Example 7: Effect of different initial administration orders when administering a combination of an oncolytic virus vaccine and a PD-1 antibody Theoretically, when JBS004 exerts its tumor lysis effect, it mainly kills tumors by recruiting T cells and infiltrating the tumor locally. After T cells reach the tumor site, they need to fight against cytokines in the tumor microenvironment, which weakens their tumor lysis killing ability to some extent. If PD-1 antibodies can be injected in advance, it may enhance the tumor lysis effect of JBS004 by effectively blocking the local immune check of the tumor. Preliminary tests and theoretical analysis have shown that the order of the first administration has a significant impact on efficacy, and the order of subsequent administrations does not have a high correlation with efficacy, so this example only shows the relationship between the order of the first administration and efficacy. The reasons why the first administration has a high correlation with efficacy are thought to be (1) the tumor volume is relatively small at the time of the first administration, so the tumor microenvironment is easily destroyed, and (2) a certain amount of PD-1 antibodies is already present in the body at the time of subsequent administrations.

[0157] A lung cancer transplanted tumor model (LLC-NY-ESO-1, mouse) was established by the method of Example 4, and the volume of the transplanted tumor was 100 mm3 When the tumor size reached about 100 mm, treatment was started. Each group had 8 mice, and JBS004 was injected intratumorally at a dose of 10 mg / kg. 8 pfu / animal, and the PD-1 antibodies were all intraperitoneally injected at a dose of 5mg / kg, once every two days for a total of three doses. A PBS group was set up, with 200μL PBS intraperitoneally injected and 50μL PBS intratumorally injected at the same time, group 1 was intraperitoneally injected with the PD-1 antibody and at the same time intratumorally injected with JBS004, group 2 was intraperitoneally injected with the PD-1 antibody first, and 6 hours later, JBS004 was intratumorally injected, and group 3 was intratumorally injected with JBS004 first, and 6 hours later, PD-1 antibody was intraperitoneally injected.

[0158] The changes in tumor volume in each group are shown in Figure 34 and Figure 35, and the metastasis of cancer cells is shown in Figure 36. When administered simultaneously, the cure rate was 62.5%, while the cure rate in the group injected with JBS004 first dropped to 37.5%, while the cure rate in the group administered with the PD-1 antibody first was high at 87.5%, and the therapeutic efficiency was significantly improved.

[0159] As can be seen from the above, the combination therapy of first providing PD-1 antibody and then providing JBS004 had the best therapeutic effect. That is, when JBS004 and PD-1 antibody are used in combination, it is more useful to improve the therapeutic effect if the PD-1 antibody is released (produces its effect) in the body before JBS004. In practical use, in order to achieve that the PD-1 antibody is released or reaches the tumor first to produce its effect, a sequential administration method or a pharmaceutical means may be adopted. When a sequential administration method is adopted, it may be administered by intravenous injection, intratumoral injection, intraperitoneal injection, subcutaneous injection, etc. When a pharmaceutical means is adopted, JBS004 and PD-1 may be manufactured together as a drug, and the order and release time of JBS004 and PD-1 may be controlled by means of coating, etc. during the manufacturing process.

[0160] (Example 8: Demonstration of the effect of combined treatment of JBS004 and PD-1 antibody against other tumors (cancers)) In this example, each group had 8 mice, and JBS004 was injected intratumorally at a dose of 10 8 pfu / mouse, and the PD-1 antibodies were administered intraperitoneally at a dose of 5 mg / kg, once every two days for a total of three doses. The administration sequence was to provide the PD-1 antibody first, followed by JBS004 6 hours later.

[0161] 1.This demonstrated the effectiveness of the combination of JBS004 and PD-1 antibody in the treatment of breast cancer (transplanted tumors).

[0162] 5 × 10 5 4T1-NY-ESO-1 cells (breast cancer cells) were orthotopically inoculated, and the volume of the transplanted tumor was 100 mm 3 The subjects were divided into three groups: a PBS group, a group injected with JBS004 alone, a group injected with PD-1 antibody alone, and a group injected with JBS004 and PD-1 antibody in combination.

[0163] The changes in tumor volume in each group are shown in Figures 37 and 38. The cure rate in the JBS004 and PD-1 antibody combination treatment group was 62.5%, and the efficacy rate was 100%, which was significantly superior to the other groups.

[0164] 2.The combination of JBS004 and PD-1 antibody showed efficacy in the treatment of colon cancer (transplanted tumors, large tumors).According to current clinical trials, all known cancer drugs are ineffective against large tumors exceeding a certain volume.

[0165] MC38-NY-ESO-1 cells (colon cancer cells) were subcutaneously inoculated into C57BL / 6 mice, and the volume of the transplanted tumor was 200–300 mm 3 There were three groups: a PBS group, a group injected with JBS004 alone, a group injected with PD-1 antibody alone, and a group injected with JBS004 and PD-1 antibody in combination.

[0166] The changes in tumor volume in each group are shown in Figures 39 and 40. It was found that even when JBS004 was administered alone, it was possible to cure large colon cancer tumors to some extent. Moreover, the inventors were pleased to find that under the conditions of this model, PD-1 antibodies also had a certain degree of efficacy against large tumors. The cure rate of the combined treatment group was as high as 75%, far superior to the use of either agent alone.

[0167] The above detailed description is provided by way of example and is not intended to limit the scope of the claims. Various modifications of the embodiments described herein will be obvious to those skilled in the art and are within the scope of the claims and their equivalents.

Claims

1. The attenuated oncolytic virus strain and an immune checkpoint inhibitor, wherein the attenuated oncolytic virus strain has a modified matrix protein M compared to a VSV MuddSummer subtype strain, the modification comprising knockout of a base encoding leucine at amino acid position 111; The amino acid sequence of the substrate protein M before modification is shown in SEQ ID NO: 2, and the immune checkpoint inhibitor is any one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, and an anti-SIGLEC15 antibody.

2. The pharmaceutical composition according to claim 1, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111.

3. The pharmaceutical composition according to claim 1 or 2, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain further comprises a mutation of methionine at amino acid position 51 to arginine.

4. The pharmaceutical composition according to claim 3, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111 and a mutation of methionine at position 51 to arginine.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain further comprises a mutation of valine at position 221 to phenylalanine.

6. The pharmaceutical composition according to claim 5, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain is knockout of the base coding for leucine at position 111 and mutation of valine at position 221 to phenylalanine.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain further comprises an amino acid mutation at amino acid position 226, which is a serine to arginine mutation.

8. The pharmaceutical composition according to claim 7, wherein the modification of the matrix protein M of the attenuated oncolytic virus strain is knockout of the base encoding leucine at amino acid position 111 and mutation of serine at position 226 to arginine.

9. The pharmaceutical composition according to claim 7, wherein the modification of the substrate protein M of the attenuated oncolytic virus strain is a knockout of the base encoding leucine at amino acid position 111, a mutation of valine at position 221 to phenylalanine, and a mutation of serine at position 226 to arginine.

10. The pharmaceutical composition according to claim 7, wherein the modifications of the matrix protein M of the attenuated oncolytic virus strain are a mutation of methionine at position 51 to arginine, a knockout of the base encoding leucine at position 111, a mutation of valine at position 221 to phenylalanine, and a mutation of serine at position 226 to arginine.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the substrate protein M of the attenuated oncolytic virus strain has any one of the amino acid sequences of the group consisting of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11.

12. A pharmaceutical composition comprising an oncolytic virus vaccine and an immune checkpoint inhibitor, wherein the oncolytic virus vaccine is an attenuated oncolytic virus strain according to any one of claims 1 to 11 into which an antigen has been inserted.

13. The pharmaceutical composition of claim 12, wherein the antigen is a specific tumor antigen.

14. The antigens include NY-ESO-1, gp33, gp100, TX103, mucin-1, WT-1, MART-1, MAGE A1, MAGE A3, MAGE A4, MAGE B2, PRAME, survivin, MART-1, col6A3, tyrosinase, T antigen, SLC45A2, VCX / Y, HPV, α-fetoprotein, carcinoembryonic antigen, CA 14. The pharmaceutical composition according to claim 12 or 13, wherein the protein is selected from the group consisting of 125, Her2, dopachrome tautomerase, BAGE protein, GAGE ​​protein, survivin, tyrosinase, SSX2, cyclin A1, KIF20A, MUC5AC, Meloe, Lengsin, kallikrein 4, IGF2B3 and glypican 3.

15. The pharmaceutical composition according to any one of claims 12 to 14, wherein the immune checkpoint inhibitor is released in the body prior to the oncolytic virus vaccine.

16. The pharmaceutical composition according to any one of claims 1 to 15 for treating antitumor or cancer.

17. 17. The pharmaceutical composition of claim 16, wherein the tumor or cancer is selected from the group consisting of head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.

18. Use of a pharmaceutical composition according to any one of claims 1 to 17 in the manufacture of an antitumor or tumor treatment medicament.

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