Anti-tumor drug combination and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses an anti-tumor combined drug, the anti-tumor combined drug comprises a two-factor oncolytic virus and an HER2 CAR-T cell, the two-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus capable of simultaneously expressing OV-OX40L and IL12, the HER2 CAR-T cell is a cell specially targeting an HER2 antigen, and the CAR structure contained in the HER2 CAR-T cell comprises Anti-HER2 scfv, CD8hinge, CD8TM, CD28-4-1BB, CD3zeta, IRES and EGFP. The CAR-T therapy and the oncolytic virus therapy are combined, the advantages of the CAR-T therapy and the oncolytic virus therapy are mutually promoted, the infiltration and the activity of the CAR-T cells in solid tumors are enhanced by the two-factor oncolytic viruses, and meanwhile, the oncolytic virus-mediated oncolytic property can be improved by the CAR-T cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-tumor combined drug and application thereof. Background Art

[0002] Malignant tumors seriously threaten human life and health and have become one of the most heavy disease burdens in today's society. Despite the continuous advancement of medical technology, the increasing number of treatment methods, and the increasing number of drugs available, the vast majority of malignant tumors are still difficult to cure, and the survival period of patients is generally short. Therefore, any method that can effectively improve the treatment effect, whether it is a new treatment method or a measure to optimize the existing treatment plan, has a practical value that cannot be ignored. In recent years, the field of tumor immunotherapy has focused on the CAR-T strategy and carried out a lot of research. Compared with traditional radiotherapy and chemotherapy, this strategy has shown significant advantages: not only outstanding treatment effects, but also long-lasting efficacy and fewer adverse reactions, which has brought new hope for overcoming tumor problems and is expected to achieve a true cure of tumors. CAR-T therapy is to extract patient T cells, genetically engineer them to carry chimeric antigen receptors, and accurately identify and efficiently kill tumor cells after re-infusion. CAR-T therapy is more specific than radiotherapy and chemotherapy, can accurately kill tumors, has fewer side effects, and can also stimulate lasting immunity. CAR-T therapy has significant results for a variety of blood tumors, such as relapsed / refractory acute lymphoblastic leukemia. Clinical trials have shown that the remission rate of some patients exceeds 80%. For example, in a study of a specific type of leukemia, the complete remission rate among patients receiving CAR-T therapy was much higher than that of traditional treatments.

[0003] In the process of gradual advancement of clinical research, CAR-T therapy has encountered a series of problems that need to be overcome in the field of anti-tumor treatment. There are many problems in the treatment of solid tumors with CAR-T therapy. First, the heterogeneity of tumor antigens. The antigens in different patients or in the same tumor vary greatly, which affects the accurate recognition of CAR-T cells. Second, the tumor microenvironment is complex, and the immunosuppressive factors, hypoxia, etc. in it will hinder the activity and function of CAR-T cells. Third, it is difficult for CAR-T cells to effectively infiltrate deep into solid tumors and cannot fully kill cancer cells, resulting in limited treatment effects. In response to the above problems, we can start from many aspects. For example, develop precise detection technology, deeply analyze tumor antigens, overcome antigen heterogeneity; design regulators for the tumor microenvironment to improve the state of immunosuppression; optimize the design of CAR-T cells and enhance their infiltration ability, so as to comprehensively improve the treatment effect and allow CAR-T therapy to play a greater role in the treatment of solid tumors.

[0004] In the exploration of improving the response rate of solid tumor patients to CAR-T therapy, some studies have attempted to combine CAR-T therapy with chemotherapy, radiotherapy or immunosuppressants (such as PD-1 / PD-L1 inhibitors). However, although this combination therapy has a certain effect, it inevitably increases the probability of treatment-related adverse events. From the perspective of clinical practice, when CAR-T therapy is combined with chemotherapy or radiotherapy, patient compliance is not ideal, and economic pressure increases accordingly. In addition, some scholars have pointed out that the cytotoxicity of chemotherapy drugs can damage rapidly proliferating blood lymphocytes, which may cause T cell exhaustion and thus weaken the anti-tumor synergistic effect of CAR-T therapy. In view of these circumstances, it is necessary to explore a new generation of combined treatment models without chemotherapy. Therefore, the development of combination drugs with excellent efficacy, high safety and the ability to enhance the effect of CAR-T therapy is not only of great significance, but also has huge commercial potential. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an anti-tumor combination drug and its application, which solves the problem that the cytotoxicity of chemotherapy drugs will damage rapidly proliferating blood lymphocytes during combined chemotherapy or radiotherapy, causing T cell exhaustion, thereby weakening the anti-tumor synergistic effect of CAR-T therapy.

[0006] To achieve the above objectives, the present invention is implemented through the following technical scheme: an anti-tumor combination drug, comprising a dual-factor oncolytic virus and HER2CAR-T cells, wherein the dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12, and the HER2CAR-T cells are cells that specifically target HER2 antigens, and the CAR structure contained in the HER2CAR-T cells includes Anti-HER2 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

[0007] Preferably, the Anti-HER2 scfv is a single-chain antibody fragment of the anti-HER2 antigen, which is formed by connecting the heavy chain variable region and light chain variable region of the immunoglobulin of the anti-HER2 antigen through a flexible connecting peptide.

[0008] Preferably, the CD8 hinge is the hinge region of CD8.

[0009] Preferably, the CD8 TM is the transmembrane region of CD8.

[0010] Preferably, the CD28-4-1BB is the intracellular region of CD28 and 4-1BB co-stimulatory factor.

[0011] Preferably, the CD3ζ is the intracellular region of the CD3ζ chain.

[0012] Preferably, the structure of the dual-factor insertion site of the OV-OX40L / IL12 oncolytic virus includes OV-OX40L, UL26, UL27, AICP47, and IL12.

[0013] Preferably, the dosage form of the anti-tumor combination drug is an injection, and the administration method includes one or more of intratumor injection and intravenous injection.

[0014] The invention discloses an application of an anti-tumor combined drug in the field of anti-tumor treatment.

[0015] Preferably, the tumor is a solid tumor and a tumor with high expression of HER2, including but not limited to pancreatic cancer and ovarian cancer, and the cells of the tumor with high expression of HER2 include human ovarian cancer cells SKOV3, mouse pancreatic cancer cells Pan02, and primary tumor cells from patients.

[0016] The present invention provides an anti-tumor combined drug and its application. It has the following beneficial effects:

[0017] 1. The present invention combines CAR-T therapy and oncolytic virus therapy, utilizing the advantages of both to promote each other. The dual-factor oncolytic virus enhances the infiltration and activity of CAR-T cells in solid tumors, while CAR-T cells can enhance the oncolysis mediated by oncolytic viruses, thereby enhancing the ability of both to clear tumors. This overcomes the limitations of traditional therapies and has better targeting, lethality and safety, thereby solving the problem that the cytotoxicity of chemotherapy drugs during combined chemotherapy or radiotherapy will damage rapidly proliferating blood lymphocytes, causing T cell exhaustion, thereby weakening the anti-tumor synergistic effect of CAR-T therapy.

[0018] 2. The present invention combines CAR-T therapy with oncolytic virus therapy. The dual-factor oncolytic virus can enhance the infiltration and activity of CAR-T cells in solid tumors. At the same time, CAR-T cells can enhance the oncolysis mediated by oncolytic virus. The two complement each other and significantly enhance the ability to clear tumors.

[0019] 3. The present invention specifically targets HER2 antigens through HER2 CAR-T cells, which can accurately identify and kill HER2-positive tumor cells, improve the targeted nature of treatment, and reduce damage to normal cells.

[0020] 4. The present invention shows that the combined treatment group has no significant weight loss compared with other groups in experiments on immunodeficient tumor-bearing mouse model and immune-complete tumor-bearing mouse model, indicating that the combined drug has no obvious toxic side effects and is beneficial to improving the patient's quality of life and treatment compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Schematic diagram of the CAR structure contained in the HER2CAR-T cell of the present invention;

[0022] Figure 2 This is a schematic diagram of the structure of the dual-factor insertion site of the OV-OX40L / IL12 oncolytic virus of the present invention;

[0023] Figure 3 A schematic diagram of the killing ability of the combined drug of the present invention on tumor cells;

[0024] Figure 4 This is a schematic diagram of the tumor cell killing activity of the present invention;

[0025] Figure 5 This is a schematic diagram of the amount of killing factor IFN-γ released by the CAR-T cells in the combination group of the present invention;

[0026] Figure 6 This is a schematic diagram of the amount of the killing factor Granzyme B released by the CAR-T cells in the combination group of the present invention;

[0027] Figure 7 A schematic diagram of the combined treatment of OV-OX40 / IL12 and HER2CAR-T cells in vivo to control the growth of pancreatic cancer;

[0028] Figure 8 Schematic diagram of the side effects of the combined treatment of OV-OX40 / IL12 and HER2CAR-T cells of the present invention;

[0029] Fig. 9 A schematic diagram comparing the treatment scheme of OV-OX40 / IL12 combined with HER2mCAR-T cells of the present invention with the monotherapy of HER2mCAR-T cells and the treatment scheme of OV combined with HER2mCAR-T cells;

[0030] Fig.10 This is another schematic diagram comparing the OV-OX40 / IL12 combined with HER2mCAR-T cell treatment regimen of the present invention with the HER2mCAR-T cell monotherapy and the OV combined with HER2mCAR-T cell treatment regimen. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Please see attached Figure 1 -Attached Fig.10 The embodiment of the present invention provides an anti-tumor combination drug, including a dual-factor oncolytic virus and a HER2CAR-T cell, wherein the dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12, and the HER2CAR-T cell is a cell that specifically targets the HER2 antigen, and the CAR structure contained in the HER2 CAR-T cell includes Anti-HER2 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP; Anti-HER2 scfv is a single-chain antibody fragment against HER2 antigen, which is formed by connecting the heavy chain variable region and the light chain variable region of the immunoglobulin against the HER2 antigen through a flexible connecting peptide; CD8 hinge is the hinge region of CD8; CD8TM is the transmembrane region of CD8; CD28-4-1BB is the intracellular region of the CD28 and 4-1BB costimulatory factors; CD3ζ is the intracellular region of the CD3ζ chain.

[0033] Specifically, Anti-HER2 scfv is a single-chain antibody fragment against HER2 antigen, which is composed of the variable region of the immunoglobulin heavy chain (VH) and the variable region of the light chain (VL) of the anti-HER2 antigen connected by a flexible connecting peptide. This enables Anti-HER2scfv to accurately identify the HER2 antigen. The VH and VL regions have specific amino acid sequences, respectively, which have been screened and optimized to give the single-chain antibody fragment a high degree of specificity and affinity. The flexible connecting peptide ensures that the VH and VL regions can swing freely in space to better bind to the HER2 antigen, just like a tailor-made "key" that can be accurately inserted into the "lock" of the HER2 antigen, laying the foundation for subsequent immune responses.

[0034] CD8 hinge is the hinge region of CD8. It plays a role of connection and buffering, connecting different parts of the CAR structure like a bridge. Its amino acid composition has a certain flexibility and elasticity, which can ensure the stability of the structure while allowing CAR to stretch and bend appropriately on the cell surface, making CAR-T cells more flexible in identifying and binding to tumor cells, and improving the targeting and attack efficiency of cells.

[0035] CD8 TM is the transmembrane region of CD8. Like an "anchor", it fixes the CAR structure on the cell membrane of HER2CAR-T cells to ensure the stable expression of CAR on the cell surface. The amino acids in the transmembrane region have special hydrophobicity, which enables it to be stably embedded in the lipid bilayer of the cell membrane, ensuring the effective connection between the CAR structure and the signal transduction pathway inside the cell, so that after the cell recognizes the tumor cell antigen, it can promptly transmit the signal to the inside of the cell and start the immune killing program.

[0036] CD28-4-1BB is the intracellular region of CD28 and 4-1BB co-stimulatory factors. These two co-stimulatory factors play a vital role in the activation of CAR-T cells. When the Anti-HER2 scFv on the surface of CAR-T cells binds to the HER2 antigen, the CD28 and 4-1BB co-stimulatory factors work together to provide additional activation signals for T cells. CD28 can promote the proliferation, survival and cytokine secretion of T cells, while 4-1BB further enhances the activity and memory function of T cells. They work together to enable CAR-T cells to quickly activate and proliferate in large numbers when encountering tumor cells, thereby enhancing their ability to kill tumor cells.

[0037] CD3ζ is the intracellular region of the CD3ζ chain. It is an important component of the T cell receptor (TCR) signaling complex. When CAR recognizes and binds to the HER2 antigen on the surface of tumor cells, the CD3ζ chain initiates a series of intracellular signaling events. It recruits a variety of signaling molecules and activates downstream signaling pathways, such as the phosphatidylinositol 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway, which ultimately leads to the activation, proliferation and release of cytotoxic substances of T cells, and directly participates in the killing process of tumor cells.

[0038] IRES (internal ribosome entry site) plays a key regulatory role in the CAR structure of HER2 CAR-T cells. It allows independent translation of multiple genes on the same transcript. In the present invention, IRES enables other elements in the CAR structure (such as CD28-4-1BB, CD3ζ, etc.) and the reporter gene EGFP to be translated and expressed on the same mRNA molecule. This not only ensures the coordinated expression of the various parts of the CAR structure, but also provides an intuitive detection method during the experiment. By detecting the expression of EGFP, it is possible to determine whether the CAR structure is successfully transfected into T cells, as well as the level of its expression, which greatly facilitates the preparation and screening of HER2 CAR-T cells.

[0039] As a reporter gene, EGFP (enhanced green fluorescent protein) has greatly facilitated the research and application of HER2 CAR-T cells. During cell culture and experiments, the distribution, number and activity changes of EGFP-labeled HER2 CAR-T cells can be visually observed with the help of equipment such as fluorescence microscopes or flow cytometers. For example, in in vitro killing experiments, by observing the changes in the fluorescence intensity of EGFP, the killing effect of HER2 CAR-T cells on tumor cells can be monitored in real time; in in vivo experiments, the migration and distribution of HER2 CAR-T cells in mice can also be tracked, providing an important basis for further understanding the mechanism of action of combined drugs and optimizing treatment plans.

[0040] The structure of the dual-factor insertion site of the OV-OX40L / IL12 oncolytic virus includes OV-OX40L, UL26, UL27, AICP47, and IL12.

[0041] Specifically, OV-OX40L is a modified oncolytic virus vector part that can express OX40L. As a co-stimulatory molecule, OX40L can bind to the OX40 receptor on the surface of T cells, activate the immune response of T cells, enhance the proliferation and survival of T cells, and provide favorable conditions for the activation and expansion of CAR-T cells in the tumor microenvironment. UL26 and UL27 are important gene fragments in the structure of oncolytic viruses themselves, and they play an indispensable role in maintaining the stability, infection ability and replication of oncolytic viruses. AICP47 is a special regulatory protein that can regulate the replication and immune escape process of oncolytic viruses in cells, so that oncolytic viruses can better survive and play a role in tumor cells, while avoiding excessive clearance by the body's immune system. IL12 is a cytokine with powerful immunoregulatory function. It can activate immune cells such as natural killer cells (NK cells) and T cells, promote the secretion of cytokines such as interferon-γ (IFN-γ), and enhance the body's anti-tumor immune response. In the OV-OX40L / IL12 oncolytic virus, these elements work together to enhance immunity and oncolysis.

[0042] The dosage form of the anti-tumor combination drug is an injection, and the administration method includes one or more of intratumor injection and intravenous injection.

[0043] Specifically, intratumoral injection can make the drug act directly on the tumor tissue, increase the local drug concentration of the tumor, and enhance the killing effect on tumor cells. When performing intratumoral injection, the appropriate injection site and dose are selected according to the size, location and type of the tumor. For example, for some small and superficial tumors, multi-point intratumoral injection can be used to ensure that the drug can be evenly distributed in the tumor tissue; for larger tumors, the injection dose needs to be accurately calculated to avoid adverse reactions caused by excessive drug. Intravenous injection has the advantages of simple operation and the ability to quickly reach the systemic blood circulation, so that combined medication can have an effect on tumor cells throughout the body, especially for patients with tumors that have metastasized. During intravenous injection, the injection speed and concentration of the drug need to be strictly controlled to prevent adverse drug reactions and allergic reactions. At the same time, in order to ensure that the drug can effectively reach the tumor tissue, some auxiliary means can be combined, such as using nanoparticles to encapsulate the drug to improve the stability and targeting of the drug in the blood circulation.

[0044] The invention discloses an application of an anti-tumor combined drug in the field of anti-tumor treatment.

[0045] The tumor is a solid tumor and a tumor with high expression of HER2, including but not limited to pancreatic cancer and ovarian cancer. The cells of the tumor with high expression of HER2 include human ovarian cancer cell SKOV3, mouse pancreatic cancer cell Pan02, and primary tumor cells from patients.

[0046] Specifically, at the first level of the present invention, a combined drug preparation for anti-tumor has been developed. The preparation consists of a dual-factor oncolytic virus (OV-OX40L / IL12) with a specific efficacy dose and HER2 CAR-T cells, wherein the dual-factor oncolytic virus refers to an OV-OX40L / IL12 oncolytic virus that can express OV-OX40L and IL12 at the same time, and HER2CAR-T cells are cells that specifically target HER2 antigens. In some specific application scenarios, OV-OX40L / IL12 and HER2CAR-T cells will each act independently as different drug delivery units; in other cases, the two will be combined to form a complete drug delivery unit.

[0047] In some embodiments, the HER2 CAR-T cell comprises a CAR structure such as Figure 1As shown in the figure, Anti-HER2 scfv is a single-chain antibody fragment (scFv) against HER2 antigen, which is composed of the immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) of the anti-HER2 antigen connected by a flexible connecting peptide; CD8 hinge is the hinge region of CD8; CD8 TM is the transmembrane region of CD8; CD28-4-1BB is the intracellular region of CD28 and 4-1BB co-stimulatory factors; CD3ζ is the intracellular region of the CD3ζ chain.

[0048] In some embodiments, the structure of the dual factor insertion site of the OV-OX40L / IL12 oncolytic virus is as follows Figure 2 shown.

[0049] Furthermore, the dosage form of the anti-tumor combination drug preparation is an injection, and the administration method includes one or more of intratumor injection and intravenous injection. Furthermore, the anti-tumor combination drug preparation is a combination of OV-OX40L / IL12 oncolytic virus and HER2 CAR-T cells.

[0050] In the second aspect of the present invention, the practical application of the above-mentioned combination of drugs in the field of anti-tumor treatment is explored. It has been found that the combination of drugs has a potential therapeutic effect on a variety of tumors, especially pancreatic cancer and ovarian cancer. The method of the present invention combines CAR-T therapy and oncolytic virus therapy, using the advantages of the two to promote each other. The dual-factor oncolytic virus enhances the infiltration and activity of CAR-T cells in solid tumors, while CAR-T cells can enhance the oncolytic virus-mediated oncolysis and enhance the ability of both to clear tumors. This treatment method not only overcomes the limitations of traditional therapies, but also has good targeting, lethality and safety, thereby solving the problem that the cytotoxicity of chemotherapy drugs during combined chemotherapy or radiotherapy will damage rapidly proliferating blood lymphocytes, causing T cell exhaustion, thereby weakening the anti-tumor synergistic effect of CAR-T therapy, and has a potential therapeutic effect on a variety of tumors, especially solid tumors with high HER2 expression such as pancreatic cancer and ovarian cancer, providing a new option for the treatment of these refractory tumors.

[0051] In some embodiments, the tumor is a solid tumor.

[0052] In some embodiments, the tumor is a HER2-high expressing tumor.

[0053] In some embodiments, the solid tumor is: HER2-positive ovarian cancer, HER2-positive pancreatic cancer.

[0054] In some embodiments, the tumor cell is a tumor cell that overexpresses HER2.

[0055] In some embodiments, the HER2 high-expressing tumor cells are: human ovarian cancer cells SKOV3, or mouse pancreatic cancer cells Pan02, or primary tumor cells from patients.

[0056] In some embodiments, a gene expressing luciferase is inserted into all SKOV3 cells by lentiviral infection, denoted as SKOV3-LUC.

[0057] In some embodiments, Pan02 cells express both human antigen HER2 and luciferase, represented as Pan02-HVEM-HER2-LUC-BFP.

[0058] In some embodiments, HER2 CAR-T represents a human type and HER2 mCAR-T represents a mouse type.

[0059] In some embodiments, the scFv in the CAR structure in the HER2 mCAR-T targets the human HER2 antigen, and the other structural components of the CAR are all of mouse origin.

[0060] In some embodiments, data are presented as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0061] The following is further introduced in conjunction with specific embodiments:

[0062] Embodiment 1:

[0063] In vitro killing effect of combined drug therapy on HER2-positive cells.

[0064] HER2 CAR-T cells and oncolytic viruses were incubated separately or simultaneously with human ovarian cancer cell SKOV3 cells for 36 hours, and the bioluminescence value was detected by an enzyme-labeled instrument to determine the killing effect of the in vitro combination therapy on HER2-positive cells.

[0065] 1. Prepare a 96-well plate and plate SKOV3-LUC cells at a density of 5000 cells / well. Set up 3 replicate wells in each group and add 100 μL of culture medium to each well.

[0066] 2. After 24 hours of cell culture, HER2 CAR-T cells and basic oncolytic virus (OV) or dual-factor oncolytic virus (OV-OX40 / IL12) were added alone or simultaneously. The ratio of HER2 CAR-T cells to target cells was 1:10, and the volume was 50 μL. Oncolytic virus was used to infect target cells at an MOI of 0.1, and the volume was 50 μL. The cells were cultured in a 37-degree incubator for 36 hours.

[0067] 3. After the culture, aspirate 100ul of supernatant from each well and detect the secretion of IFN-γ and Granzyme B by ELISA.

[0068] 4. Discard the excess liquid in each well, add 100ul of D-luciferin sodium salt (Yeasen Biotechnology, 40901ES03) with a working concentration of 150ug / ml, and use a microplate reader to read the fluorescence intensity of each well at a speed of 0.05s. The formula for calculating the cell viability (Cell index) is: fluorescence intensity of the experimental group / fluorescence intensity of the positive control group × 100%. The results are as follows Figure 3 As shown, the combination of drugs has the best ability to kill tumor cells.

[0069] At the same time, two-dimensional co-culture experiments were performed using the xCELLigence real-time cell analysis (RTCA) system according to the above-mentioned cell and virus doses to evaluate the anti-tumor efficacy of monotherapy or combination therapy. When HER2 CAR-T cells were used in combination with OV-OX40L / IL12 oncolytic virus, they showed excellent tumor cell killing activity ( Figure 4 ). In addition, Figure 5 and Figure 6 As shown in the combined group, CAR-T cells released the largest amount of killing factors IFN-γ and Granzyme B. In summary, the dual-factor oncolytic virus can enhance the cytotoxicity of CAR-T in vitro.

[0070] Embodiment 2:

[0071] The anti-tumor ability of the combination therapy was evaluated using an immunodeficient tumor-bearing mouse model.

[0072] Female NSG mice aged 6-8 weeks were subcutaneously inoculated with primary human pancreatic cancer cells derived from patients that were partially HER2 antigen-positive. When the tumor size grew to 100-350 mm, 3 The mice were randomly divided into PBS group, HER2 CAR-T group, OV-OX40 / IL12 group, HER2 CAR-T and OV combination group, and HER2 CAR-T and OV-OX40 / IL12 combination group, with 6 mice in each group. The OV group, HER2 CAR-T and OV combination group, OV-OX40 / IL12 group, and HER2 CAR-T and OV-OX40 / IL12 combination group were intratumorally administered with 2x10 5 pfu of oncolytic virus, and on the second day, the HER2 CAR-T group, the HER2 CAR-T and OV-OX40 / IL12 combination group, and the HER2 CAR-T and OV combination group were injected with HER2 CAR-T cells by tail vein, with 1x10 per mouse 7The tumor size and body weight were measured with a vernier caliper every two days.

[0073] like Figure 7 As shown in the results, in vivo, the combination of OV-OX40 / IL12 and HER2 CAR-T cells was significantly superior to monotherapy and the combination of OV and HER2 CAR-T cells in controlling the growth of pancreatic cancer. Compared with other groups, the combination of OV-OX40 / IL12 and HER2 CAR-T cells did not significantly reduce body weight, indicating no obvious toxic side effects ( Figure 8 ).

[0074] Embodiment 3:

[0075] The anti-tumor ability of the combination therapy was explored using an immune-intact tumor-bearing mouse model.

[0076] 6-8 week old female C57BL / 6 mice were subcutaneously inoculated with 1×106 Pan02-HVEM-HER2-LUC-BFP cells. When the tumor size grew to 30-100 mm 3 The mice were randomly divided into PBS group, mouse CAR-T (HER2 mCAR-T) group, OV-OX40 / IL12 group, OV group, HER2 mCAR-T and OV combination group, and HER2 mCAR-T and OV-OX40 / IL12 combination group, with 6 mice in each group. The OV group, HER2 mCAR-T and OV combination group, OV-OX40 / IL12 group, and HER2 mCAR-T and OV-OX40 / IL12 combination group were given 5x10 4 pfu of oncolytic virus, and the second injection of oncolytic virus was given intratumorally 7 days later. After 3 days, the HER2 mCAR-T group, the HER2 mCAR-T combined with OV-OX40 / IL12 group, and the HER2 mCAR-T combined with OV group were given by tail vein, 2x10 per mouse 6 The tumor size was measured with a vernier caliper every two days, and the mouse survival curve was drawn using GraphPadPrism software.

[0077] like Fig. 9 and Fig.10 In terms of inhibiting pancreatic cancer growth and prolonging survival time, the treatment regimen of OV-OX40 / IL12 combined with HER2mCAR-T cells is significantly more effective than HER2 mCAR-T cell monotherapy and OV combined with HER2 mCAR-T cells.

[0078] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An anti-tumor combination drug, comprising a dual-factor oncolytic virus and HER2CAR-T cells, characterized in that: The dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12, the HER2CAR-T cell is a cell that specifically targets the HER2 antigen, and the CAR structure contained in the HER2 CAR-T cell includes Anti-HER2scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

2. The anti-tumor combination drug according to claim 1, characterized in that: The Anti-HER2 scfv is a single-chain antibody fragment of the anti-HER2 antigen, which is formed by connecting the heavy chain variable region and the light chain variable region of the immunoglobulin of the anti-HER2 antigen through a flexible connecting peptide.

3. The anti-tumor combination drug according to claim 1, characterized in that: The CD8 hinge is the hinge region of CD8.

4. The anti-tumor combination drug according to claim 1, characterized in that: The CD8 TM is the transmembrane region of CD8.

5. The anti-tumor combination drug according to claim 1, characterized in that: The CD28-4-1BB is the intracellular region of CD28 and 4-1BB co-stimulatory factors.

6. The anti-tumor combination drug according to claim 1, characterized in that: The CD3ζ is the intracellular region of the CD3ζ chain.

7. The anti-tumor combination drug according to claim 1, characterized in that: The structure of the dual-factor insertion site of the OV-OX40L / IL12 oncolytic virus includes OV-OX40L, UL26, UL27, AICP47, and IL12.

8. The anti-tumor combination drug according to claim 1, characterized in that: The dosage form of the anti-tumor combination drug is an injection, and the administration method includes one or more of intratumor injection and intravenous injection.

9. Use of the anti-tumor combined drug according to claim 1 in the field of anti-tumor treatment.

10. The use of an anti-tumor combined drug according to claim 9 in the field of anti-tumor treatment, characterized in that: The tumor is a solid tumor and a tumor with high expression of HER2, including but not limited to pancreatic cancer and ovarian cancer. The cells of the tumor with high expression of HER2 include human ovarian cancer cell SKOV3, mouse pancreatic cancer cell Pan02, and primary tumor cells from patients.

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