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, CD8 hinge, CD8 TM, CD28-4-1BB, CD3 zeta, 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] This invention relates to the field of biomedical technology, specifically to a combination anti-tumor drug and its application. Background Technology

[0002] Malignant tumors pose a serious threat to human life and health, becoming one of the heaviest disease burdens in society today. Despite continuous advancements in medical technology, increasingly diverse treatment methods, and a growing number of available drugs, the vast majority of malignant tumors remain incurable, and patients generally have short survival rates. Therefore, any method that can effectively improve treatment outcomes, whether a completely new treatment or an optimization of existing treatment plans, has undeniable practical value. In recent years, the field of tumor immunotherapy has focused on the CAR-T strategy, conducting extensive research. Compared to traditional radiotherapy and chemotherapy, this strategy has shown significant advantages: not only is the treatment effect outstanding, but the efficacy is also long-lasting, with fewer adverse reactions, bringing new hope to the fight against cancer and potentially achieving a true cure for tumors. CAR-T therapy involves extracting the patient's T cells, genetically engineering them to carry chimeric antigen receptors, and then reinfusing them to precisely identify and efficiently kill tumor cells. CAR-T therapy is more specific than radiotherapy and chemotherapy, accurately killing tumor cells with fewer side effects and stimulating long-lasting immunity. CAR-T therapy has shown significant efficacy against various hematological malignancies, such as relapsed / refractory acute lymphoblastic leukemia, with clinical trials showing remission rates exceeding 80% in some patients. For example, in studies targeting specific types of leukemia, the complete remission rate of patients receiving CAR-T therapy is much higher than that of traditional treatments.

[0003] As clinical research progresses, CAR-T therapy has encountered a series of pressing challenges in the field of anti-tumor treatment. CAR-T therapy for solid tumors presents several problems. First, tumor antigen heterogeneity, with significant differences in antigens among different patients or even within the same tumor, affects the precise recognition by CAR-T cells. Second, the complex tumor microenvironment, including immunosuppressive factors and hypoxia, hinders the activity and function of CAR-T cells. Third, CAR-T cells are limited by tumor microenvironment inhibition and insufficient targeting, making it difficult for cells to effectively infiltrate deep into solid tumors and comprehensively kill cancer cells, thus limiting treatment efficacy. In exploring ways to improve 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, while such combination therapy has shown some efficacy, it inevitably increases the probability of treatment-related adverse events. From a clinical perspective, when CAR-T therapy is combined with chemotherapy or radiotherapy, patient compliance is often unsatisfactory, and the economic burden also increases. Furthermore, some scholars have pointed out that the cytotoxicity of chemotherapy drugs can damage rapidly proliferating hemolymphocytes, potentially leading to T cell depletion and thus weakening the anti-tumor synergistic effect of CAR-T therapy. Given these circumstances, exploring next-generation combination therapy modalities that eliminate chemotherapy is essential. Intratumoral injection of open-cell immunotherapy (OV) can directly lyse tumor cells, releasing antigens and immune-stimulating factors, reshaping the tumor microenvironment, and transforming "cold tumors" into "hot tumors," creating favorable conditions for CAR-T cell infiltration and activation, and potentially enhancing their killing efficacy.

[0004] Furthermore, intratumoral administration of OV is highly invasive and cannot reach metastatic or distant tumors, limiting its clinical application. Intravenous injection, as an alternative, is easily intercepted by the liver and neutralized by the body's immune system, making it difficult to achieve targeted enrichment of OV in tumors. The shortcomings of existing administration methods severely restrict the potential of OV combined with CAR-T therapy. The CAR-T loaded OV strategy, as a targeted solution, holds great promise. This strategy relies on the inherent tumor antigen targeting and recognition capabilities of CAR-T cells to precisely deliver OV to the tumor lesion site, achieving targeted enrichment and controlled release of OV in the tumor microenvironment, thus effectively addressing the core drawbacks of traditional administration methods. Simultaneously, this strategy can further enhance the synergistic anti-tumor effect of both: OV continuously enhances the activity and proliferation capacity of CAR-T cells by reshaping the tumor immune microenvironment; CAR-T cells can specifically kill both OV-infected and uninfected tumor cells, compensating for the limited tumor-killing range of OV alone, forming a highly efficient and synergistic anti-tumor pattern.

[0005] In summary, developing efficient and safe CAR-T loaded OV combination strategies and researching complementary synergistic combination drugs has become an urgent research direction and clinical need in the field of solid tumor immunotherapy. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a combination of anti-tumor drugs and their application, solving the problem that the cytotoxicity of chemotherapy drugs can damage rapidly proliferating blood lymphocytes during combined chemotherapy or radiotherapy, leading to T cell depletion and thus weakening the anti-tumor synergistic effect of CAR-T therapy.

[0007] To achieve the above objectives, the present invention provides the following technical solution: An anti-tumor combination drug is achieved through the following technical solution: an anti-tumor combination drug comprising a dual-factor oncolytic virus and HER2 CAR-T cells, wherein the dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12, and the HER2 CAR-T cells are cells specifically targeting the HER2 antigen, and the CAR structure contained in the HER2 CAR-T cells includes Anti-HER2 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

[0008] Preferably, the Anti-HER2 scfv is a single-chain antibody fragment against the HER2 antigen, which is composed of the variable regions of the heavy chain and light chain of the immunoglobulin against the HER2 antigen linked by a flexible linker peptide. The amino acid sequence of the flexible linker peptide is (Gly4Ser)3, as shown in SEQ ID NO.4.

[0009] SEQ ID NO.4 GGGGSGGGGSGGGGS.

[0010] Preferably, the CD8 hinge is the hinge area of ​​CD8.

[0011] Preferably, the CD8 TM is the transmembrane region of CD8.

[0012] Preferably, CD28-4-1BB is the intracellular region of CD28 and 4-1BB co-stimulatory factors.

[0013] Preferably, the CD3ζ is the intracellular region of the CD3ζ chain.

[0014] Preferably, the structure of the two-factor insertion site of the OV-OX40L / IL12 oncolytic virus includes OV-OX40L, UL26, UL27, and IL12.

[0015] Preferably, the recombinant oncolytic virus is herpes simplex virus type 1 (HSV1).

[0016] Preferably, when the OV-OX40L-encoded nucleic acid and the IL-12-encoded nucleic acid are provided by different oncolytic viruses: ① When the -OX40 encoded nucleic acid is inserted into the ICP34.5 site of the HSV1 virus, it is preferable to insert two copies into the two ICP34.5 sites of the virus; ② When the IL12-encoded nucleic acid is inserted into the ICP34.5 site of the HSV1 virus, it is preferable to insert two copies into the two ICP34.5 sites of the virus.

[0017] Preferably, when the OV-OX40L-encoded nucleic acid and the IL-12-encoded nucleic acid are provided by the same oncolytic virus, the OV-OX40L-encoded nucleic acid and the IL-12-encoded nucleic acid are inserted at different HSV1 genomic locations. When the OV-OX40L-encoded nucleic acid and the IL-12-encoded nucleic acid are provided by the same oncolytic virus, they are inserted at different locations in the HSV1 genome: ① When the -OX40L-encoded nucleic acid is inserted into the ICP34.5 site of the HSV1 virus, it is preferable to insert two copies into both ICP34.5 sites of the virus; ②-IL12-encoded nucleic acid is inserted at different locations in the HSV1 viral genome; for example, the intergenic region between UL26 and UL27 or the intergenic region between UL3 and UL4, preferably the intergenic region between UL26 and UL27.

[0018] Preferably, the dosage form of the combined antitumor drug is an injection, and the administration method includes one or more of intratumoral injection and intravenous injection.

[0019] The present invention also provides the application of the above-mentioned combination of antitumor drugs in the field of antitumor treatment.

[0020] Preferably, the combined drug formulation for the tumor is an intravenous injection prepared by incubating a dual-factor oncolytic virus and CAR-T cells.

[0021] Preferably, the tumor is a solid tumor and is a tumor that highly expresses HER2, including but not limited to pancreatic cancer and ovarian cancer. The cells of the tumor that highly expresses HER2 include human ovarian cancer cells SKOV3, mouse pancreatic cancer cells Pan02, and primary tumor cells from the patient.

[0022] The present invention also provides a combination antitumor drug, comprising a dual-factor oncolytic virus and CAR-T cells, wherein the dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12, and the CAR-T cells are cells that specifically target GPC3 or CLDN18.2 antigens. The CAR structures contained in the GPC3 CAR-T cells include Anti-GPC3 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP; The CAR structures contained in the CLDN18.2 CAR-T cells include Anti-CLDN18.2 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

[0023] The present invention also provides the application of the above-mentioned combination of antitumor drugs in the field of antitumor treatment.

[0024] Preferably, the tumor includes a solid tumor.

[0025] Preferably, the solid tumor is a solid tumor that highly expresses GPC3 or CLDN18.2.

[0026] Preferably, tumors that highly express GPC3 or CLDN18.2 include any one of the following: human oral squamous cell carcinoma cell line SCC-15, human glioma cell line SHG-44, human breast cancer cell line MCF-7, human colon cancer cell line HT-29, or human fibrosarcoma cell line HT-1080.

[0027] Preferably, the dosage form of the antitumor combination drug is an injection, and the administration method includes one or more of intratumoral injection and intravenous injection; wherein intratumoral injection is the preferred method, and the recommended dose is 1×10⁻⁶. 4 ~1×10 6 PFU / mouse (mice), clinical conversion dose can be calculated based on body surface area as 1×10⁻⁶. 8 ~1×10 10 pfu / person (cited from page 18 of the instruction manual CN202280076656.4); the dosage of HER2 CAR-T cells is 1×10 6 ~1×10 8 The clinical conversion dose was 1 × 10⁻⁶ cells / kg body weight (mouse model). 7 ~5×10 8 The dosage per kg of body weight can be adjusted according to the patient's tumor burden and physical condition.

[0028] Preferably, the criteria for determining "HER2 high expression" are: HER2 protein expression score of ≥3+ in tumor tissue detected by immunohistochemistry (IHC) (strong and intact cell membrane staining, proportion of stained cells ≥10%), or HER2 gene amplification fold of ≥2.0 detected by fluorescence in situ hybridization (FISH) (HER2 / CEP17 ratio ≥2.0). The tumor cells with high HER2 expression include human ovarian cancer cells SKOV3, mouse pancreatic cancer cells Pan02, and primary tumor cells from patients.

[0029] This invention provides a combination of antitumor drugs and their application. It has the following beneficial effects: 1. This invention combines CAR-T therapy and oncolytic virus therapy, leveraging their respective advantages 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, thereby strengthening the tumor-clearing ability of both. This overcomes the limitations of traditional therapies and possesses better targeting, killing effect, and safety. It solves the problem that the cytotoxicity of chemotherapy drugs can damage rapidly proliferating hemolymphocytes during combined chemotherapy or radiotherapy, leading to T cell depletion and weakening the anti-tumor synergistic effect of CAR-T therapy.

[0030] 2. This 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, while CAR-T cells can enhance the oncolytic virus-mediated oncolysis. The two complement each other and significantly enhance the ability to eliminate tumors.

[0031] 3. This invention utilizes HER2 CAR-T cells to specifically target the HER2 antigen, enabling precise identification and killing of HER2-positive tumor cells, thereby improving the specificity of treatment and reducing damage to normal cells.

[0032] 4. In experiments using immunodeficient tumor-bearing mouse models and immunointact tumor-bearing mouse models, the combined treatment group showed no significant decrease in body weight compared to other groups, suggesting that the combined medication has no obvious toxic side effects and is beneficial to improving patients' quality of life and treatment compliance. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the CAR structure contained in the HER2 CAR-T cells of the present invention; Figure 2 This is a schematic diagram of the structure of the two-factor insertion site of the OV-OX40L / IL12 oncolytic virus of the present invention; Figure 3 This is a schematic diagram illustrating the killing effect of the combined drug on tumor cells according to the present invention; Figure 4 This is a schematic diagram illustrating the tumor cell killing activity of the present invention; Figure 5 This is a schematic diagram showing the amount of IFN-γ, the cytotoxic factor, released by CAR-T cells in the combination therapy group of the present invention. Figure 6 This is a schematic diagram showing the amount of Granzyme B, a cytotoxic factor, released by CAR-T cells in the combination therapy group of the present invention. Figure 7 This is a schematic diagram illustrating the combined in vivo treatment of OV-OX40L / IL12 and HER2 CAR-T cells to control the growth of pancreatic cancer according to the present invention. Figure 8 This is a schematic diagram illustrating the side effects of the combined treatment of OV-OX40L / IL12 and HER2 CAR-T cells according to the present invention; Figure 9 This is a schematic diagram comparing the OV-OX40L / IL12 combined with HER2 mCAR-T cell therapy regimen of the present invention with HER2 mCAR-T cell monotherapy and the OV combined with HER2 mCAR-T cell therapy regimen; Figure 10 This is another comparative schematic diagram of the OV-OX40L / IL12 combined with HER2 mCAR-T cell therapy regimen of the present invention, HER2 mCAR-T cell monotherapy, and OV combined with HER2 mCAR-T cell therapy regimen; Figure 11 Experimental results showing that CAR-T cells can be loaded with oncolytic viruses; Figure 12 For CAR-T HSV-OX40L / IL12 A schematic diagram of the experimental results showing the killing effect on HER2-positive cells; Figure 13 For CAR-T HSV-OX40L / IL12 A schematic diagram of the experimental results on the anti-tumor ability; Figure 14 This demonstrates the potent anti-tumor effects of CAR-T combined with oncolytic virus therapy in clinical treatment. Detailed Implementation

[0034] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Please see the appendix Figure 1 -Appendix Figure 10This invention provides a combination antitumor drug including a dual-factor oncolytic virus and CAR-T cells. The dual-factor oncolytic virus is a recombinant oncolytic virus (OV-OX40L / IL12) constructed based on the herpes simplex virus type 1 (HSV-1) backbone. Its genome has two copies of the ICP34.5 and ICP47 genes deleted to enhance tumor targeting and antigen presentation capabilities (cited from pages 14-15 of CN202280076656.4 specification). The virus simultaneously expresses OV-OX40L and IL12, and the dual-factor insertion site is located in the region between the UL26 and UL27 genes.

[0036] HER2 CAR-T cells are cells that specifically target the HER2 antigen. The CAR structures contained in the HER2 CAR-T cells include Anti-HER2 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

[0037] CAR-T cells also include cells that specifically target the GPC3 or CLDN18.2 antigen, and the CAR structures contained in the GPC3 or CLDN18.2 CAR-T cells include scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

[0038] The Anti-HER2 scfv is a single-chain antibody fragment against the HER2 antigen, composed of the variable region (VH) and variable region (VL) of the immunoglobulin heavy chain against the HER2 antigen linked by a flexible linker peptide. The amino acid sequence of the flexible linker peptide is (Gly4Ser)3 (as shown in SEQ ID NO.4), which ensures the spatial conformational flexibility of the VH and VL regions and antigen-binding activity. The scfv also includes Anti-GPC3 scfv and Anti-CLDN18.2 scfv.

[0039] The VH and VL regions each possess specific amino acid sequences, which, through screening and optimization, endow the single-chain antibody fragments with high specificity and affinity. The flexible linker peptide ensures that the VH and VL regions can move freely in space to better bind to the HER2 antigen, acting like a custom-made "key" that can accurately insert itself into the "lock" of the HER2 antigen, laying the foundation for subsequent immune responses.

[0040] The nucleic acid sequence of HER2 scfv-VH is shown in SEQ ID NO.5; the nucleic acid sequence of HER2 scfv-VL is shown in SEQ ID NO.6; the nucleic acid sequence of CLDN18.2 scfv-VH is shown in SEQ ID NO.7; the nucleic acid sequence of CLDN18.2 scfv-VL is shown in SEQ ID NO.8; the nucleic acid sequence of GPC3 scfv-VH is shown in SEQ ID NO.9; and the nucleic acid sequence encoding GPC3 scfv-VL is shown in SEQ ID NO.10.

[0041] The amino acid sequence of HER2 scfv-VH is shown in SEQ ID NO.11; the amino acid sequence of HER2 scfv-VL is shown in SEQ ID NO.12; the amino acid sequence of CLDN18.2 scfv-VH is shown in SEQ ID NO.13; the amino acid sequence of CLDN18.2 scfv-VL is shown in SEQ ID NO.14; the amino acid sequence of GPC3 scfv-VH is shown in SEQ ID NO.15; and the amino acid sequence encoding GPC3 scfv-VL is shown in SEQ ID NO.16.

[0042] SEQ ID NO.5 Gaggtgcagctggtggagtctggaggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctgggttcaatattaaggacacttacatccactgggtccgccaggctccagggaaggggctggagtgggtcgcacgtatttatcctaccaatggttacacacgctac gcagactccgtgaagggccgattcaccatctccgcagacacttccaagaacacggcgtatcttcaaatgaacagcctgagagccgaggacacggccgtgtattactgttcgagatggggcggtgacggcttctatgccatggactactggggccaaggaaccctggtcaccgtgtctcct.

[0043] SEQ ID NO.6 Gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcaggatgtgaataccgcggtcgcatggtatcagcagaaaccagggaaagcccctaagctcctgatctattctgcatccttcttgtatagtggggtcccatcaaggttcagtggcagtagatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagcattacactacccctccgacgttcggccaaggtaccaagcttgagatcaaa。

[0044] SEQ ID NO.7 CAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCTCTGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCACAGGATATAATATGAACTGGGTGAGGCAGGCCCCAGGACAGGGACTGGAGTGGATGGGCAATATCGATCCCTATTATGGCGGCACCTCTTACAATCAGAAGTTTAAGGGAAGGGTGACCATGACAATCGATAAGAGCACCTCTACCGTGTACATGGAGCTGAGCTCCCTGCGGAGCGAGGACACAGCCGTGTACTATTGTGCCCGGATGTATCATGGCAACGCCTTCGATTATTGGGGCCAGGGCACCACAGTGACAGTGTCTAGC。

[0045] SEQ ID NO.8 GACATCGTGATGACCCAGTCTCCTGACTCCCTGGCCGTGTCTCTGGGCGAGCGGGCCACAATCAATTGCAAGTCTAGCCAGTCTCTGCTGAACAGCGGCAATCTGAAGAACTACCTGACCTGGTATCAGCAGAAGCCAGGCCAGCCCCCTAAGCTGCTGATCTACTGGGCCTCCACAAGGAAGTCTGGAGTGCCAGACAGATTCTCCGGAAGCGGATCCGGAACAGACTTCACCCTGACAATCTCCTCTCTGCAGGCCGAGGACGTGGCCGTGTACTATTGTCAGAATGATTACAGCTATCCCCTGACCTTTGGCGGCGGCACAAAGGTGGAGATCAAG。

[0046] SEQ ID NO.9 GAGGTGCAGCTGGTGGAGACCGGCGGCGGCCTGGTTCAACCTGGGGGCAGCCTGCGCCTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGACACCTACATGCACTGGGTGCGCCAGGCTCCCGGCAAGGGCCTGGAGTGGATGGGCGGCATCAATCCCAGCAACGGCGGCACCAACTACGCCCAGAAGTTCAGCGGCAGAGTGACCATGACCACCGACACCTCCACCTCCACCGCCTACATGGAGCTGAGCAGCCTGCGCAGCGAGGACACCGCCGTGTACTACTGCGCCCGGGGCGGCTTCGACATCGCCATGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGAGCAGC SEQ ID NO.10 GACATCCAGATGACCCAGAGCCCGTCCTCCCTGAGCGCCAGCGTGGGGGACAGAGTGACCATCACCTGCAGATCTAGCCAGAGCCTGGTGCACAGCAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACAAGGTGAGCAACCGCTTCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCGGAGGACTTCGCCACCTACTACTGCTCTCAATCTTACGACAGCAGCCTGAGCTTCGGCCAGGGC ACCAAGGTCGAGATCAAG。

[0047] SEQ ID NO.11 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSP。

[0048] SEQ ID NO.12 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKLEIK。

[0049] SEQ ID NO.13 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYNMNWVRQAPGQGLEWMGNIDPYYGGTSYNQKFKGRVTMTIDKSTSTVYMELSSLRSEDTAVYYCARMYHGNAFDYWGQGTTVTVSS。

[0050] SEQ ID NO.14 DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNLKNYLTWYQQKPGQPPKLLIYWASTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGGGTKVEIK.

[0051] SEQ ID NO.15 EVQLVETGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVGGINPSNGGTNYAQKFQGRVTMTTDTSSTAYMELSSLRSEDTAVYYCARGGFDIAMDYWGQGTLVTVSS.

[0052] SEQ ID NO.16 DIQMTQSPSSSLSASVGDRVTITCRSSQSLVHSNGNTYLHWYLQKPGKAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCSQSYDSSLSFGQGTKVEIK.

[0053] CD8 hinge is the hinge region of CD8; CD8 TM is the transmembrane region of CD8; CD28-4-1BB is the intracellular region of the CD28 and 4-1BB co-stimulatory factors, where the intracellular region of CD28 corresponds to amino acid positions 186-220 (GenBank accession number: NP_006130.1), and the intracellular region of 4-1BB corresponds to amino acid positions 214-255 (GenBank accession number: NP_001552.2). The two are fused together via a GS linker (Gly-Ser) to form a co-stimulatory structure; CD3ζ is the intracellular region of the CD3ζ chain, corresponding to amino acid positions 52-164 (GenBank accession number: NP_932170.1), containing three immune receptor tyrosine activation motifs (ITAMs), which are core components of T cell activation signal transduction.

[0054] The CD8 hinge acts as a connector and buffer, like a bridge, linking different parts of the CAR structure. Its amino acid composition has a certain degree of flexibility and elasticity, which allows the CAR to stretch and bend appropriately on the cell surface while ensuring structural stability. This makes CAR-T cells more flexible in recognizing and binding to tumor cells, improving cell targeting and attack efficiency.

[0055] CD8™ acts as an "anchor," securing the CAR structure to the cell membrane of HER2 CAR-T cells and ensuring stable CAR expression on the cell surface. The amino acids in the transmembrane region possess unique hydrophobicity, allowing them to be stably embedded in the lipid bilayer of the cell membrane. This ensures effective connection between the CAR structure and intracellular signaling pathways, enabling the cell to promptly transmit signals into the cell after recognizing tumor cell antigens, thus initiating the immune killing process.

[0056] CD28-4-1BB is the intracellular region of the CD28 and 4-1BB co-stimulatory factors. The CD28 intracellular region corresponds to amino acid positions 186-220 (GenBank accession number: NP_006130.1), and the 4-1BB intracellular region corresponds to amino acid positions 214-255 (GenBank accession number: NP_001552.2). They are fused together via a GS linker (Gly-Ser) to form a co-stimulatory structure. These two co-stimulatory factors play a crucial role in activating CAR-T cells. When the Anti-HER2 scfv on the surface of CAR-T cells binds to the HER2 antigen, CD28 and 4-1BB co-stimulatory factors work synergistically to provide additional activation signals to T cells. CD28 promotes T cell proliferation, survival, and cytokine secretion, while 4-1BB further enhances T cell activity and memory function. Together, they enable CAR-T cells to rapidly activate and proliferate upon encountering tumor cells, enhancing their ability to kill tumor cells.

[0057] CD3ζ is the intracellular region of the CD3ζ chain, corresponding to amino acid positions 52-164 (GenBank accession number: NP_932170.1). It contains three immune receptor tyrosine activation motifs (ITAMs) and is a core element of T cell activation signaling. It is an important component of the T cell receptor (TCR) signaling complex. When CARs recognize and bind to the HER2 antigen on the surface of tumor cells, the CD3 chain initiates a series of intracellular signaling events. It recruits various signaling molecules, activating downstream signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway, ultimately leading to T cell activation, proliferation, and the release of cytotoxic substances, directly participating in the killing of tumor cells.

[0058] The IRES (Internal Ribosome Entry Site) plays a crucial regulatory role in the CAR structure of HER2 CAR-T cells. It allows for the independent translation of multiple genes on the same transcript. In this invention, the 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 different parts of the CAR structure but also provides a direct detection method during experiments. By detecting EGFP expression, it is possible to determine whether the CAR structure has been successfully transfected into T cells and its expression level, greatly facilitating the preparation and screening of HER2 CAR-T cells.

[0059] EGFP (Enhanced Green Fluorescent Protein), as a reporter gene, has greatly facilitated the research and application of HER2 CAR-T cells. During cell culture and experiments, the distribution, quantity, and activity changes of EGFP-labeled HER2 CAR-T cells can be directly observed using instruments such as fluorescence microscopy or flow cytometry. For example, in in vitro killing experiments, changes in EGFP fluorescence intensity can be observed to monitor the killing effect of HER2 CAR-T cells on tumor cells in real time; in in vivo experiments, the migration and distribution of HER2 CAR-T cells in mice can also be tracked, providing important evidence for further understanding the mechanism of action of combined drug therapy and optimizing treatment regimens.

[0060] OV-OX40L is a modified oncolytic virus vector portion capable of expressing trimerized OX40L. The trimerized OX40L is a fusion polypeptide containing, from the N-terminus to the C-terminus, the trimerization domain of human TRAF2 (as shown in SEQ ID NO. 17: DQDKIEALSSKVQQLERSIGLKDLAMADLEQKVLEMEAST) and the extracellular domain of human OX40L (amino acid sequences 51-183 as shown in SEQ ID NO. 18). QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGE LILIHQNPGEFCVLASVDYKDDDDKGSTSGSGKPGSGEGSTKGENLYFQGDLNAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR) and the PDGFR transmembrane domain (such as SEQ Shown as ID NO.1, SEQ ID NO.1: LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR); OX40L, as a co-stimulatory molecule, can efficiently bind to the OX4 receptor on the cell surface after trimerization modification, significantly enhancing cell proliferation, survival, and cytokine secretion, providing a key signal for the activation and expansion of CAR-T cells in the tumor microenvironment (cited from CN202280076656.4, Example 3.2). Figure 9-10 ).

[0061] UL26 and UL27 are important gene segments in the structure of oncolytic viruses, and they play an indispensable role in maintaining the stability, infectivity and replication of oncolytic viruses.

[0062] IL12 is an IL-12p70 heterodimer, formed by the p35 subunit (GenBank accession number: NP_000873.1, as shown in SEQ ID NO. 2) and the p40 subunit (GenBank accession number: NP_002178.2, as shown in SEQ ID NO. 3) linked by disulfide bonds, possessing complete immune-activating activity (cited from pages 10-11 of the specification CN202280076656.4). Its expression is driven by the CMV promoter, and the synergistic expression of the p35 and p40 subunits is achieved through the IRES sequence. After secretion, it can activate NK cells and T cells, promote the release of anti-tumor cytokines such as IFN-γ, and reshape the tumor immune microenvironment (cited from Example 3.3 of CN202280076656.4). Figure 13 In OV-OX40L / IL12 oncolytic virus, these elements work together to enhance immunity and promote oncolysis.

[0063] The OV-OX40L / IL12 oncolytic virus possesses broad-spectrum tumor-killing activity. In vitro experiments have confirmed its effective infection and lysis of various solid tumor cell lines, including human oral squamous cell carcinoma (SCC-15), human glioma (SHG-44), human breast cancer (MCF-7), human colon cancer (HT-29), and human fibrosarcoma (HT-1080) (cited from CN202280076656.4, Example 2.2, Figure 4E). Killing experiments on primary oral cancer cells and primary tumor tissues from patients showed that the virus significantly inhibited tumor cell viability within an MOI range of 0.01–100 and could efficiently amplify (2–8-fold) in tumor tissues, further enhancing the oncolytic effect (cited from CN202280076656.4, Example 2.2). Figure 4 AD).

[0064] The dosage form of the combination antitumor drugs is injection, and the administration methods include one or more of intratumoral injection and intravenous injection; among which, intratumoral injection is the preferred method, and the recommended dose is 1×10⁻⁶. 4 ~1×10 6 PFU / mouse (mice), clinical conversion dose can be calculated based on body surface area as 1×10⁻⁶. 8 ~1×10 10 pfu / person (cited from page 18 of CN202280076656.4 instruction manual); CAR-T cell administration dose 1×10 6 ~1×10 8 The clinical conversion dose was 1 × 10⁻⁶ cells / kg body weight (mouse model). 7 ~5×10 8 The number of patients per kg of body weight can be adjusted according to the patient's tumor burden and physical condition. Specifically, intratumoral injection allows drugs to act directly on tumor tissue, increasing local drug concentration and enhancing the killing effect on tumor cells. When performing intratumoral injection, the appropriate injection site and dosage are selected based on the size, location, and type of the tumor. For example, for smaller, superficial tumors, multiple intratumoral injections can be used to ensure uniform drug distribution within the tumor tissue; for larger tumors, precise dosage calculation is necessary to avoid overdose and adverse reactions. Intravenous injection offers advantages such as ease of operation and rapid drug delivery to the systemic bloodstream, enabling combined drug therapy to act on tumor cells throughout the body, especially suitable for patients with metastatic tumors. During intravenous injection, strict control of the injection rate and concentration is crucial to prevent adverse drug reactions and allergic reactions. Furthermore, to ensure effective drug delivery to tumor tissue, auxiliary methods can be employed, such as encapsulating the drug with nanoparticles to improve drug stability and targeting in the bloodstream.

[0065] In some implementations, the CAR structure contained in HER2 CAR-T cells is as follows: Figure 1 As shown, Anti-HER2 scfv is a single-chain antibody fragment (scFv) against the HER2 antigen, which is composed of the variable region (VH) of the heavy chain and the variable region (VL) of the immunoglobulin against the HER2 antigen linked by a flexible linker peptide. The amino acid sequence of the flexible linker peptide is (Gly4Ser)3 (i.e., GGGGSGGGGSGGGGS); CD8 hinge is the hinge region of CD8; CD8 TM is the transmembrane region of CD8; CD28-4-1BB is the intracellular region of the co-stimulatory factors of CD28 and 4-1BB, where the intracellular region of CD28 corresponds to amino acid positions 186-220 (GenBank accession number: NP_006130.1), and the intracellular region of 4-1BB corresponds to amino acid positions 214-255 (GenBank accession number: NP_001552.2). The two are linked by GS... The linker (Gly-Ser) is tandemly linked to form a fusion co-stimulatory structure; CD3ζ is the intracellular region of the CD3ζ chain, corresponding to amino acid positions 52-164 (GenBank accession number: NP_932170.1), and contains 3 immune receptor tyrosine activation motifs (ITAMs).

[0066] In some embodiments, the structure of the two-factor insertion site of the OV-OX40L / IL12 oncolytic virus is as follows: Figure 2 As shown.

[0067] Furthermore, the dosage form of the combination antitumor drug is an injection, and the administration route includes one or more of intratumoral injection and intravenous injection; the recommended dose is 1×10⁻⁶. 4 ~1×10 6 PFU / mouse (mice), clinical conversion dose can be calculated based on body surface area as 1×10⁻⁶. 8 ~1×10 10 pfu / person; HER2 CAR-T cell administration dose was 1×10 6 ~1×10 8 / kg body weight (mouse model), clinical conversion dose is 1×10 7 ~5×10 8 The dosage per kg of body weight can be adjusted according to the patient's tumor burden and physical condition. Furthermore, the combination anti-tumor drug preparation is a combination of OV-OX40L / IL12 oncolytic virus and HER2 CAR-T cells.

[0068] In a second aspect, the practical application of the aforementioned combined drug therapy in the field of anti-tumor treatment was explored. Studies have found that this combined drug therapy has potential therapeutic effects on various tumors, especially pancreatic cancer and ovarian cancer. The method of this invention combines CAR-T therapy and oncolytic virus therapy, utilizing their respective advantages 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 oncolytic virus-mediated oncolysis, thereby strengthening the tumor-clearing ability of both. This treatment approach not only overcomes the limitations of traditional therapies but also possesses good targeting, killing power, and safety. This solves the problem that the cytotoxicity of chemotherapy drugs can damage rapidly proliferating hemolymphocytes during combined chemotherapy or radiotherapy, leading to T cell depletion and weakening the anti-tumor synergistic effect of CAR-T therapy. Furthermore, it has potential therapeutic effects on various tumors, especially HER2-high expressing solid tumors such as pancreatic cancer and ovarian cancer, providing a new option for the treatment of these refractory tumors.

[0069] In some embodiments, the combined drug for the tumor is in the form of an intravenous injection prepared by incubating a dual-factor oncolytic virus and CAR-T cells.

[0070] In some embodiments, the tumor is a solid tumor and is a tumor that highly expresses HER2, including but not limited to pancreatic cancer and ovarian cancer, wherein the cells of the HER2-highly expressing tumor include human ovarian cancer cells SKOV3, mouse pancreatic cancer cells Pan02, and primary tumor cells from the patient.

[0071] On the other hand, a combination anti-tumor drug is also provided, including a dual-factor oncolytic virus and CAR-T cells. The dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12. The CAR-T cells are cells that specifically target GPC3 or CLDN18.2 antigen. The CAR structures contained in the GPC3 CAR-T cells include Anti-GPC3 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP; The CAR structures contained in the CLDN18.2 CAR-T cells include Anti-CLDN18.2 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

[0072] On the other hand, it also provides the application of the above-mentioned combination of anti-tumor drugs in the field of anti-tumor treatment.

[0073] In some implementations, the tumor is a solid tumor.

[0074] In some implementations, solid tumors include tumors that highly express GPC3 or CLDN18.2.

[0075] In some embodiments, tumors that highly express GPC3 or CLDN18.2 include any one of the following: human oral squamous cell carcinoma cell line SCC-15, human glioma cell line SHG-44, human breast cancer cell line MCF-7, human colon cancer cell line HT-29, or human fibrosarcoma cell line HT-1080.

[0076] In some implementations, all SKOV3 cells were infected with a lentiviral gene expressing fireflyluciferase (GenBank accession number: MK484108.1) with an MOI of 5. Stable expression strains were obtained after 2 weeks of selection with puromycin (final concentration 2 μg / mL), denoted as SKOV3-LUC.

[0077] In some embodiments, Pan02 cells were sequentially inserted with the human HER2 gene (GenBank accession number: NM_004448.3), firefly luciferase gene, and BFP gene (blue fluorescent protein) via double lentiviral infection, and co-expressed the HVEM helper molecule (GenBank accession number: NM_003820.3) to enhance viral infection efficiency. Stable expression strains were obtained after 3 weeks of dual selection with puromycin (final concentration 2 μg / mL) and hygromycin (final concentration 50 μg / mL), denoted as Pan02-HVEM-HER2-LUC-BFP.

[0078] In some implementations, HER2 CAR-T represents the human type, and HER2 mCAR-T represents the mouse type.

[0079] In some implementations, the scfv in the CAR structure of HER2 mCAR-T targets the human HER2 antigen, while the other structural components of the CAR are of murine origin.

[0080] In some implementations, the data are presented as mean ± standard deviation. *p < 0.05,**p < 0.01,***p < 0.001,****p < 0.0001.

[0081] The following is a further description with reference to specific embodiments: Example 1: The killing effect of combined in vitro drug administration on HER2-positive cells.

[0082] After co-incubating HER2 CAR-T cells and oncolytic viruses with human ovarian cancer cells SKOV3 cells for 36 hours, the bioluminescence value was measured using an enzyme-linked immunosorbent assay (ELISA) reader to determine the killing effect of the combined drug on HER2-positive cells in vitro.

[0083] 1. Preparation of Human CAR-T Cells: First, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood using density gradient centrifugation (800 × g for 20 minutes). The isolated PBMCs were resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS; #A5669701, Gibco) and 1% penicillin-streptomycin (P / S; #PB180120 Procell) and cultured at 37°C and 5% CO2. T cells were activated using Dynabeads human T cell activator CD3 / CD28 (#111.31D, Invitrogen) and human IL-2 (100 U / mL) in the medium. Forty-eight hours after activation, the activated T cells were transduced by centrifugation using a third-generation lentiviral vector (containing VSV-G envelope protein, Rev, and tat regulatory elements) at a titer of 1 × 10⁻⁶. 8 ~1×10 9 TU / mL, MOI=2, centrifuged at 800×g, 32℃ for 1 hour to improve transduction efficiency; lentivirus was pretreated with Polybrene (final concentration 10μg / mL) before transduction to enhance viral binding to T cell membranes. 12 hours after transduction, the virus-containing medium was removed by centrifugation, and cells were resuspended in complete medium containing IL-2 (initial concentration 100U / mL) for approximately 8 days of expansion culture; from day 3 of culture, the IL-2 concentration was adjusted to 50 U / mL, and half of the medium was replaced every 2 days with fresh IL-2 to maintain a cell density of 1×10⁶ cells / mL. 6 ~2×10 6 The cells / mL were cultured at 37°C, 5% CO2, and saturated humidity throughout the entire process.

[0084] 2. Prepare a 96-well plate, seed SKOV3-LUC cells at a density of 5000 cells / well, set up 3 replicates per group, and add 100 μL of culture medium to each well.

[0085] 3. After culturing cells for 24 hours, HER2 CAR-T cells and basic oncolytic virus (OV) or dual-factor oncolytic virus (OV-OX40L / 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. The oncolytic virus was used to infect the target cells at an MOI of 0.1, with a volume of 50 μL. The cells were then incubated at 37 degrees Celsius for another 36 hours.

[0086] 4. After the culture is completed, 100 μL of supernatant was aspirated from each well, and the secretion of IFN-γ and Granzyme B was detected by ELISA.

[0087] 5. Discard excess liquid from each well, add 100 μL of D-fluorescein sodium salt (Yeasen Biotechnology, 40901ES03) at a working concentration of 150 μg / ml, and read the fluorescence intensity of each well using a microplate reader at a speed of 0.05 s. Cell viability (Cell index) is calculated as: (Experimental group fluorescence intensity / Positive control group fluorescence intensity) × 100%. Results are as follows... Figure 3 As shown, combined drug therapy has the best killing effect on tumor cells.

[0088] We constructed CAR-T cells targeting GPC3 and CLDN18.2 (their CAR structures, except for the specific scF, were identical to those of HER2CAR), and conducted in vitro killing experiments with oncolytic viruses on their respective target cell lines: GPC3 CART used the human hepatocellular carcinoma cell line HepG2, and CLDN18.2 CART used the human pancreatic carcinoma cell line BxPC3. The in vitro co-culture procedures and conditions were consistent with the previous HER2 CART combined with oncolytic virus system. Cell viability assays showed that GPC3 CART and CLDN18.2 CART combined with OVOX40L / IL12 oncolytic virus significantly enhanced tumor cell killing effects compared to CART alone or oncolytic virus alone. Figure 3 ).

[0089] Simultaneously, a two-dimensional co-culture experiment was conducted using the xCELLigence Real-Time Cell Analysis (RTCA) system at the aforementioned cell and virus dosages to evaluate the antitumor efficacy of single or combination therapy. HER2 CAR-T cells, when used in combination with OV-OX40L / IL12 oncolytic virus, exhibited superior tumor cell killing activity. Figure 4 Additionally, such as Figure 5 and Figure 6 The combined group showed the highest levels of cytotoxic factors IFN-γ and Granzyme B released by CAR-T cells. In conclusion, dual-factor oncolytic virus can enhance the cytotoxicity of CAR-T cells in vitro.

[0090] Example 2: The antitumor efficacy of combination therapy was evaluated using an immunodeficient tumor-bearing mouse model.

[0091] Female NSG mice aged 6-8 weeks were selected and subcutaneously inoculated with HER2-positive human pancreatic cancer primary cells derived from patients. When the tumor size reached 100-350 mm... 3Mice were randomly assigned to four groups: PBS group, HER2 CAR-T group, OV-OX40L / IL12 group, HER2 CAR-T combined with OV group, and HER2 CAR-T combined with OV-OX40L / IL12 group, with six mice in each group. The OV group, HER2 CAR-T combined with OV group, OV-OX40L / IL12 group, and HER2 CAR-T combined with OV-OX40L / IL12 group received intratumoral administration of 2×10⁻⁶ styrene. 5 Oncolytic virus (PFU / mouse) was administered to mice on the second day. HER2 CAR-T cells were then injected via tail vein into the HER2 CAR-T group, the HER2 CAR-T combined with OV-OX40L / IL12 group, and the HER2 CAR-T combined with OV group. Each mouse received 1 × 10⁻⁶ PFU / mouse. 7 Each HER2 CAR-T cell was used, and tumor size was measured with calipers and body weight was checked every two days.

[0092] like Figure 7 As shown, in vivo, the combination therapy of OV-OX40L / IL12 and HER2 CAR-T cells was significantly superior to monotherapy and the combination therapy of OV and HER2 CAR-T cells in controlling the growth of pancreatic cancer. The combination therapy of OV-OX40L / IL12 and HER2 CAR-T cells did not result in significant weight loss compared to other groups, suggesting no significant toxic side effects. Figure 8 ).

[0093] Example 3: To explore the anti-tumor efficacy of combination therapy using an immune-intact tumor-bearing mouse model.

[0094] Female C57BL / 6 mice aged 6-8 weeks were subcutaneously injected with 1×10 6 Pan02-HVEM-HER2-LUC-BFP cells, when the tumor size reaches 30-100 mm 3 Mice were randomly assigned to four groups: PBS group, mouse CAR-T (HER2 mCAR-T) group, OV-OX40L / IL12 group, OV group, HER2 mCAR-T combined with OV group, and HER2 mCAR-T combined with OV-OX40L / IL12 group, with six mice in each group. The OV group, HER2 mCAR-T combined with OV group, OV-OX40 / IL12 group, and HER2 mCAR-T combined with OV-OX40L / IL12 group received intratumoral administration of 5 × 10⁻⁶ mg / L of PBS. 4The mice were given pfu / mouse of oncolytic virus. Seven days later, a second dose of oncolytic virus was administered intratumorally. Three days later, the mice were given HER2 mCAR-T, HER2 mCAR-T combined with OV-OX40L / IL12, and HER2 mCAR-T combined with OV via tail vein. Each mouse received 2 × 10⁻⁶ pfu / mouse of oncolytic virus. 6 HER2 mCAR-T cells were collected and tumor size was measured with calipers every two days. Survival curves for mice were plotted using GraphPad Prism software.

[0095] like Figure 9 and Figure 10 In terms of inhibiting pancreatic cancer growth and prolonging survival time, the OV-OX40L / IL12 combined with HER2 mCAR-T cell therapy is significantly more effective than HER2 mCAR-T cell monotherapy and OV combined with HER2 mCAR-T cell therapy.

[0096] Example 4: In vitro immunofluorescence assays confirmed that CAR-T cells can be loaded with oncolytic viruses.

[0097] HER2 CAR-T cells were activated with CD3 and CD28 activating antibodies for 24 hours, and 1×10⁶ cells were placed in each well of a 6-well plate. 6 CAR-T cells were prepared by adding dual-factor oncolytic virus at an MOI of 5 to 6-well plates, with a working concentration of 1 μg / ml of polybrene. The cells were centrifuged at 800g with a slow intensification and deintensity of 5°C for 1 hour, then incubated overnight at 37°C. The next day, 1×10⁻⁶ cells were collected. 6 Suspension T cells (viability > 90%) were centrifuged at 1500 rpm for 5 min, washed twice with PBS containing 2% FBS, resuspended in 200 μL PBS, transferred to a centrifuge, and centrifuged at 800 rpm for 5 min to allow cells to adhere to a glass slide. After drying at room temperature, the cells were fixed with 4% PFA for 15 min and washed three times with PBS. Then, 5% BSA blocking solution was added and incubated at room temperature for 30 min. After discarding the blocking solution, primary antibody working solution (anti-HSV-1gD 1:200) was added and incubated at room temperature for 2 h. The cells were washed three times with PBS, and 1:500 fluorescent secondary antibody was added and incubated in the dark for 1 h. After rinsing, the slides were mounted with anti-fluorescence quenching mounting solution and photographed using a confocal microscope. The experimental results are as follows: Figure 11 As shown.

[0098] Example 5: The killing effect on HER2-positive cells was verified by in vitro co-culture.

[0099] CAR-T cells, a hybrid system (CAR-T cells: CAR-T...) HSV-OX40L / IL12The cells were mixed in a 9:1 ratio, i.e., CAR-T 10% OV; CAR-T cells:CAR-T HSV-OX40L / IL12 The cells were mixed in a 5:5 ratio (i.e., CAR-T 50% OV) and CAR-T... HSV-OX40L / IL12 The in vitro killing effect of the combined drug on HER2-positive cells was determined by co-incubating CAR-T 100% OV with human ovarian cancer cells SKOV3 for 36 hours and then measuring the bioluminescence value using an enzyme-linked immunosorbent assay (ELISA) reader.

[0100] 1. Prepare a 96-well plate, seed SKOV3-LUC cells at a density of 5000 cells / well, set up 3 replicates per group, and add 100 μL of culture medium to each well.

[0101] 2. After 24 hours of cell culture, HER2 CAR-T cells, HER2 CAR-T 10% OV, HER2 CAR-T 50% OV, and HER2 CAR-T 100% OV were added separately. The ratio of HER2 CAR-T cells to target cells was 1:10, and the volume was 100 μL. The cells were then cultured in a 37°C incubator for another 36 hours.

[0102] 3. Discard excess liquid from each well, add 100 μL of D-fluorescein sodium salt (Yeasen Biotechnology, 40901ES03) at a working concentration of 150 μg / mL, and read the fluorescence intensity of each well using a microplate reader at a speed of 0.05 s. Cell viability (Cell index) is calculated as: (Experimental group fluorescence intensity / Positive control group fluorescence intensity) × 100%. Results are as follows... Figure 12 As shown, there are hybrid systems and CAR-T. HSV-OX40L / IL12 The cytotoxicity against SKOV3-LUC cells was significantly enhanced.

[0103] Example 6: Evaluation of CAR-T in immunodeficient tumor-bearing mouse models HSV-OX40L / IL12 Its anti-tumor ability.

[0104] Female NSG mice aged 6-8 weeks were subcutaneously inoculated with SKOV3 cells. When the tumor size reached 100-350 mm... 3 Mice were randomly assigned to four treatment groups: a PBS control group, an HSV-OX40L / IL12-only group, a HER2 CAR-T-only group, and a combination therapy group (HER2 CAR-T and HER2 CAR-T). HSV-OX40L / IL12 The ratio was 4:1 (CAR-T 20% OV), and the administration method was tail vein injection, 1×10⁻⁶ per mouse. 7 CAR-T cells, oncolytic virus 2×10 per cell 5PFU, the size of the tumor was measured with calipers every other day.

[0105] like Figure 13 As shown, tumors progressed rapidly in the PBS group and the OHSV-only group; the HER2 CAR-T-only group showed moderate tumor growth inhibition, while the mixed therapy group achieved the most significant anti-tumor effect.

[0106] In addition, after the tumor was punctured, tumor cells were taken for immunohistochemical staining, and the intratumoral content of HSV and CAR-T cells was detected using HSV-1gD and CD3 antibodies.

[0107] Specific steps: After dewaxing and rehydration of paraffin-embedded sections, they were placed in EDTA buffer (pH 9.0) for heat-induced antigen retrieval for 30 minutes, cooled naturally, and washed with PBS. Subsequently, the following steps were performed in sequence: incubation with 3% hydrogen peroxide (H2O2) at room temperature for 10 minutes to block endogenous peroxidase activity; blocking with 3% bovine serum albumin (BSA) at room temperature for 30 minutes to block non-specific binding sites; CD3 antibody; incubation with HSV-1gC at 4°C overnight (equilibration at room temperature after incubation, followed by washing with PBS); incubation with species-matched horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 30 minutes; and DAPI incubation at room temperature for 10 minutes for nuclear counterstaining. Finally, the slides were mounted with anti-fluorescence quenching mounting solution and imaged by whole-slide scanning.

[0108] Further validation of the treatment mechanism through tumor tissue immunostaining showed that HSV antigen positive signals and CD3 were simultaneously detected in the tumor tissue of the mixed treatment group. + T cell infiltration; in contrast, the HSV-only group showed only weak local viral signals, while the CAR-T-only group showed predominantly T cell infiltration. Figure 14 In summary, these results confirm that CAR-T cells can target and deliver HSV to tumor sites in vivo, achieving a potent anti-tumor effect in clinical treatment through the combination of CAR-T and oncolytic virus therapy.

[0109] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A combination antitumor drug comprising dual-factor oncolytic virus and HER2 CAR-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 HER2 CAR-T cells are cells that specifically target the HER2 antigen. The CAR structures contained in the HER2 CAR-T cells include Anti-HER2scfv, CD8 hinge, CD8™, CD28-4-1BB, CD3ζ, IRES, and EGFP.

2. The combination antitumor drug according to claim 1, characterized in that: The Anti-HER2 scfv is a single-chain antibody fragment against the HER2 antigen, which is composed of the variable regions of the heavy chain and light chain of the immunoglobulin against the HER2 antigen linked by a flexible linker peptide.

3. The combination antitumor drug according to claim 1, characterized in that: The CD8 TM is the transmembrane region of CD8.

4. The combination antitumor drug according to claim 1, characterized in that: The structure of the two-factor insertion site of the OV-OX40L / IL12 oncolytic virus includes OV-OX40L, UL26, UL27, and IL12.

5. The combination antitumor drug according to claim 1, characterized in that: The dosage form of the combined antitumor drug is an injection, and the administration method includes one or more of intratumoral injection and intravenous injection.

6. The application of the combination antitumor drug according to claim 1 in the field of antitumor treatment.

7. The application of the combination antitumor drug according to claim 6 in the field of antitumor treatment, characterized in that: The combined drug formulation for the tumor is an intravenous injection prepared by incubating a dual-factor oncolytic virus and CAR-T cells.

8. The application of the combination antitumor drug according to claim 6 in the field of antitumor treatment, characterized in that: The tumor is a solid tumor, and is a tumor that highly expresses HER2, including but not limited to pancreatic cancer and ovarian cancer. The cells of the tumor that highly expresses HER2 include human ovarian cancer cells SKOV3, mouse pancreatic cancer cells Pan02, and primary tumor cells from the patient.

9. A combination antitumor drug comprising a dual-factor oncolytic virus and CAR-T cells, characterized in that: The dual-factor oncolytic virus is an OV-OX40L / IL12 oncolytic virus that simultaneously expresses OV-OX40L and IL12, and the CAR-T cells are cells that specifically target GPC3 or CLDN18.2 antigens. The CAR structures contained in the GPC3 CAR-T cells include Anti-GPC3 scfv, CD8 hinge, CD8 TM, CD28-4-1BB, CD3ζ, IRES, and EGFP; The CAR structures contained in the CLDN18.2 CAR-T cells include Anti-CLDN18.2 scfv, CD8 hinge, CD8TM, CD28-4-1BB, CD3ζ, IRES, and EGFP.

10. The application of a combination antitumor drug as described in claim 9 in the field of antitumor treatment.

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