GM-CSF loaded tumor cell vaccine and application thereof in prevention and treatment of liver cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a GM-CSF loaded tumor cell vaccine and application thereof in prevention and treatment of liver cancer. In order to enhance the immunogenicity of the existing tumor cell vaccine, a liver cancer cell line is modified through genetic engineering, and a key cell factor GM-CSF capable of regulating and controlling differentiation, maturation, migration and functions of DCs is introduced, so that the GM-CSF is stably overexpressed, and the whole-cell tumor vaccine capable of efficiently secreting the GM-CSF is successfully constructed. And the antigen presentation and T cell starting capability of the DCs are further enhanced through ionization irradiation. Experiments prove that the GM-CSF loaded tumor cell vaccine can effectively activate specific T cell immune response, induce long-acting immune memory, inhibit immune tolerance and obviously inhibit growth of liver cancer tumors and tumor recurrence, and is high in safety. The method is hopeful to enter the development and application of clinical tumor vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to tumor cell vaccines loaded with GM-CSF and their application in the prevention and treatment of liver cancer. Background Technology

[0002] Liver cancer is the sixth most common malignant tumor worldwide, and primary liver cancer mainly includes hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Currently, the traditional treatment strategy for primary liver cancer is primarily surgery, supplemented by comprehensive treatment. For early-stage HCC patients, radical treatments include surgical resection, local ablation therapy, and liver transplantation, and for some patients, transarterial chemoembolization (TACE) is recommended. However, even with these standard treatments, the tumor recurrence rate in liver cancer patients remains high. For advanced HCC patients, although various treatment options exist, such as TACE, radiotherapy, and targeted drugs (e.g., tyrosine kinase inhibitors sorafenib and lenvatinib), overall efficacy remains limited. High tumor recurrence rates, poor response to systemic therapy, and high drug resistance lead to poor overall prognosis. Therefore, developing more effective, durable, and immunologically effective novel treatment strategies has become an urgent need in the field of liver cancer research.

[0003] Immunotherapy has become a hot research topic in tumor biotherapy in recent years. Immunotherapy activates the host's immune system to recognize and eliminate tumor cells, encompassing various treatment strategies, including immune checkpoint inhibitors (ICIs), adoptive cell therapy (ACT), and tumor vaccines. These therapies have been extensively studied in the field of liver cancer. The IMbrave150 clinical trial demonstrated the significant efficacy of the combination of the anti-programmed cell death ligand 1 (PD-L1) antibody atezolizumab and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab in HCC patients, and therefore this regimen is now recommended as a first-line systemic treatment for advanced HCC. In addition, ACT therapy, particularly chimeric antigen receptor T-cell (CAR-T) therapy targeting tumor-associated antigens (TAAs), has shown encouraging results in early clinical trials.

[0004] Among numerous immunotherapies, tumor vaccines, by delivering targeted antigens (usually in combination with adjuvants) to activate or enhance the host's immune system (especially T-cell immunity), possess unique advantages in immunotherapy due to their ability to target a wider range of tumor antigens and induce more durable immune responses. Tumor vaccines are mainly divided into two categories: therapeutic and prophylactic. The former induces a potent cellular immune response to eliminate existing cancer cells and establishes lasting immune memory to prevent tumor recurrence, while the latter stimulates the immune system of tumor-free individuals to produce antibodies and immune memory cells, thereby reducing the risk of cancer development. Current research on HCC tumor vaccines primarily focuses on peptide vaccines targeting antigens such as morphogenetic receptor glycoprotein-3 (GPC3), and dendritic cell (DC) vaccines loaded with tumor antigens or tumor-specific antigens (TSA). Unlike vaccines targeting a single specific antigen, whole-cell tumor vaccines can cover all potential antigens, thereby circumventing HLA restrictions and reducing the risk of immune escape due to antigen loss. To date, several whole-cell tumor vaccines have entered clinical trials. Examples include inactivated autologous tumor cell-Bacillus Calmette-Guérin (BCG) vaccines used to treat colon cancer or melanoma, and engineered autologous tumor cell vaccines, such as gemogenovatucel-T, developed for ovarian cancer. However, research on cell vaccines for liver cancer remains limited, and no such products have yet entered the clinical evaluation stage.

[0005] Whole-cell tumor vaccines are selected from autologous or allogeneic tumor cells, which are then inactivated through physical (e.g., ionizing radiation), chemical, or biological methods to ensure both immunogenicity and safety. Ionizing radiation (IR) is a classic method for preparing whole-cell tumor vaccines, and irradiated tumor cell vaccines have been widely studied and applied in numerous preclinical and clinical studies. IR not only effectively inactivates tumor cells but also induces a strong oxidative stress response, leading to a large accumulation of reactive oxygen species (ROS) within tumor cells, resulting in mitochondrial oxidative damage and the release of mitochondrial DNA (mtDNA). Notably, oxidized mitochondrial DNA (ox-mtDNA) in IR-treated tumor cells has been shown to be a key damage-associated molecular pattern (DAMP). ox-mtDNA can activate innate immune pathways through multiple mechanisms, including the nuclear factor-κB (NF-κB) pathway, the NOD-like receptor family Pyrin domain-containing 3 (NLRP3) inflammasome pathway, and the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which has attracted much attention in recent years. Previous studies have shown that IR-induced antitumor immune responses are partly attributed to the activation of the STING pathway, which promotes the production of type I interferon (IFN-I), thereby enhancing antigen presentation by dendritic cells (DCs) and T cell initiation.

[0006] The activation of the antitumor response to tumor vaccines depends on dendritic cells (DCs), which, as professional antigen-presenting cells (APCs), play a crucial role in initiating antigen-specific immunity and immune tolerance. DCs can efficiently take up and process tumor antigens and present them to naive T cells, thereby activating antigen-specific CD4+. + and CD8 +T-cell responses bridge the gap between innate and adaptive immunity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key cytokine regulating dendritic cell (DC) differentiation, maturation, migration, and function, and has been widely used as an adjuvant to enhance vaccine immunogenicity. Based on this principle, tumor vaccines constructed using GM-CSF have shown promising therapeutic effects in various solid tumors. For example, talimogene laherparepvec (Imlygic, T-VEC) is a genetically engineered attenuated oncolytic herpes simplex virus expressing human GM-CSF. This vaccine has been shown to induce anti-tumor immune responses and improve survival rates in patients with advanced melanoma. T-VEC is administered via intratumoral injection only. It selectively replicates in tumor cells, directly causing tumor cell death and secreting human GM-CSF to promote an immune response, thereby inducing an anti-tumor response. Although it can induce distant effects, when used as a monotherapy, the intensity of the systemic immune response it induces is insufficient to effectively control widespread metastases. Furthermore, although T-VEC has relatively low toxicity, it still carries the risk of causing herpesvirus infection-related symptoms (such as fever and flu-like symptoms), while tumor cell vaccines are inactivated cells, have better safety, and pose no risk of infection or replication. However, there are currently no clinical studies on tumor cell vaccines using GM-CSF as an adjuvant for the prevention and treatment of liver cancer. Summary of the Invention

[0007] To enhance the immunogenicity of existing tumor cell vaccines, this invention utilizes genetic engineering to modify liver cancer cell lines, introducing the key cytokine GM-CSF, which regulates the differentiation, maturation, migration, and function of dendritic cells (DCs). This allows for stable overexpression of GM-CSF, successfully constructing a whole-cell tumor vaccine capable of efficiently secreting GM-CSF. Ionizing irradiation further enhances the antigen presentation and T-cell initiation capabilities of DCs. Experimental verification shows that the GM-CSF-loaded tumor cell vaccine of this invention effectively activates specific T-cell immune responses, induces long-term immune memory, and suppresses immune tolerance. It also significantly inhibits tumor growth and recurrence, exhibiting high safety.

[0008] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a tumor cell vaccine loaded with GM-CSF for the prevention and / or treatment of liver cancer, which is obtained by ionizing radiation from a liver cancer cell line expressing GM-SCF.

[0009] Furthermore, the ionizing irradiation is performed using rays capable of inactivating cells.

[0010] Preferably, the radiation includes at least one of ultraviolet rays, X-rays, and gamma rays.

[0011] Furthermore, the ionizing radiation dose is a sublethal dose.

[0012] Preferably, the dose of the ionizing irradiation is 50–150 Gy.

[0013] Furthermore, the nucleotide sequence of the GM-SCF is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0014] SEQ ID NO:1 cDNA sequence of the mGM-CSF encoding gene atgtggctgcagaatttacttttcctgggcattgtggtctacagcctctcagcaccccacccgctcacccatcactgtcacccggccttggaagcatgtagaggcca tcaaagaagccctgaacctcctggatgacatgcctgtcacgttgaatgaagaggtagaagtcgtctctaacgagttctccttcaagaagctaacatgtgtgcagacc cgcctgaagatattcgagcagggtctacggggcaatttcaccaaactcaagggcgccttgaacatgacagccagctactaccagacatactgccccccaactccgg aaacggactgtgaaacacaagttaccacctatgcggatttcatagacagccttaaaacctttctgactgatatcccctttgaatgcaaaaaaccaggccaaaaatga SEQ ID NO:2 cDNA sequence of the hGM-CSF encoding gene atgtggctgcagagcctgctgctcttgggcactgtggcctgcagcatctctgcacccgcccgctcgcccagccccagcacgcagccctgggagcatgtgaatgccatccaggaggcccggcgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatctcagaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaagcagggcctgcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccactacaagcagcactgccctccaaccccggaaacttcctgtgcaacccagattatcacctttgaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggagccagtccaggagtga Preferably, the amino acid sequence of the GM-SCF is as shown in SEQ ID NO:3 or SEQ ID NO:4.

[0015] Amino acid sequence of SEQ ID NO:3 mGM-CSF mwlqnllflgivvyslsaptrspitvtrpwkhveaikealnllddmpvtlneevevvsnefsfkkltcvqtrlkifeqglrgnftklkgalnmtasyyqtycpptpetdcetqvttyadfidslktfltdipfeckkpgqk Amino acid sequence of SEQ ID NO:4 hGM-CSF Mwlqsllllgtvacsisaparspspstqpwehvnaiqearrllnlsrdtaaemnetvevisemfdlqeptclqtrlelykqglrgsltklkgpltmmashykqhcpptpetscatqiitfesfkenlkdfllvipfdcwepvqe Furthermore, the liver cancer cell line is selected from at least one of the following: Hepa 1-6, H22, BNL CL.2, Hepa1c1c7, RIL-175, MHCCl97-H, HepG2, Huh-7, Hep3B, PLC / PRF / 5, MHCC97-H, MHCC97-L, SMMC-7721, Li-7, SK-HEP-1, SNU series, or JHH series cell lines.

[0016] Furthermore, the tumor cell vaccine loaded with GM-CSF is prepared by the following method: (1) Construct GM-CSF overexpression plasmid, then mix it with packaging plasmid, transfect cells and collect viral supernatant, filter and concentrate to obtain lentivirus; (2) Lentiviral cells were transfected into liver cancer cell lines, and after screening and identification, stable cell lines expressing GM-SCF were obtained; (3) The stable cell line expressing GM-SCF is obtained by ionizing radiation.

[0017] Preferably, the GM-CSF overexpression plasmid is obtained by inserting the target gene GM-CSF into the plasmid development reading frame region and inserting a promoter before the target gene.

[0018] More preferably, the promoter is selected from at least one of EF-1α, CMV, CAG or Ub.

[0019] Most preferably, the EF-1α promoter nucleotide sequence is shown in SEQ ID NO:5.

[0020] SEQ ID NO:5 cDNA sequence of the EF-1α promoter In a second aspect, the present invention provides a combined medicament for the prevention and / or treatment of liver cancer, comprising the aforementioned tumor cell vaccine loaded with GM-CSF and other anti-tumor drugs.

[0021] Furthermore, the other antitumor drugs are selected from at least one of chemotherapy drugs or immune response modulators.

[0022] Preferably, the immune response modulator is at least one of cytokines, class II HLA protein-binding helper molecules, CD40 agonists, checkpoint receptor antagonists, B7 co-stimulatory molecules, FLt3 agonists, or CD40L agonists.

[0023] More preferably, the other antitumor drugs are selected from at least one of the following drugs: (1) Targeted therapy: lenvatinib, sorafenib, donafenib, regorafenib, cabozantinib, ramoximab; (2) Combination immunotherapy: atezolizumab + bevacizumab, sintilimab + bevacizumab analog, pembrolizumab + lenvatinib, durvalumab + trimelimab; (3) PD-1 / PD-L1 inhibitor monotherapy: pembrolizumab, nivolumab, sintilimab; (4) Immunotherapy: Nivolumab, pembrolizumab, camrelizumab or ipilimumab + nivolumab; (5) Targeted + Immunotherapy combination: apatinib + camrelizumab; (6) Cytotoxic chemotherapy: FOLFOX4 regimen: oxaliplatin + fluorouracil + leucovorin; (7) Anti-angiogenesis: bevacizumab.

[0024] Thirdly, the present invention provides the use of the above-mentioned tumor cell vaccine loaded with GM-CSF or its combination with other drugs in the prevention and / or treatment of liver cancer.

[0025] Furthermore, the liver cancer is selected from at least one of hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICCA), and mixed hepatocellular carcinoma (cHCC-CCA).

[0026] Furthermore, the treatment method of the drug is at least one of the following: intradermal single-point injection, intradermal multiple-point injection, subcutaneous single-point injection, subcutaneous multiple-point injection, intravenous injection, peritumoral injection, intratumoral injection, pleural injection, intraperitoneal injection, subarachnoid injection, intra- or peri-lymph node injection, or intramuscular injection.

[0027] The above-mentioned injection treatments can be used alone depending on the specific situation, or in combination if necessary.

[0028] Furthermore, the present invention also provides a method for treating liver cancer or complications arising from liver cancer. The method comprises administering a therapeutically effective amount of the aforementioned GM-CSF-loaded tumor cell vaccine or a combination thereof to a diseased mammal.

[0029] Furthermore, the mammals mentioned in the above methods are rats, dogs, monkeys, or humans.

[0030] Furthermore, the above methods can also be used in the treatment of liver cancer, including radiotherapy, chemotherapy, immunotherapy, and surgery combined with other tumor treatments.

[0031] Beneficial Effects: To enhance the immunogenicity of tumor cell vaccines against tumors, this invention prepared a tumor cell vaccine expressing GM-CSF based on a hepatocellular carcinoma cell line. Furthermore, ionizing radiation was used to improve the antitumor effect of the vaccine, ultimately obtaining a tumor cell vaccine loaded with GM-CSF. The antitumor activity and mechanism of action of the GM-CSF-loaded tumor cell vaccine were investigated in vivo and in vitro experiments. Finally, the safety of the vaccine was evaluated. Experimental results demonstrate that the ionized irradiated tumor cell vaccine expressing GM-CSF (Hepa 1-6-mGM-CSF) prepared in this invention has the following advantages: (1) The Hepa 1-6-mGM-CSF vaccine significantly promoted the maturation of DCs and increased activated CD4. + and CD8 + The proportion of T cells enhanced T CM and T EM The vaccine promotes cell generation and enhances humoral immune responses by increasing the number of Tfh and GCB cells. Furthermore, by extracting and culturing splenic lymphocytes in vitro, the Hepa 1-6-mGM-CSF vaccine increased TNF-α expression and promoted T cell proliferation. Finally, the Hepa 1-6-mGM-CSF vaccine suppressed the number of Tregs in the spleen. Therefore, the prophylactic immunization provided by this invention can synergistically mobilize innate adaptive immune pathways, induce long-term immune memory, and suppress immune tolerance.

[0032] (2) In in vitro experiments, the Hepa 1-6-mGM-CSF vaccine promoted the maturation and activation of DCs, manifested by the upregulation of co-stimulatory molecule expression and increased secretion of inflammatory factors. In in vivo experiments, the Hepa 1-6-mGM-CSF vaccine not only promoted the maturation of DCs but also promoted their recruitment and migration to draining lymph nodes. It is evident that the vaccine of the present invention can promote the maturation, recruitment, and migration of DCs, enhance T cell-mediated immune responses, induce long-term immune memory, and inhibit immune tolerance.

[0033] (3) Prophylactic and rechallenge immunization experiments on the Hepa 1-6 subcutaneous tumor model revealed that simple irradiation of Hepa 1-6 cells could induce a significant anti-tumor immune response and effectively inhibit the growth of the primary tumor. Based on this, the tumor cell vaccine Hepa 1-6-mGM-CSF loaded with mGM-CSF further enhanced the immune protection effect, provided more durable immune memory, and effectively prevented tumor recurrence.

[0034] (4) By analyzing the potential mechanisms by which the vaccine inhibits tumor growth and prevents tumor recurrence, it was found that the Hepa1-6-mGM-CSF vaccine can significantly remodel the tumor microenvironment (TME): on the one hand, it promotes the infiltration of mature DCs and M1 macrophages in tumor tissue, and on the other hand, it effectively inhibits the local recruitment of MDSCs.

[0035] (5) Preliminary findings indicate that the vaccine has no significant toxicity based on the appearance and weight of mice during immunization, as well as blood biochemistry and morphological examination of major organs after immunization. Therefore, the vaccine of this invention demonstrates good safety.

[0036] In summary, the irradiated tumor cell vaccine loaded with GM-CSF prepared by this invention provides a new approach for the development of tumor vaccines. Based on its efficacy and safety demonstrated in a mouse model of liver cancer, this vaccine platform, after optimization and improvement, is expected to enter the development and application of clinical tumor vaccines. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the construction of a stable cell line expressing mGM-SCF by lentivirus transfection as described in Example 1.

[0038] Figure 2 This study aims to detect the expression levels of mGM-CSF gene RNA and protein in the Hepa 1-6-mGM-CSF stably transfected cell line (vaccine group) and the empty vector group in Example 1.

[0039] Figure 3 Figure 1 shows the results of promoting the maturation of lymph node DCs using the tumor cell vaccine loaded with mGM-CSF in Experiment Example 1: (A) Mature DCs (CD80) in inguinal lymph node cells of mice in different experimental groups. + CD86 + (A) The proportion of (B) the corresponding bar chart statistical analysis.

[0040] Figure 4 Figure 1 shows the results of T cell activation promoted by the tumor cell vaccine loaded with mGM-CSF in Experiment 1: (A) and (B) show the activated CD4+ cells in mouse inguinal lymph node cells in different experimental groups. + T cells and CD8 +Flow cytometry scatter plot of T cells; (C) and (D) are the corresponding bar charts for statistical analysis.

[0041] Figure 5 The results of the tumor cell vaccine loaded with mGM-CSF significantly increased the proportion of memory T cells in Experiment Example 1 are shown in the figure: (A) shows the proportion of memory T cells in mouse inguinal lymph node cells in different experimental groups detected by flow cytometry; (B), (C), (D) and (E) are the corresponding bar charts for statistical analysis.

[0042] Figure 6 The results of the tumor cell vaccine loaded with mGM-CSF significantly increased the proportion of Tfh cells in Experiment Example 1 are shown in the figure: (A) The proportion of Tfh cells in mouse inguinal lymph nodes in different experimental groups was detected by flow cytometry; (B) The corresponding bar chart statistical analysis.

[0043] Figure 7 The results of the tumor cell vaccine loaded with mGM-CSF significantly increased the proportion of GCB cells in Experiment Example 1 are shown in the figure: (A) The proportion of GCB cells in mouse inguinal lymph nodes in different experimental groups was detected by flow cytometry; (B) The corresponding bar chart statistical analysis.

[0044] Figure 8 The tumor cell vaccine loaded with mGM-CSF in Experiment 1 significantly increased Granzyme B. + Cell proportion results: (A) Granzyme B in mouse spleen lymphocytes from different experimental groups detected by flow cytometry. + (A) Cells; (B) Corresponding bar chart statistical analysis.

[0045] Figure 9 The tumor cell vaccine loaded with mGM-CSF in Experiment 1 significantly increased TNF-α. + Cell proportion results: (A) shows the TNF-α levels in mouse spleen lymphocytes from different experimental groups detected by flow cytometry. + The proportion of cells; (B) and (C) are the corresponding bar charts for statistical analysis.

[0046] Figure 10 The results of reducing the proportion of Tregs in the tumor cell vaccine loaded with mGM-CSF in Experiment Example 1 are shown in the figure: (A) The proportion of Tregs in mouse spleen cells in different experimental groups was detected by flow cytometry; (B) The corresponding bar chart statistical analysis.

[0047] Figure 11The results of the tumor cell vaccine loaded with mGM-CSF promoting T cell proliferation in Experiment 1 are shown in the following figures: (A) shows the fluorescence intensity of CFSE staining in mouse spleen lymphocytes in different experimental groups detected by flow cytometry. (B) and (C) are the corresponding bar charts for statistical analysis.

[0048] Figure 12 This is a flow cytometry result showing the changes in the expression levels of surface co-stimulatory molecules after stimulating BMDCs in different experimental groups in Experiment Example 2.

[0049] Figure 13 The image shows the ELISA results of pro-inflammatory cytokines secreted by BMDCs in the supernatant after co-culturing cells from different experimental groups with BMDCs in Experiment Example 2.

[0050] Figure 14 In Experiment 2, after subcutaneous injection of different experimental group cells into the paws of mice, flow cytometry was used to detect CD11c in the popliteal lymph node cells of mice. + The figure shows the proportion of CD80, CD86, and CD40 positive cells in DCs.

[0051] Figure 15 In Experiment 2, after subcutaneous injection of different experimental group cells into the local skin of mice, flow cytometry was used to detect DCs (CD11c) isolated from the injection site skin cells. + MHCⅡ + CD45 + )Proportion.

[0052] Figure 16 In Experiment 2, after subcutaneous injection of different experimental group cells into the paws of mice, flow cytometry was used to detect migrating dendritic cells (DCs) (CD11c) in the popliteal lymph node cells of mice. + CCR7 + (A) The proportion of (B) the corresponding bar chart statistical analysis.

[0053] Figure 17 The anti-tumor effect of the tumor cell vaccine loaded with mGM-CSF in Experiment Example 3 is shown in the following figures: (A) Tumor growth curve; (B) Survival curve of tumor-bearing mice.

[0054] Figure 18 The images show a gross view (A) and a tumor weight (B) of the tumor cell vaccine prophylactic immunization model loaded with mGM-CSF in Experiment Example 3. Figure 19 This is a diagram showing the anti-tumor effect of the tumor cell vaccine loaded with mGM-CSF in Experiment Example 3 after re-challenge.

[0055] Figure 20Figure 4 shows the effective increase in the proportion of mature DCs in tumors by the tumor cell vaccine loaded with mGM-CSF in Experiment Example 4: (A) Flow cytometry analysis of mature DCs (CD80) in tumor tissues isolated from different experimental groups after inoculation with Hepa 1-6 tumor cells. + CD86 + (A) The proportion of (B) the corresponding bar chart statistical analysis.

[0056] Figure 21 Results of increasing the proportion of M1 macrophages in the tumor cell vaccine loaded with mGM-CSF in Experiment Example 4; (A) Detection of the proportion of M1 macrophages in tumor tissues isolated after different experimental groups were inoculated with Hepa 1-6 tumor cells by flow cytometry; (B) Statistical analysis of the corresponding bar chart.

[0057] Figure 22 Results of reducing the proportion of MDSCs in the tumor cell vaccine loaded with mGM-CSF in Experiment Example 4; (A) Detection of the proportion of MDSCs in tumor tissues isolated after different experimental groups were inoculated with Hepa 1-6 tumor cells by flow cytometry; (B) Statistical analysis of the corresponding bar charts.

[0058] Figure 23 Results of the increased proportion of memory T cells in the tumor cell vaccine loaded with mGM-CSF in Experiment Example 4: (A) Flow cytometry analysis of T cells in spleens isolated after inoculation with Hepa 1-6 tumor cells in different experimental groups. EM (B) shows the proportion; (C) shows the corresponding bar chart statistical analysis; (D) shows the T in tumor tissue. EM Statistical analysis of proportions using bar charts.

[0059] Figure 24 The results show the weight changes of mice immunized with the tumor cell vaccine loaded with mGM-CSF in Experiment 5.

[0060] Figure 25 The results show the changes in blood biochemical parameters of mice immunized with the tumor cell vaccine loaded with mGM-CSF in Experiment Example 5.

[0061] Figure 26 HE staining results of various tissues and organs of mice immunized with the tumor cell vaccine loaded with mGM-CSF in Experiment Example 5.

[0062] A p < 0.05 is considered statistically significant and is indicated by "*"; p < 0.01 is indicated by "**"; p < 0.001 is indicated by "***"; and p < 0.0001 is indicated by "****". Detailed Implementation

[0063] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0064] To enhance the immunogenicity of cell tumor vaccines, in one embodiment of the present invention, the key cytokine GM-CSF, which can regulate the differentiation, maturation, migration and function of DCs, is introduced to stably overexpress GM-CSF, thus successfully constructing a whole-cell tumor vaccine that can efficiently secrete GM-CSF.

[0065] In the initial screening, irradiation treatment was found to induce apoptosis in tumor cells and trigger a significant oxidative stress response within the cells. Furthermore, ionizing radiation was found to significantly increase the production of ROS within tumor cells. Due to the structural differences between mtDNA and nDNA, mtDNA is more sensitive to ROS-induced oxidative damage. This conclusion was confirmed by the co-localization of the oxidative DNA damage marker 8-OHdG and the mitochondrial marker TOMM20 in irradiated hepatocellular carcinoma cells in immunofluorescence images. Flow cytometry results also showed an increase in 8-OHdG in irradiated tumor cells. It is evident that irradiation can induce tumor cells to produce oxidized mitochondrial DNA. Previous experiments also confirmed that irradiation significantly enhances the efficiency of hepatocellular carcinoma cells being phagocytosed by dendritic cells (DCs). Therefore, in some preferred embodiments of this invention, whole-cell tumor vaccines secreting GM-CSF were subjected to ionizing irradiation to obtain ionized GM-CSF-expressing whole-cell tumor vaccines.

[0066] In some specific experimental examples of the present invention, the anti-tumor immune response induced by the tumor vaccines of the present invention, which were irradiated and expressed or did not express GM-CSF, was investigated by flow cytometry analysis of mouse lymph node cells after prophylactic immunization.

[0067] In some specific experiments of this invention, given the close relationship between GM-CSF and DC development and function, and the important role of DCs in initiating T cell-mediated anti-tumor immune responses, the effects of the irradiated GM-CSF-expressing cell tumor vaccine of this invention on DC function were systematically evaluated.

[0068] In some specific experimental examples of the present invention, based on a subcutaneous tumor model of liver cancer cell lines, the cell tumor vaccine loaded with GM-CSF of the present invention was subjected to preventive and rechallenge immunization experiments, and the potential mechanisms by which the vaccine inhibits tumor growth and prevents tumor recurrence were further analyzed.

[0069] In some specific experimental examples of this invention, it was studied that tumor cells treated with irradiation alone can activate dendritic cells (DCs) and generate an anti-tumor immune response. Further research revealed that integrating GM-CSF into the cell vaccine resulted in a more significant immunostimulatory effect. Therefore, the potent anti-tumor effect of the irradiated GM-CSF-expressing cell tumor vaccine of this invention may stem from the synergistic effect between irradiated tumor cells and GM-CSF.

[0070] In some specific experimental examples of this invention, the effects of different treatment conditions—irradiation and mGM-CSF-expressing cells—on the phagocytic capacity of DCs were studied.

[0071] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0072] The experimental cell lines used in the following examples and test cases, namely human renal epithelial 293T cell line and mouse hepatocellular carcinoma cell line Hepa 1-6, were purchased from the American Type Culture Collection (ATCC) in the United States and cultured and cryopreserved at the National Key Laboratory of Biotherapy, Sichuan University.

[0073] The C57BL / 6 mice used in the following examples and experiments were all 6-week-old females, weighing approximately 16-18 g. All mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in the specific pathogen-free (SPF) animal facility of the State Key Laboratory of Biotherapy, Sichuan University. All animal experiments in this study were conducted in strict accordance with the guidelines approved by the Animal Protection and Use Committee of Sichuan University.

[0074] The technical operations or reagents used in the following examples and test cases, such as cell extraction, culture, separation, processing and detection methods, are all performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0075] Example 1: Preparation of a tumor cell vaccine expressing mGM-CSF The mGM-CSF overexpression plasmid was mixed with two packaging plasmids, pSPAX2 and pMD2.G, and transfected into 293T cells. The viral supernatant was collected. After filtration and concentration, the resulting lentivirus was used to transfect Hepa 1-6 cell lines (mouse hepatocellular carcinoma cell lines) to construct stable cell lines expressing mGM-SCF. Puromycin was used for selection during the experiment. Figure 1 The specific steps are as follows: 1. Construction of a stable mGM-CSF transfected cell line (1) Construction of mGM-CSF overexpression plasmid: The overexpression plasmid is constructed by inserting the target gene mGM-CSF (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:3) into the open reading frame (ORF) region of the plasmid, and inserting the EF-1α promoter (nucleotide sequence as shown in SEQ ID NO:5) before the target gene. This plasmid contains a puromycin resistance gene and can be used for resistance screening of stable cell lines after plasmid transfection.

[0076] (2) Transformation and amplification of mGM-CSF overexpression plasmid: Take 100 μl of competent DH5α cells and thaw them on ice. Add 20 ng of DNA solution to the DH5α cell suspension, noting that the added mGM-CSF overexpression plasmid DNA solution should not exceed 1 / 10 of the DH5α cell suspension volume. Gently mix and incubate on ice for 30 minutes. Heat shock the mixed solution in a 42℃ water bath for 90 seconds, then quickly remove the sample and place it on ice for 1-2 minutes. Add 900 μl of antibiotic-free sterile LB liquid medium to the mixture and incubate at 37℃ and 220 rpm for 1 hour with shaking. Spread an appropriate amount of sample onto solid LB medium containing ampicillin (100 μg / ml) and then incubate overnight at 37℃. After 16 hours of incubation, a single colony of appropriate size was inoculated from the plate onto LB liquid medium containing ampicillin (100 μg / ml) using a pipette tip for further culture.

[0077] (3) Large-scale extraction of mGM-CSF overexpression plasmid DNA: After operating according to the instructions of the EndoFreemini Plasmid Kit II from Tiangen Company, the extracted mGM-CSF overexpression plasmid solution was collected into centrifuge tubes and stored at -20℃.

[0078] (4) Lentiviral Packaging Preparation: 293T cells were passaged, and transfected when the cell confluence reached 70-80%. The transfection reagent mixture included: pSPAX2, 3 μg; pMD2.G, 2 µg; mGM-CSF overexpression plasmid, 5 µg; opti-MEM, 700 μl; PEI, 40 μl. The transfection reagent mixture was added to the 293T cells and cultured for 6-8 hours. The old medium was discarded, and then 10 ml / plate of fresh cell culture medium was added. The cells were incubated at 37°C in a 5% CO2 incubator for 48 hours. The cell status was observed, and the mGM-CSF-rich lentiviral supernatant was collected, filtered and concentrated using a 0.45 μm filter, aliquoted, and stored at -80°C.

[0079] (5) Lentiviral infection of cells and screening of stable lines: Mouse hepatocellular carcinoma cell line Hepa 1-6 was infected with 5×10⁻⁶ cells and screened. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and cultured overnight. After 24 hours, the medium was replaced with fresh medium containing 0, 1, 2, 4, or 8 μg / ml puromycin. The lowest puromycin concentration that killed cells 48 hours after the addition of puromycin was the selection concentration. The selection concentration for Hepa 1-6 cells was 2 μg / ml.

[0080] The selection process for stable mGM-CSF transfected cell lines is as follows: Hepa 1-6 cells were cultured at 5 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell per well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Cell status was observed, and the old culture medium was aspirated. 2 ml of viral supernatant and 2 μl of Polybrene (1000×) infection reagent were added to each well, mixed, and then incubated. Lentiviral cells overexpressing mGM-CSF from step (4) were transfected with fresh cell culture medium 24 hours later. 72 hours after transfection, cells were screened using cell culture medium containing 2 μg / ml puromycin (as verified in previous experiments), and thereafter cultured in medium containing 2 μg / ml puromycin.

[0081] 2. Identification of stably transfected cell lines overexpressing lentiviruses To verify whether the constructed stable transfected cell line successfully expressed mGM-CSF, RNA was extracted from Hepa1-6-mGM-CSF cells selected with puromycin and from untransfected Hepa1-6 control cells using the Foregene Cell Total RNA Isolation Kit. Reverse transcription (TaKaRa PrimeScript RT Reagent Kit) was performed, followed by RT-qPCR detection. Simultaneously, the culture supernatant of the corresponding cells was collected, and the secretion level of mGM-CSF protein was detected by ELISA. Figure 2 As shown, compared with the control group, the Hepa 1-6-mGM-CSF stably transfected cell line (vaccine group) showed higher levels of mRNA ( Figure 2 A) and protein levels ( Figure 2 B) Both cells significantly expressed mGM-CSF, confirming that the cell line can stably transcribe and secrete mGM-CSF.

[0082] The PCR reaction for RT-qPCR detection uses the following primers: Actin-F (SEQ ID NO:6): AGAGGGAAATCGTGCGTGAC; Actin-R (SEQ ID NO:7): CAATAGTGATGACCTGGCCGT; mGM-CSF-F (SEQ ID NO:8): GCTCTAGAAGATCACCGGCGAAGGA; mGM-CSF-R (SEQ ID NO:9): TATCGGCCGCTTCCTCATTTTTGGCC.

[0083] 3. Preparation of tumor cell vaccines expressing mGM-CSF 100 μl of identified Hepa 1-6-mGM-CSF stably transfected cell suspension (5 × 10⁶ cells / year) was used to transfect the cell suspension. 6 (cells / ml) and Hepa1-6 cell suspension (5×10⁻⁶) 6 The cells (each cell / ml) were irradiated with 100 Gy X-rays to obtain either irradiated Hepa 1-6-mGM-CSF cells or irradiated Hepa 1-6 cells, which are the tumor cell vaccine loaded with mGM-CSF (vaccine group) and the tumor cell vaccine without mGM-CSF (empty group) described in this invention.

[0084] Experimental Example 1: Effects of mGM-CSF-loaded tumor cell vaccine on specific immune responses in mice Detecting the activation level of tumor-specific immune responses is an important indicator for evaluating the efficacy of tumor vaccines. Specific immunity, also known as acquired immunity or adaptive immunity, is immune response that targets only specific antigens and possesses immunological memory; it can be divided into cellular immune responses and humoral immune responses. Therefore, we will examine the level of specific immune response activated in mice by a tumor cell vaccine loaded with mGM-CSF from multiple perspectives to evaluate the vaccine's efficacy.

[0085] Six-week-old female C57BL / 6 mice were randomly divided into three groups of five mice each. On days 0, 14, and 28, the C57BL / 6 mice were subcutaneously injected with 100 μl PBS (control group) and 5 × 10⁶ PBS (control group) into the left back. 5 Irradiated Hepa 1-6 cells (empty vector group) or 5×10 5 Mice were irradiated with Hepa 1-6-mGM-CSF cells (vaccine group) at a dose of 100 Gy via X-ray irradiation, and were immunized three times. Inguinal lymph node cells or spleen cells were extracted from mice on day 42 and lymphocytes were isolated. After stimulation with irradiated and inactivated Hepa 1-6 cells for 48 or 72 hours in vitro, the following assays were performed: (1) The effect of tumor cell vaccines loaded with mGM-CSF on DC maturation APCs play a crucial role in both innate and adaptive immune responses, and dendritic cells (DCs), as important APCs, are essential in initiating antigen-specific immunity and immune tolerance. The cytokine GM-CSF is considered a key factor in DC development under homeostatic or inflammatory conditions. Therefore, on day 14 after the third prophylactic immunization in mice, cells were extracted from the inguinal lymph nodes of mice, and CD80 levels were detected by flow cytometry. + CD86 + Cells account for CD11c + The proportion of cells. For example... Figure 3 As shown, CD80 in the control group, empty vector group, and vaccine group + CD86 + The increasing proportion of cells indicates that Hepa 1-6 irradiation can effectively promote DC maturation, and the vaccine loaded with the mGM-CSF gene has a better effect on activating DCs.

[0086] (2) Effect of mGM-CSF-loaded tumor cell vaccines on T cell activity After maturation, dendritic cells (DCs) activate T cells by interacting with corresponding receptors on the surface of T cells through co-stimulatory molecules on their surface. CD69 is a marker molecule for T cell activation. Cells from the inguinal lymph nodes of mice were analyzed by flow cytometry on day 14 of the third prophylactic immunization. Figure 4As shown, activated CD4 in the control group, empty vector group, and vaccine group + and CD8 + T cells (CD69) + CD4 + and CD69 + CD8 + The proportions increased sequentially and showed statistical differences. This result indicates that the vaccine loaded with the mGM-CSF gene promoted T cell activation more effectively than the PBS control group and irradiated empty tumor cells.

[0087] (3) Effects of tumor cell vaccines loaded with mGM-CSF on memory T cells Memory T cells (T cells) M Memory T cells differentiate from naive T cells after antigen stimulation. Upon re-exposure to the original antigen, they can trigger a sustained and enhanced immune response. Memory T cells can be divided into two major subsets: central memory T cells (T cells...). CM ) and effector memory T cells (T cells) EM T CM It mainly circulates in lymphatic organs and possesses stem cell-like characteristics, meaning it can differentiate and proliferate in large numbers after receiving appropriate signals (from antigens or cytokines), and it maintains immune memory for a relatively long time. T... EM The cells mainly migrate to non-lymphoid tissues, and can quickly trigger an immune response upon re-exposure to the antigen, but their immune memory is short-lived.

[0088] To investigate the effect of the vaccine on memory T cells, C57BL / 6 mice were prophylactically immunized. On day 14 of the third prophylactic immunization, inguinal lymph nodes were extracted from the mice, and T cells were detected by flow cytometry. CM and T EM The proportion. The results are as follows: Figure 5 As shown, unloaded group CD4 + T CM The proportion of cells was slightly higher than that of the control group, but the difference was not statistically significant, but CD8... + T CM The proportion of cells was statistically higher in the vaccine group compared to the control group. + T CM Cells and CD8 + T CM The proportion of cells was significantly higher in the empty vector group than in the empty vector group. (The empty vector group contained CD4+.) + T EM and CD8 + T EM The proportion of cells was higher than that of the control group, but CD4 + TEM There were statistically significant differences in the cell groups, while CD8... + T EM There was no statistically significant difference in the cell group. (Vaccine group CD4) + T EM The cell ratio was significantly different from that of the empty vector group, while CD8 + T EM The cell ratio decreased slightly, but the CD8+ group in the vaccine group... + T EM The proportion of cells was higher than that of the control group, showing a statistically significant difference. This indicates that the tumor cell vaccine loaded with mGM-CSF, compared with simple irradiation of tumor cells, can better enhance the long-term specific immune memory against tumors, thereby inhibiting tumor growth.

[0089] (4) Effects of tumor cell vaccines loaded with mGM-CSF on humoral immune responses Follicular helper T cells (Tfh cells) originate from naïve T cells and are CD4+ cells. + T cells are a specific subset of T cells found in lymphatic tissues such as tonsils and lymph nodes. Tfh cells participate in humoral immunity, playing a crucial auxiliary role in B cell proliferation, somatic hypermutation (SHM), class-switch recombination (CSR), and differentiation into antibody-producing plasma cells. Therefore, on day 14 after the third prophylactic immunization of C57BL / 6 mice, we extracted inguinal lymph nodes and analyzed the proportion of Tfh cells using flow cytometry. The results are as follows: Figure 6 As shown, the proportion of Tfh cells in the control group, empty vector group, and vaccine group increased sequentially, and the differences were statistically significant.

[0090] Germinal centers (GCs) develop within B cell follicles in secondary lymphoid tissues and are microscopic anatomical structures formed during the immune response. Germinal center B cells (GCBs) differentiate from naïve B cells after antigen stimulation and are mainly found in the germinal centers of lymph nodes and the spleen. GCBs can complete the cytokine-mediated immune response (CSR) process under the influence of cytokines secreted by Tfh cells and differentiate into plasma cells and memory B cells, thereby participating in humoral immunity. Therefore, we simultaneously examined the proportion of GCB cells in inguinal lymph node cells. The results are as follows: Figure 7As shown, the proportion of GCB cells in the empty vector group was significantly higher than that in the control group, while the proportion of GCB cells in the tumor cell vaccine group loaded with the mGM-CSF gene was even higher than that in the empty vector group, and the difference was statistically significant. All of the above results indicate that irradiation of tumor cells alone can promote humoral immune responses in vivo, while tumor cell vaccines loaded with mGM-CSF can enhance their efficacy against tumor humoral immune responses.

[0091] (5) Tumor cell vaccines loaded with mGM-CSF increase the expression of T cell anti-tumor effector molecules. Upon activation by the T cell receptor (TCR), naive T cells exhibit upregulated CD44 expression, and this high expression level is maintained in memory cells. Therefore, this marker is often used to identify T cells that have undergone antigen stimulation. Cytotoxic T lymphocytes (CTLs), as important cells in the immune system, can kill tumor cells and clear pathogens. After activation and recognition of target cells, CTLs primarily guide cell death through the perforin / granzyme (PRF / GZM) system and the death receptor / ligand system. Among all GZMs secreted by mouse and human cytotoxic lymphocytes, most studies agree that granzyme B has the highest cytotoxicity. Granzyme B belongs to the serine protease family and is also an important indicator of CTL activation. Therefore, we administered prophylactic immunizations to C57BL / 6 mice. On day 14 of the third prophylactic immunization, spleen cells were extracted and lymphocytes were isolated. After stimulation with irradiated Hepa1-6 cells for 48 hours in vitro, Granzyme B-positive CD44 expression was detected. hi CD8 + The proportion of T cells. Results as follows: Figure 8 As shown, the Granzyme B vaccine group + The proportion of cells in the empty vector group was significantly higher than that in the control group, and the number of Granzyme B-positive cells in the empty vector group was also statistically higher than that in the control group.

[0092] Tumor necrosis factor-α (TNF-α) has both cytotoxic and inhibitory effects on tumor cells and is a key cytokine in the type 1 helper T cell (Th1) signaling pathway. Therefore, we also detected that TNF-α-positive cells accounted for a significant proportion of CD44 cells. hi CD4 + T cells and CD44 hi CD8 + The proportion of T cells. Results as follows: Figure 9As shown, although the empty group TNF-α + The proportion of cells did not increase compared to the control group, and even decreased slightly. However, the proportion of TNF-α in the vaccine group was different from that in the control group and the empty vector group. + CD44 hi CD4 + T cells and TNF-α + CD44 hi CD8 + All T cell counts were significantly increased. All these results indicate that the tumor cell vaccine loaded with the mGM-CSF gene can further increase GranzymeB expression in CTLs and also promote CD4+ expression. + T cells and CD8 + T cells express TNF-α, thereby enhancing the anti-tumor effect of tumor cell vaccines.

[0093] (6) Tumor cell vaccines loaded with mGM-CSF can suppress immune tolerance. Regulatory T cells (Tregs) are CD4+ cells. + A subset of T cells, Tregs function to suppress excessive immune activation and are an important factor in maintaining immune tolerance. Tregs primarily regulate the immune response by inhibiting the activation and proliferation of effector T cells through contact inhibition. Therefore, we administered three prophylactic immunizations to C57BL / 6 mice. On day 14 after the third prophylactic immunization, we used flow cytometry to detect the proportion of Tregs in the spleen cells of the mice. The results are as follows... Figure 10 As shown, the proportion of Tregs in the empty vector group was slightly lower than that in the control group, but the difference was not statistically significant. However, the proportion of Tregs in the vaccine group was significantly lower than that in the empty vector group. These results indicate that tumor cell vaccines loaded with mGM-CSF can reduce the number of Tregs, thereby overcoming immune tolerance and enhancing the protective effect of tumor vaccines.

[0094] (7) Tumor cell vaccines loaded with mGM-CSF can promote the proliferation of T cells. CFSE is a long-acting live-cell dye that covalently binds to proteins in the cytoplasm of cells. During cell division, the dye is evenly distributed to daughter cells, allowing flow cytometry to detect its fluorescence intensity and thus investigate cell proliferation. In our study, on day 14 after the third prophylactic immunization of C57BL / 6 mice, spleen cells were extracted and lymphocytes were separated. The spleen cells were stained with CFSE and then co-cultured with irradiated and inactivated Hepa 1-6 cells for 72 hours before fluorescence intensity was measured. The results are as follows: Figure 11 As shown, CD4 of the simple unloaded group + T cells and CD8 +The proportion of T cell proliferation was slightly higher than that of the control group, but the difference was not statistically significant. Meanwhile, the CD4+ of the vaccine group loaded with the mGM-CSF gene was significantly higher. + T cells and CD8 + T cells exhibited the strongest proliferative activity. This result demonstrates that the vaccine can significantly promote T cell proliferation, thereby enhancing the anti-tumor immune response.

[0095] Experimental Example 2: Tumor cell vaccine loaded with mGM-CSF promotes DC cell maturation, recruitment and migration. In prophylactic immunization models, it has been demonstrated that the mGM-CSF-loaded tumor cell vaccine of this invention can promote DC maturation in vivo, and GM-CSF plays an important role in DC development, differentiation, and functional regulation. Therefore, the effects of the constructed mGM-CSF-loaded tumor cell vaccine on DC maturation and function were further investigated in in vitro and in vivo experiments.

[0096] (1) Tumor cell vaccines loaded with mGM-CSF promote DC maturation When dendritic cells (DCs) are stimulated by infection or inflammation, they undergo a series of maturation processes, including the upregulation of MHC-II molecules and co-stimulatory molecules (including CD80, CD86, and CD40). To assess whether a vaccine can effectively deliver to immature DCs and stimulate DC maturation, we first stimulated BMDCs (BMDCs to cells) in vitro with the vaccine (BMDCs:cell ratio of 1:3), and then detected the expression levels of maturation molecular markers CD80, CD86, and CD40 on the surface of BMDCs by flow cytometry. The results are as follows: Figure 12 As shown, compared with the control group, the co-stimulatory molecules CD80 and CD86 on the surface of BMDCs were significantly increased in the empty vector group. In contrast, the tumor cell vaccine group loaded with the mGM-CSF gene showed a better effect in promoting the maturation of BMDCs (CD80, CD86, and CD40 were all significantly increased).

[0097] Mature dendritic cells (DCs) not only increase the expression of various co-stimulatory molecules but also promote the secretion of pro-inflammatory cytokines, including IL-6, IL-12p70, and TNF-α. To assess the effects of different treatments on DC maturation, we collected culture supernatants from BMDCs co-cultured with PBS, irradiated Hepa 1-6 cells, or irradiated Hepa 1-6-mGM-CSF vaccine (BMDCs:cell ratio 1:3) for 24 hours and detected the concentrations of IL-6, IL-12p70, and TNF-α using ELISA. The results are as follows: Figure 13As shown, the levels of pro-inflammatory cytokines, namely IL-6, IL-12p70, and TNF-α, measured in the control group, empty vector group, and vaccine group increased sequentially, indicating that the constructed tumor cell vaccine was more effective in activating BMDCs in vitro.

[0098] Immature dendritic cells (DCs) migrate to secondary lymphoid organs after taking up antigens, presenting them to helper T cells or effector T cells, and promoting T lymphocyte activation through co-stimulatory molecules. Therefore, we subcutaneously injected PBS into the paws of mice, irradiated Hepa 1-6 cells, or irradiated Hepa 1-6-mGM-CSF cells. Twenty-four hours later, we extracted popliteal lymph node cells from the mice and examined the expression levels of co-stimulatory molecules (including CD80, CD86, and CD40) in DCs to investigate their activation degree. The results are as follows: Figure 14 As shown, CD80 and CD40 levels were significantly higher in the empty vector group than in the control group, while CD80, CD86, and CD40 levels were all upregulated in the vaccine group compared to the empty vector group, and the differences were statistically significant. These results confirm that the empty vector group without protein loading can promote DC maturation to some extent, while the tumor cell vaccine loaded with mGM-CSF can more effectively drive DCs to transform into the mature phenotype.

[0099] (2) Subcutaneous injection of tumor cell vaccines loaded with mGM-CSF effectively recruited DCs Most immature dendritic cells (DCs) are widely distributed on the mucosal surface and exist as sentinel cells in the skin and parenchymal organs, recognizing antigens. Based on this, we used flow cytometry to detect the recruitment of DCs in the skin at the injection site after subcutaneous injection of PBS, irradiation of Hepa 1-6 cells, or irradiation of Hepa 1-6-mGM-CSF cells in mice. The results are as follows: Figure 15 As shown, compared with the control group and the empty vector group, the number of DCs (CD11c) in the skin of mice in the vaccine group was significantly higher. + MHCⅡ + CD45 + The proportion of subcutaneous vaccines increased significantly, and the difference was statistically significant, indicating that subcutaneous vaccination can effectively promote the recruitment of dendritic cells (DCs) to the injection site, laying the foundation for their subsequent antigen uptake, activation, and initiation of adaptive immune responses.

[0100] (3) Subcutaneous injection of tumor cell vaccine loaded with mGM-CSF induced DC migration. In addition to the upregulation of MHC-II and co-stimulatory molecules, mature dendritic cells (DCs) also show increased expression of chemokine receptor 7 (CCR7) (also known as CD197). Another important characteristic of DCs in vivo is homing and migration: they migrate from the blood to tissues to capture antigens, then mature under inflammatory stimulation and leave the tissues, migrating to draining lymphoid organs to initiate naïve T cells. CCR7 and its ligands play a crucial role in the homing of DCs to lymph nodes and the spleen. Therefore, we also examined the expression level of CCR7 in DCs from the popliteal lymph nodes of mice after subcutaneous injection of the vaccine into the paw. The results are as follows: Figure 16 As shown, after subcutaneous injection of the vaccine, CD11c in the popliteal lymph node cells of mice... + CCR7 + The proportion of cells was significantly higher than that of the control group and the empty vector group, indicating that the tumor cell vaccine loaded with mGM-CSF can effectively promote DC migration.

[0101] Experimental Example 3: Study on the anti-tumor effect of mGM-CSF-loaded tumor cell vaccine in mice The results of Experiments 1 and 2 demonstrate that the mGM-CSF-loaded tumor cell vaccine effectively promotes anti-tumor-specific immune responses in various ways and can promote the maturation, recruitment, and migration of dendritic cells (DCs). Next, we further investigated whether the vaccine had a stronger anti-tumor effect in mice compared to the control group and the empty vector group using the Hepa 1-6 subcutaneous tumor model. Additionally, we conducted a re-challenge experiment to explore whether the vaccine could induce long-term immunity, thereby inhibiting tumor growth in the long term.

[0102] (1) Prophylactic antitumor effect of tumor cell vaccines loaded with mGM-CSF On days 0, 14, and 28, C57BL / 6 mice were subcutaneously injected with 100 μl PBS and 5 × 10⁶ ppm of PBS into the left back. 5 One irradiated Hepa1-6 cell or 5 × 10⁶ cells 5 Five × 1010 Hepaa 1-6-mGM-CSF cells were subcutaneously seeded on the right back of mice on day 42 at an irradiation dose of 100 Gy. 6 Six Hepa tumor cells were collected. The mice were then observed for their condition and tumor growth. The tumor growth curve is shown below. Figure 17 As shown in Figure A, both the empty vector group and the vaccine group exhibited anti-tumor effects compared to the control group, but there was no statistically significant difference between the two groups. Mouse survival curves are shown below. Figure 17As shown in Figure B, the survival rate of the control group was 80% on day 25 after tumor inoculation; on day 28 after tumor inoculation, the survival rate of the control group dropped to 0%. However, the survival rate of mice in both the empty vector group and the vaccine group was 100% until day 28 after tumor inoculation. On day 14 after tumor inoculation, mice were euthanized and the tumors were removed, as shown in Figure B. Figure 18 As shown, the tumor volume and weight in the empty vector group were significantly smaller than those in the control group, while the tumor volume and weight in the vaccine group were slightly lower than those in the empty vector group, but there was no statistical difference.

[0103] (2) Tumor cell vaccines loaded with mGM-CSF challenge anti-tumor efficacy again The tumor rechallenge experiment can assess whether a vaccine can induce immune memory. On day 14 after the third prophylactic immunization in C57BL / 6 mice, Hepa 1-6 tumor cells were injected. On day 70, after complete tumor regression in both the empty vector and vaccine groups, 1.5 × 10⁻⁶ tumor cells were injected on the contralateral side of the previously injected tumor. 7 The mice were treated with Hepa 1-6 tumor cells, and their condition and tumor growth were observed. The tumor growth curves are shown below. Figure 19 As shown, on day 22 after tumor inoculation, all tumors in the vaccine group mice had regressed, the empty vector group showed a regression trend but no mice had completely regressed tumors, and the tumors in the control group showed a trend of continuous growth.

[0104] Experimental Example 4: The Effect of mGM-CSF-Loaded Tumor Cell Vaccines on the Tumor Microenvironment The results of Experiment 3 show that both the empty vector group and the vaccine group effectively inhibited the growth of subcutaneous tumors in mice, and the vaccine exhibited stronger long-lasting immunogenicity. To further investigate the anti-tumor mechanism of the mGM-CSF-loaded tumor cell vaccine, we used flow cytometry to examine the tumor microenvironment (TME) in mice after immunization. The tumor microenvironment refers to the microenvironment surrounding tumor cells, composed of cellular and non-cellular components. Cellular components include T cells, dendritic cells (DCs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs).

[0105] Dendritic cells (DCs) act as a bridge between the innate and adaptive immune systems by presenting antigens and activating T cells. However, in a tumor immunosuppressive environment, DCs' ability to activate T cells decreases, leading to T cell dysfunction and Treg recruitment, thereby promoting immune tolerance. We conducted a three-stage prophylactic immunization experiment on mice. On day 14 after the third immunization, mice were inoculated with Hepa1-6 tumor cells. On day 14 after tumor inoculation, tumor cells were isolated, and flow cytometry was used to detect mature DCs (CD80) in the tumor.+ CD86 + The proportion of ). The results are as follows Figure 20 As shown, unloaded group CD80 + CD86 + The proportion of DCs in the vaccine group was higher than that in the control group, but there was no statistically significant difference. However, the proportion of mature DCs in the vaccine group was significantly higher than that in the empty vector group, and this was statistically significant.

[0106] Tumor macrophages (TAMs) are an important component of the tumor microenvironment (TME), exhibiting high plasticity and heterogeneity. Based on phenotype and function, TAMs can be broadly classified into the pro-inflammatory and anti-tumor type M1 and the anti-inflammatory and pro-tumor type M2. Therefore, we used flow cytometry to detect the proportion of M1 macrophages in tumors inoculated with mice after three prophylactic immunizations. The results are as follows: Figure 21 As shown, the number of M1 macrophages increased sequentially in the control group, empty vector group, and vaccine group, and the differences were statistically significant.

[0107] MDSCs are a class of pathologically activated, heterogeneous, immature cells with potent immunosuppressive activity. They are widely distributed in the spleen and tumor tissue of tumor-bearing mice, or in the peripheral blood and tumor sites of cancer patients. Therefore, we used flow cytometry to detect the proportion of MDSCs in tumor cells using the above-described process to investigate the effect of vaccines on MDSCs in tumors. The results are as follows: Figure 22 As shown, although the proportion of MDSCs in the empty vector group increased significantly compared with the control group, the proportion of MDSCs in the tumor cells of mice loaded with the mGM-CSF gene was significantly lower than that in both the control group and the empty vector group, and this was statistically significant.

[0108] To further investigate the impact of vaccines on long-term immune memory, we isolated the spleen and tumors of mice, prepared single-cell suspensions, and analyzed the proportion of memory T cells using flow cytometry. The results are as follows: Figure 23 As shown, the empty vector group T in mouse spleen EM The proportion was lower in the vaccine group than in the control group, while it was slightly higher in tumors than in the control group, but the difference was not statistically significant. Correspondingly, the proportion of T cells in the spleen and tumors of the vaccine group was lower. EM The proportions of mGM-CSF-loaded tumor cells were significantly higher in both the control and empty groups, and these results were statistically significant. All these results demonstrate that the tumor cell vaccine loaded with mGM-CSF can promote anti-tumor immune responses in mice, reduce the generation of suppressive immune cells, and induce long-term immune memory.

[0109] Example 5: Safety assessment of tumor cell vaccines loaded with mGM-CSF To ensure the feasibility of using vaccines in clinical treatment of cancer patients, a comprehensive and scientific evaluation of their safety is an indispensable and crucial step. We will evaluate the safety of the vaccine from three aspects: changes in body weight, changes in blood biochemical indicators, and morphological changes in various organs of mice after immunization.

[0110] (1) Changes in the apparent weight of mice after immunization C57BL / 6 mice were prophylactically immunized three times with a tumor cell vaccine loaded with mGM-CSF. Starting from day 1 post-immunization, the mice's phenomenology was observed and recorded every three days, including changes in fur, behavior, growth, and weight. Results are as follows: Figure 24 As shown, the weight of mice was not significantly affected after vaccination, there was no significant difference in weight change among groups, and all mice were in good condition throughout the immunization process, with no hair loss or dullness, no behavioral changes, no impact on growth and development, and no changes in fecal characteristics.

[0111] (2) Detection of blood biochemical indicators in mice after immunization C57BL / 6 mice were given three prophylactic immunizations with a tumor cell vaccine loaded with mGM-CSF. Seven days after the third immunization, blood was collected from the mice's eyeballs, and serum was separated for blood biochemistry analysis. Results are as follows: Figure 25 As shown, there were no significant differences in ALT and AST (liver function indicators), ALP (bile secretion), CREA and UREA (kidney function indicators) among the immunized mice.

[0112] (3) Observation of the morphology of various organs in mice after immunization After mice received three prophylactic immunizations, on day 7 of the third immunization, the mice were sacrificed, and tissue samples from various organs (including heart, liver, lungs, spleen, and kidneys) were collected for H&E staining. Figure 26 As shown, no inflammatory cell infiltration or cell necrosis was observed in the organs of any group of mice. In conclusion, the tumor cell vaccine loaded with mGM-CSF has good safety in mice.

Claims

1. A tumor cell vaccine loaded with GM-CSF, characterized in that: It was obtained from a liver cancer cell line expressing GM-SCF through ionizing radiation.

2. The tumor cell vaccine loaded with GM-CSF according to claim 1, characterized in that: The ionizing irradiation is performed using rays capable of inactivating cells; preferably, the rays include at least one of ultraviolet rays, X-rays, and gamma rays; more preferably, the dose of the ionizing irradiation is 50 to 150 Gy.

3. The tumor cell vaccine loaded with GM-CSF according to claim 1 or 2, characterized in that: The nucleotide sequence of the GM-SCF is shown in SEQ ID NO:1 or SEQ ID NO:2; preferably, the amino acid sequence of the GM-SCF is shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. The tumor cell vaccine loaded with GM-CSF according to any one of claims 1 to 3, characterized in that: The liver cancer cell line is selected from at least one of the following: Hepa 1-6, H22, BNL CL.2, Hepa1c1c7, RIL-175, MHCCl97-H, HepG2, Huh-7, Hep3B, PLC / PRF / 5, MHCC97-H, MHCC97-L, SMMC-7721, Li-7, SK-HEP-1, SNU series, or JHH series cell lines.

5. The tumor cell vaccine loaded with GM-CSF according to any one of claims 1 to 4, characterized in that, The tumor cell vaccine loaded with GM-CSF is prepared by the following method: (1) Construct GM-CSF overexpression plasmid, then mix it with packaging plasmid, transfect cells and collect viral supernatant, filter and concentrate to obtain lentivirus; (2) Lentiviral cells were transfected into liver cancer cell lines, and after screening and identification, stable cell lines expressing GM-SCF were obtained; (3) The stable cell line expressing GM-SCF is obtained by ionizing radiation.

6. The tumor cell vaccine loaded with GM-CSF according to claim 5, characterized in that: The GM-CSF overexpression plasmid is obtained by inserting the target gene GM-CSF into the plasmid development reading frame region and inserting a promoter before the target gene; preferably, the promoter is selected from at least one of EF-1α, CMV, CAG or Ub.

7. A combination drug for the prevention and / or treatment of liver cancer, characterized in that: Tumor cell vaccines and other antitumor drugs containing GM-CSF as described in any one of claims 1 to 6.

8. The combination drug according to claim 7, characterized in that: The other antitumor drugs are selected from at least one of chemotherapy drugs or immune response modulators; preferably, the other antitumor drugs are selected from at least one of the following drugs: (1) Targeted therapy: lenvatinib, sorafenib, donafenib, regorafenib, cabozantinib, ramoximab; (2) Combination immunotherapy: atezolizumab + bevacizumab, sintilimab + bevacizumab analog, pembrolizumab + lenvatinib, durvalumab + trimelimab; (3) PD-1 / PD-L1 inhibitor monotherapy: pembrolizumab, nivolumab, sintilimab; (4) Immunotherapy: Nivolumab, pembrolizumab, camrelizumab or ipilimumab + nivolumab; (5) Targeted + Immunotherapy combination: apatinib + camrelizumab; (6) Cytotoxic chemotherapy: FOLFOX4 regimen: oxaliplatin + fluorouracil + leucovorin; (7) Anti-angiogenesis: bevacizumab.

9. The use of the tumor cell vaccine loaded with GM-CSF as described in any one of claims 1 to 6 or the combination drug as described in any one of claims 7 to 8 in the prevention and / or treatment of liver cancer.

10. The application according to claim 9, characterized in that: The liver cancer is selected from at least one of hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and mixed liver cancer.

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