A novel tumor antigen vaccine system that specifically labels heterologous proteins and its applications

Abstract

This invention discloses a tumor novel antigen vaccine system that specifically labels heterologous proteins and its applications. The vaccine system consists of a heterologous protein mRNA vaccine and a recombinant oncolytic virus. The heterologous protein mRNA vaccine induces the generation of heterologous protein-specific memory T cells in the body, and the recombinant oncolytic virus promotes the expression of microbial-derived tumor novel antigens in tumors. When pre-immunized memory T cells specifically identify these novel antigens, they are rapidly activated to kill tumor cells expressing the novel antigens and promote antigen release from the tumor itself. Dendritic cells phagocytose the antigens, further activating the antitumor response and activating a systemic antitumor immune effect through antigen epitope spreading. This therapeutic policy can be applied to the manufacture of tumor immunotherapy drugs to achieve rapid identification and killing of tumor cells and activation of antitumor immune effects.

Description

[Technical Field]

[0001] The present invention relates to an antitumor vaccine system, more particularly to a novel tumor antigen vaccine system that specifically labels heterologous proteins, and more further to applications of the above vaccine system. [Background technology]

[0002] Immunotherapy is a milestone advance in the history of oncology and has significantly transformed clinical oncology. Novel oncological antigen vaccines, possessing unique tumor targeting properties, are currently a hot spot in the field of immunotherapy. These novel antigens, also called tumor-specific antigens, are characterized by their expression only in tumors and not in normal tissues. By activating the patient's immune system, they can induce tumor-specific T cells and suppress tumor growth. While novel oncological antigens offer advantages such as higher tumor specificity compared to conventional tumor-associated antigens, multiple clinical trials both in China and internationally have revealed that novel oncological antigen vaccines have low therapeutic response rates against multiple types of tumors. For example, the personalized novel oncological antigen vaccine RO7198457, developed by BioNTech, has an objective response rate of only 8% against multiple types of tumors, including melanoma, breast cancer, and colorectal cancer. Existing novel oncological antigen vaccines still face the following challenges: low immunogenicity of novel antigens, high heterogeneity in their expression, and suppression of the therapeutic response to novel oncological antigen vaccines by TIME (Time Inhibition).

[0003] Oncolytic virus therapy is a new treatment method for tumors. Oncolytic viruses are a general term for natural or genetically modified viruses that selectively infect and kill tumors without affecting normal cells. However, oncolytic viruses have significant limitations in tumor treatment. Because the immunogenicity of tumor antigens is usually lower than that of proteins derived from viral vectors, the body generates a strong antiviral immune response, suppressing viral replication and tumor lysis, resulting in insufficient tumor therapeutic effect. Oncolytic viruses modified to express immune checkpoint inhibitors such as PD-1, dendritic cell (DC) activators such as GM-CSF, and cytokines such as IL-2 enhance the antitumor immune effect of the oncolytic virus to some extent, but still rely on the de novo induction of the immune response by tumor-derived antigens. Therefore, they are limited by the suppressive effect of the host immune system on the virus, and significant tumor therapeutic effects cannot be obtained. [Overview of the project] [Problems that the invention aims to solve]

[0004] Objective of the Invention: The objective of the present invention is to provide a tumor antigen vaccine system that efficiently and specifically labels heterologous proteins, and applications for the above vaccine system, which allows memory T cells induced by the mRNA vaccine to identify and kill tumor cells that have been accurately labeled with a novel antigen derived from a heterologous protein, and further activate a systemic antitumor immunotherapy effect. [Means for solving the problem]

[0005] Technical proposal: The present invention provides a tumor novel antigen vaccine system that specifically labels a heterologous protein, comprising a heterologous protein mRNA vaccine and a recombinant oncolytic virus, wherein the heterologous protein mRNA vaccine induces the generation of specific memory T cells against a precisely labeled novel antigen, and the recombinant oncolytic virus is used to precisely label a novel antigen that is highly immunogenic to tumor cells.

[0006] Preferably, the heterologous protein is one or more of the following: hepatitis B virus-derived protein (HBsAg), human papillomavirus-derived proteins (E6, E7), EBV-derived protein (PP65), influenza virus-derived protein (MP), tetanus toxoid protein (TT), Staphylococcus aureus-derived protein (C2), or flagellin from Salmonella typhi.

[0007] Preferably, the oncolytic virus is one or more of vaccinia virus, herpes simplex virus, or adenovirus.

[0008] Preferably, the heterologous protein mRNA vaccine is produced by constructing, synthesizing, and amplifying an mRNA vaccine recombinant plasmid, and then encapsulating the mRNA vaccine recombinant plasmid with lipid nanomaterials (LNPs).

[0009] Preferably, the recombinant oncolytic virus is produced by constructing a recombinant oncolytic virus that expresses a novel antigen, and then performing plaque purification and amplification purification.

[0010] Applications in the manufacture of the aforementioned vaccine-based tumor immunotherapy drugs.

[0011] Preferably, it is used to enhance tumor immunotherapy. A heterologous protein mRNA vaccine is used to induce the generation of heterologous protein-specific memory T cells in the body, and a recombinant oncolytic virus is used to cause tumor cells to express a novel tumor antigen derived from a microorganism. When pre-immunized memory T cells detect the specific novel antigen, they are rapidly activated to kill the tumor cells expressing the novel antigen and promote antigen release from the tumor itself. The antitumor immune response is further activated when dendritic cells (DCs) phagocytose the antigen, thereby activating a systemic antitumor immune effect.

[0012] Preferably, the administration order is to administer the heterologous protein mRNA vaccine first, followed by the recombinant oncolytic virus, or to administer the heterologous protein mRNA vaccine and the recombinant oncolytic virus simultaneously.

[0013] Preferably, in the aforementioned administration, the heterologous protein mRNA vaccine induces the generation of immune memory in the living body via intramuscular injection, intranasal administration or intravenous administration, and the recombinant oncolytic virus accurately labels tumor neoantigens with high immunogenicity against tumor cells via intratumoral administration or intravenous administration.

[0014] Preferably, the aforementioned tumor is a solid tumor or a metastatic tumor associated with a solid tumor, including melanoma, colorectal cancer, breast cancer, liver cancer, glioma, cholangiocarcinoma or pancreatic cancer.

Effects of the Invention

[0015] Principle of the Invention: To address the three issues that cause insufficient therapeutic response of neoantigen vaccines, namely "low immunogenicity of neoantigens", "tumor heterogeneity (spatiotemporal heterogeneity of neoantigen expression within tumors)" and "limitation of TIME", the present invention constructs a tumor neoantigen vaccine system composed of a heterologous protein mRNA vaccine and a recombinant oncolytic virus, which specifically labels heterologous proteins. On the one hand, by independently constructing tumor neoantigens derived from heterologous proteins with high immunogenicity, the problems of "low immunogenicity" and "expression heterogeneity" of conventional neoantigens derived from tumor gene mutations are solved. On the other hand, a heterologous protein mRNA vaccine is used to induce the generation of neoantigen-specific memory T cells in the living body and break through the limitation of TIME, so as to activate the anti-tumor immune effect through dual actions. Aiming to independently construct highly immunogenic tumor neoantigens and induce the breakthrough of TIME by neoantigen-specific memory T cells, the present invention improves the therapeutic response rate of neoantigen vaccines, thereby solving the following three problems existing in the prior art.

[0016] (1) Solving the problems of "low immunogenicity" and "expression heterogeneity" in existing tumor neoantigens: Using a gene-deficient oncolytic virus to specifically label neoantigens derived from heterologous proteins with high safety and strong immunogenicity for tumor cells. The low immunogenicity of neoantigens derived from tumor mutations and the presence of heterogeneity in their expression are the core bottlenecks leading to an insufficient therapeutic response rate of neoantigen vaccines against solid tumors, which requires an urgent solution. In addition, the present invention utilizes the property that gene-deficient oncolytic viruses replicate only within tumors, to specifically label "neoantigens derived from heterologous proteins with high safety and strong immunogenicity" for tumor cells, thereby solving the problems of "low immunogenicity" and "expression heterogeneity" of conventional mutation-derived neoantigens. More importantly, since the heterologous protein constructed in the present invention has known highly immunogenic T cell epitopes, there is no need to go through time-consuming and costly steps such as sequencing and screening of neoantigens, and it can be rapidly applied to tumor treatment.

[0017] (2) Solving the problem of TIME-mediated suppression of the therapeutic response of tumor neoantigen vaccines: Using an mRNA vaccine to induce the production of neoantigen-specific memory T cells (not restricted by TIME) and specifically labeled neoantigens in the living body, so as to rapidly and efficiently kill tumors and activate the anti-tumor immune effect. Even if neoantigen-specific T cells derived from tumor mutations are generated, TIME can make these neoantigen T cells unable to recognize and kill tumors, for example, by exhausting the neoantigen-specific T cells. In contrast, immune memory T cells, particularly heterologous protein-specific memory T cells, have a low activation threshold during antigen recognition and are not restricted by TIME. The present invention takes advantage of the property that heterologous protein-specific memory T cells are not restricted by TIME, and the specifically labeled neoantigens derived from heterologous proteins, to rapidly and efficiently kill tumors and release the antigens of the tumors themselves (epitope spreading), further activate the anti-tumor immune effect, and improve the therapeutic response rate of neoantigen vaccines.

[0018] (3) Solving the scientific problem of insufficient antitumor immune effect by oncolytic viruses: Theoretically, oncolytic viruses can eliminate tumors by lysing tumor cells and releasing tumor antigens, thereby activating the antitumor immune response. However, since the immunogenicity of tumor antigens is usually lower than that of viruses, the body generates a stronger antiviral immune response, which limits the oncolytic effect of the virus, resulting in insufficient antitumor immune effect due to reduced tumor lysis and antigen release. Oncolytic viruses expressing PD-L1, GM-CSF, novel antigens, etc., can enhance the antitumor immune response to some extent, but still depend on immune induction by tumor antigens. Therefore, they are limited by the host's antiviral immunity. To solve this problem, the applicant, on the one hand, improves the immunogenicity of tumors by delivering novel tumor antigens derived from heterologous proteins, and on the other hand, uses mRNA-H vaccines to pre-induce the generation of a large number of novel antigen-specific memory T cells in the body. These heterologous protein-specific memory T cells can rapidly and efficiently kill tumors expressing novel antigens, releasing tumor antigens and activating antitumor immune responses. Thus, the present invention also solves the problem of "insufficient antitumor immune responses of oncolytic viruses due to limitations imposed by the host immune system."

[0019] Most microbial proteins, such as human papillomavirus proteins E6 and E7, EBV virus-derived protein PP65, influenza virus-derived protein, tetanus toxoid protein, Staphylococcus aureus-derived protein, or Salmonella typhi flagellin, are heterologous microbial proteins and possess known highly immunogenic epitopes. Their immunogenicity is stronger than that of novel antigens derived from single tumor gene mutations. Therefore, by using mRNA vaccines corresponding to these proteins (which generate heterologous protein-derived novel antigen-specific memory T cells and overcome the TIME limitation) in combination with oncolytic viruses, and accurately labeling the novel antigen against tumor cells (solving the problems of "low immunogenicity" and "heterogeneity of expression" of tumor-derived mutant antigens), the same therapeutic effect as when labeling the aforementioned HBsAg-derived novel antigen can be achieved. Similar to vaccinia virus, oncolytic viruses can specifically infect tumor cells and not infect surrounding normal cells. Therefore, even when oncolytic viruses such as herpes simplex virus or adenovirus are used as delivery vectors for the accurate labeling of novel antigens derived from heterologous proteins, they can accurately label tumor cells with the novel antigens derived from heterologous proteins, thus achieving the same effect as oncolytic vaccinia virus.

[0020] The vaccine system of the present invention solves the problems of "low immunogenicity" and "heterogeneity of expression" of existing neotumor antigens by accurately labeling tumor cells with highly immunogenic heterogeneous proteins using oncolytic viruses and constructing highly immunogenic neotumor antigens. At the same time, the mRNA vaccine can pre-induce the generation of spontaneously labeled new antigen-specific T cells (not limited by time) in the living body. By utilizing the characteristics of accurately labeled highly immunogenic new antigens and memory T cells that are not limited by time, real tumors are killed rapidly and efficiently. This dual action activates the antitumor immune effect and improves the therapeutic response rate of tumor vaccines. Furthermore, memory T cells induced by the mRNA vaccine efficiently solve the problem of oncolytic viruses that cannot exert a strong antitumor immune effect due to limitations imposed by the host immune system. They rapidly identify and kill tumor cells before the living body's immune system can identify and eliminate the tumor, thereby activating the antitumor immune effect. Furthermore, recombinant oncolytic viruses are genetically deficient vaccinia viruses that replicate only within tumor tissue and not in normal tissue, thus enabling precise delivery of heterologous proteins.

[0021] Beneficial effects: Compared to the prior art, the present invention has the following remarkable advantages.

[0022] (1) The heterologous protein mRNA vaccine in the present invention induces a strong memory T cell response in the living body, and compared to commercially available recombinant protein vaccines, mouse spleen cells of the mRNA-H group, i.e., the heterologous protein HBsAg mRNA vaccine group, react with IFN-γ after antigen restimulation. + The cells increased 150-fold, and IFN-γ + CD8 + T cell counts increase at least 25-fold, effectively overcoming the tumor immunosuppressive microenvironment and improving the therapeutic response rate of the new antigen vaccine. This high response rate, combined with recombinant oncolytic virus, achieves a more precise and efficient therapeutic effect.

[0023] (2) When the vaccine system of the present invention was used in mouse B16F10 melanoma experiments, tumor growth in the treatment group was clearly suppressed, and according to the results of detection by flow cytometry, tumor infiltration CD8 in the treatment group was suppressed. + T cells and CD4 + T cells increased by at least 10 times and 4 times, respectively, and effector memory T cells (T) in the spleen EM ) also increases significantly.

[0024] (3) In three distinct types of solid tumors with different characteristics—B16F10-OVA melanoma, 4T1 breast cancer (low immunogenicity, high malignancy, and prone to spontaneous metastasis), and CT26 (immunocytic tumor)—we successfully labeled a novel antigen derived from a heterologous protein using the recombinant oncolytic virus VV-H of the present invention, activated specific memory T cells, synergistically enhanced the effect, and obtained therapeutic effects for solid tumors with significant differences. [Brief explanation of the drawing]

[0025] [Figure 1] This is a process flowchart and explanation of the background technology. [Figure 2] This is a flowchart illustrating the application of the vaccine system of the present invention to tumor immunotherapy. Steps a-b are steps in which mice are immunized with the mRNA-H vaccine (intramuscular injection) to induce the generation of HBsAg memory T cells in the body; step c is a step in which VV-H is injected into the tumor to specifically label tumor cells with the new HBsAg antigen; step d is a step in which the new HBsAg antigen induces the activation of memory T cells, rapidly killing tumor cells and releasing tumor-specific antigens; step e is a step in which epitope spreading occurs; and step f is a step in which a systemic anti-tumor immune effect is induced. [Figure 3] This is a map of the pUC-HBsAg recombinant plasmid. [Figure 4] These are the average particle size, surface potential, and average dispersion coefficient of LNP HBsAg mRNA. [Figure 5] These are the quantitative results of serum HBsAb levels after the first immunization with mRNA-H vaccine (a) and 7 days after the second immunization (b). [Figure 6] These are the results of characterizing HBsAg memory T cells in the spleen after immunization with HBsAg vaccine. a-b are the quantitative results of the number of IFN-γ+ cells after stimulation with an HBsAg peptide library using the ELISPOT method, and c-d are the gating policy and quantitative results of flow cytometry for detecting IFNγ+CD8+ T cells after restimulation with an HBsAg peptide library. [Figure 7] This is a study verifying the preventive and therapeutic effect of mRNA-H vaccines on B16-HBsAg tumors. 'a' shows the results of monitoring tumor volume (n=5), 'b' shows the results of monitoring body weight of tumor-bearing mice during the treatment period (n=5), and 'c' shows the statistical results of mouse survival rates after immunization with different vaccines (n=6). HepB-CHO is a commercially available recombinant HBsAg hepatitis B vaccine. [Figure 8] The results of evaluating the immunogenicity of the new antigen HBsAg are shown, representing tumor volume and body weight monitoring results (n=10) for B16-HBsAg, B16-OVA, and B16F10, respectively. [Figure 9] This is an evaluation of the epitope spreading effect by the novel antigen HBsAg. The number of IFN-γ+ cells was quantified by the ELISPOT method after stimulating B16-HBsAg tumor-carrying mice with a melanoma peptide library. [Figure 10] This shows the plasmid map and gene construction process of an oncolytic virus. A is the plasmid map of vaccinia virus, and B is the gene construction process of the said virus. [Figure 11] This is a red fluorescence image taken when recombinant viruses were screened and cultured using Hela-s3 cells. [Figure 12] This image shows the results of identifying the expression of target genes in recombinant oncolytic vaccinia virus using the PCR method. PCR was performed using HBsAg and GM-CSF gene primers; the left image shows HBsAg, and the right image shows GM-CSF. [Figure 13]This histogram shows the results of evaluating the ability of VV-H to induce HBsAg and GM-CSF expression in B16F10 tumor cells using the ELISA method. [Figure 14] This is the result of identifying TK gene expression using gDNA from recombinant oncolytic vaccinia virus after plaque purification. [Figure 15] This is the result of measuring the HBsAg content in Hela-s3 tumor cells at 24h, 48h, and 72h after infection with VV-H at different MOIs using the ELISA method. [Figure 16] This invention provides a therapeutic protocol for activating antitumor immune effects using the vaccine system of the present invention, where sc indicates subcutaneous injection and it indicates intratumor injection. [Figure 17] This is an evaluation of the therapeutic effect of HBsAg memory T cells activated by VV-H on melanoma (B16F10). a is the result of monitoring tumor volume, b is the result of measuring the body weight of tumor-bearing mice, c-d are the results of quantitative analysis of CD3+CD8+ and CD3+CD4+ T cells in the tumor (Gate:CD45+cell,cd), and e is a representative flow cytometry gating policy. [Figure 18] This is an evaluation of the therapeutic effect of HBsAg memory T cells activated by VV-H against B16-OVA. a-c are the results of monitoring tumor volume and body weight, d is the tumor-bearing status of mice at the end of treatment, e is the measurement result of tumor weight of tumor-bearing mice, and f-g are the results of quantitative analysis of CD3+CD8+ and CD3+CD4+ T cells in tumor tissue. [Figure 19] This is an evaluation of the therapeutic effect of combining HBsAg memory T cells activated by VV-H with the immune checkpoint inhibitor PD-L1 on colorectal cancer (CT26). 'a' shows the results of tumor volume monitoring, and 'b' shows the results of body weight measurement of tumor-bearing mice. [Figure 20]This is an evaluation of the therapeutic effect of HBsAg memory T cells activated by VV-H on breast cancer (4T1). 'a' shows the results of tumor volume monitoring, 'b' shows the statistical results of mouse survival rates, and 'c' shows the statistical results of mouse tumor recurrence rates. [Figure 21] This is an immunofluorescence image showing the results of in vivo induction of HBsAg expression in tumor cells using VV-H. After injecting VV-H into the tumor, tumor tissue was excised, and immunofluorescence staining for HBsAg (HBsAg: green) and VV (mcherry, the red fluorescence of the virus itself) were performed. [Figure 22] This is the safety evaluation result after combination therapy with VV-H + mRNA-H. 'a' shows the results of measuring serum biochemical indicators after treatment, including alanine aminotransferase (ALT), total protein (TP), creatine kinase isozymes (CKI), and blood urea nitrogen (BUN). 'b' shows the HE staining results for major organs after treatment. [Modes for carrying out the invention]

[0026] The technical aspects of the present invention will be further explained below with reference to the drawings.

[0027] Example 1 Manufacturing of heterologous protein mRNA vaccines 1. mRNA-H construction: (1) Construction, synthesis, and amplification of mRNA vaccine recombinant plasmids The HBsAg, T7 promoter, and UTR sequence were conjugated to a vector to construct the pUC-HBsAg recombinant plasmid. Plasmid mapping was performed by GeneWith, Inc., to synthesize E. coli containing the recombinant plasmid, as shown in Figure 3. An appropriate amount of E. coli suspension containing the HBsAg plasmid was added and incubated overnight in a 37°C incubator. Three monoclonal plaques were placed in centrifuge tubes containing 5 mL of culture medium and incubated at 37°C at 220 rpm for 14-18 hours. The pHBsAg plasmid was extracted and quantified.

[0028] (2) HBsAg plasmid PCR, purification and identification After adding the PCR reaction system and running the PCR program, a 1% agarose gel was prepared, the PCR product was mixed in, three times the volume of Buffer DC was added and mixed further, and the mixture was transferred to an adsorption column. Centrifugation was performed at 12000 rpm for 1 minute at room temperature, and the filtrate was discarded. Buffer WB was added to the adsorption column and allowed to stand for 2 minutes. Centrifugation was performed at 12000 rpm for 30 seconds at room temperature, and the filtrate was discarded. Sterile water was dropped onto the center of the adsorption column, and the sample was collected by centrifugation.

[0029] (3) In vitro transcription (IVT) and capping The IVT reaction system was added and thoroughly mixed, then the reaction was carried out in a PCR instrument at 37°C for 2 hours. DNase I was added, and sterile water was added to the PCR product to 50 μL. The mixture was mixed by pipetting, and the PCR reaction was carried out at 37°C for 15 minutes to remove the DNA template. RNA cleanup binding buffer was added to 100 μL of the sample, followed by ethanol, and mixed by pipetting. The liquid was transferred to a purification column, centrifuged at 12000 rpm at room temperature for 1 minute, and the filtrate was discarded. RNA cleanup wash buffer was added, and the column was centrifuged at 12000 rpm at room temperature for 1 minute. The above steps were repeated once, and the column was transferred to an RNase-free centrifuge tube. An appropriate amount of sterile water was added, and after standing for 5 minutes, the sample was collected by centrifugation. Quantitative dilution was performed, and agarose gradient electrophoresis was carried out.

[0030] 2. Manufacturing of heterologous protein mRNA vaccines Each lipid component was mixed according to the molar ratio of Lipid / DSPC / Cholesterol / PEG-DMG = 50 / 10 / 38.5 / 1.5. Then, each Lipid solution was mixed with anhydrous ethanol to prepare a 2.85 mg / mL Lipid mixture. An aqueous phase solution containing mRNA was prepared. 100 μg of mRNA was diluted in 2 mL of 10 mM citrate-sodium citrate buffer to obtain an mRNA dilution. mRNA LNPs were prepared by passing the aqueous phase containing mRNA and the above alcohol phase containing lipids through a microfluidic device at a flow rate ratio of 3:1. After ultrafiltration centrifugation, the particle size and zeta potential of the mRNA LNPs were measured.

[0031] As shown in Figure 4, the physical parameters of the heterologous protein mRNA vaccine mRNA-H were appropriate and favorable for entering cells and expressing HBsAg. The plasmid nucleotide sequence of HBsAg mRNA was SEQ ID NO.1.

[0032] Example 2 Detection of anti-HBsAg antibody (HBsAb) in serum after mRNA-H immunization. The immunizing effects of a prepared mRNA-H vaccine and a commercially available HBsAg hepatitis B vaccine (HepB-CHO) were preliminarily evaluated by detecting HBsAb in serum. Six-week-old C57BL / 6 mice were randomly divided into three groups: 1. Saline (physiological saline), 2. mRNA-H vaccine group, and 3. HepB-CHO group. After group division, the mice were immunized twice by intramuscular injection on day 0 and day 14. Serum HBsAb levels were measured seven days after each immunization.

[0033] As shown in Figure 5, on day 7 after the second immunization, serum HBsAb levels in the mRNA-H group were much higher than in the blank group (HBsAb ≥ 10 mIU / mL was sufficient to prevent hepatitis B).

[0034] Example 3 Detection of the ability of mRNA-H vaccines to induce the formation of splenic memory T cells. ELISPOT method: Mice were immunized using the method of Example 3. On day 30 after the final immunization, spleens were isolated under sterile conditions to prepare a single-cell suspension. The suspension was centrifuged at 350g for 5 min, and the supernatant was discarded. 1 mL of erythrocyte lysate was added for lysis for 10 min, then 3 mL of buffer was added to stop erythrocyte lysis, followed by centrifugation at 350g for 5 min to collect cells. 4 mL of 1640 medium was added to resuspend the cells, the cell count was determined, and the cells were adjusted to 2×10 6 / mL. Activation of pre-coated plates: RPMI-1640 medium (5% FBS) was added at 200 μL / well, allowed to stand for 10 min, then discarded. ELISPOT plating was performed. The negative control consisted of 100 μL of cells + 5 μL of PBS. The HBsAg protein group consisted of 100 μL of cells + 10 μL of HBsAg epitope peptide library. The positive group consisted of 100 μL of cells + 10 μL of positive stimulant. After addition, the plate was covered and incubated for 16-24 h. After the incubation, spots were detected and statistical analysis was performed. In addition, flow cytometry was used to verify the ability of memory T cells in the spleen to secrete IFN-γ after stimulation with the HBsAg epitope antigen library.

[0035] As shown in Figure 6, compared with the recombinant HBsAg vaccine group, the HBsAg-specific memory T cells induced by the mRNA-H vaccine had at least 150-fold more IFN-γ + CD8 + T cell numbers increased by at least 25-fold.

[0036] Example 4 Evaluation of the preventive effect on B16-HBsAg tumor development by memory T cells induced by mRNA-H Six-week-old C57BL / 6 female mice were randomly divided into three groups: 1. Blank, 2. mRNA-H, and 3. HepB-CHO. The mice were immunized according to the method of Example 3. In addition, B16F10 tumors expressing HBsAg (B16-HBsAg) were constructed using slow viruses, and B16-HBsAg was inoculated 30 days after final immunization. The condition of the mice was observed, and tumor volume was monitored. Survival statistics for duplicate treatments were also performed.

[0037] As shown in Figure 7, after treatment completion, all mice in the mRNA-H group were tumor-free, and the average tumor volume of mice immunized with the control group (Saline group) and HepB-cho was 1000 mm². 3 The threshold was exceeded. Furthermore, there was no significant change in the body weight of mice treated with mRNA-H. In addition, all control mice died on day 26 due to excessive tumor burden, but all mRNA vaccine-immunized mice survived until day 33 and showed no tumor growth.

[0038] The therapeutic effect of mRNA-H vaccine-induced memory T cells against B16-HBsAg tumors was investigated using a B16-HBsAg tumor model. The results showed that mice immunized with the mRNA-H vaccine had 100% prevention of B16-HBsAg tumor growth, compared to 0% in the control group. Furthermore, there was no significant change in the body weight of the mRNA-vaccinated mice, indicating good safety. The results suggest that heterologous protein (HBsAg) memory T cells can completely eliminate B16-HBsAg tumors, providing strong evidence for the subsequent killing of tumors expressing novel antigens derived from heterologous proteins.

[0039] Example 5 Immunogenicity evaluation of the new antigen HBsAg Six-week-old C57BL / 6 female mice were divided into three groups: 1. B16-HBsAg, 2. B16-OVA, and 3. B16-Neo. Each group was immunized with mRNA-H, mRNA-OVA, and mRNA-Neo, respectively, and after two immunizations, B16-HBsAg, B16-OVA, and B16F10 cells were inoculated one week after the final immunization. The mice's condition was observed, and tumor volume was measured.

[0040] As shown in Figure 8, the B16-HBsAg group significantly suppressed tumors, demonstrating that mRNA-H can effectively activate memory T cells and kill tumors by labeling the novel HBsAg antigen. Simultaneously, it was shown that heterologous proteins derived from microorganisms possessed higher immunogenicity than the novel antigen and other highly immunogenic proteins, enabling rapid recognition and killing by memory cells.

[0041] Example 6 Evaluation of epitope spreading of the novel antigen HBsAg According to the method of Example 5, the average tumor volume of the B16-Neo group was 2000 mm². 3 When the target was reached, the spleen of the B16-HBsAg group of mice was removed under sterile conditions, prepared as a single-cell suspension, and 350g was centrifuged for 5 minutes. The supernatant was discarded, 1 mL of erythrolysis solution was added and the cells were decomposed for 10 minutes, then 3 mL of buffer was added to stop erythrolysis, and 350g was centrifuged for 5 minutes to collect the cells. 4 mL of 1640 medium was added to resuspend the cells, and the number of cells was counted to 2 × 10⁶. 6 The solution was adjusted to the recommended concentration of PBS / mL. ELISPOT plating was performed. Specifically, the following was done: The negative control was 100 μL of cells + 100 μL of PBS. The HBsAg protein group was 100 μL of cells + 100 μL of HBsAg epitope peptide library. The M30 group was 100 μL of cells + 100 μL of M30 peptide. The M27 group was 100 μL of cells + 100 μL of M27 peptide. After addition, the plate was covered and incubated for 16-24 hours. After incubation was complete, the spots were measured and statistical analysis was performed.

[0042] As shown in Figure 9, tumors labeled with the novel HBsAg antigen induced and amplified a secondary antigen immune response under sustained killing of memory T cells, effectively achieving epitope spreading and contributing to overall therapeutic outcomes.

[0043] Example 7 Construction and characterization of recombinant oncolytic viruses expressing novel antigens: (1) Construction and synthesis of HBsAg shuttle plasmid VACWR093(TK-R) and VACWR095(TK-L), which are adjacent sequences to TK(VACWR094), were used as homologous regions. mCherry (fluorescent group) and xanthine-guanine phosphoribosyltransferase (gpt) were used as screening genes. HBsAg expression was induced using the Pse / l promoter, and the vector's drug resistance was set to AmpR (ampicillin), and the pVV-mcherry-HBsAg plasmid was constructed. In addition, a blank vector of vaccinia virus VV-C and vaccinia virus VV-G expressing GM-CSF (granulocyte-macrophage colony-stimulating factor) (at the same position as HBsAg on the plasmid) were constructed and their synthesis was commissioned to GenScript. The plasmid map is shown in Figure 10. The nucleotide sequence of the recombinant viral plasmid was SEQ ID NO.2.

[0044] (2) Construction and screening of HBsAg-expressing recombinant oncolytic virus (VV-H) 293T cells were cultured, detached by trypsin treatment, and counted. 5 × 10⁶ cells were then measured. 5Cells were seeded in a 6-well plate at a rate of cells / well and cultured at 37°C under 5% CO2 conditions. When the cells reached 90% confluence, they were infected with wild-type avirus (VV) at an MOI of 1. After 2 hours of infection, the medium was changed to DMEM, and a plasmid of 1 μg / well was transfected. Red fluorescence was observed and recorded 24 to 72 hours after transfection. The culture supernatant was removed, 500 μL of DMEM medium without FBS was added, and the cells were collected in a centrifuge tube by pipetting. After three freeze-thaw cycles at -80°C / 37°C, the cells were centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected. Hela-S3 cells were seeded in a 6-well plate, and after reaching 90% confluence, 10 μL and 100 μL of virus were added to three wells each. After 2 hours, the supernatant was removed, and screening culture was performed using a screening medium containing xanthine, hypoxanthine, and mycophenolic acid. mCherry fluorescence was observed 24–72 hours after adding the screening medium, and the fluorescence images are shown in Figure 11. After 72 hours, the screening medium was carefully removed, 500 μL of DMEM medium was added to collect the cells, and the supernatant was collected by centrifugation. The other steps were the same procedure. Recombinant viruses were screened for 3–4 rounds, and the screening results are shown in Figure 11.

[0045] 10 μL and 100 μL of the virus after screening were used to infect cells in two 6-well plates, respectively. DNA was extracted from the infected cells, and the presence of recombinant viruses was identified by PCR using target gene primers. The results are shown in Figure 12. Simultaneously, the target proteins in the cell degradation products were detected using an ELISA kit, and the results are shown in Figure 13. VV-H and VV-G were able to induce the expression of HBsAg and GM-CSF, respectively, in tumor cells. After identification at the genomic DNA level and protein level was completed, recombinant viruses were amplified using 1× screening medium.

[0046] (3) Plaque purification of HBsAg recombinant oncolytic virus (VV-H) Hela-S3 cells were seeded and infected with the virus in the same manner as described above. Plaque purification was performed by adding various volumes of virus solution to 1× screening medium to remove wild-type virus. After 24 hours, fluorescence was observed, and in wells with an appropriate number of plaques, the medium was replaced with 2× screening medium (1 mL), and 1 mL of 2% low-melting-point agarose was added as a top layer. The cells were then cultured in a 37°C, 5% CO2 incubator. Based on the fluorescence observation results after 24 hours, Hela-S3 cells were plated in a 24-well plate (24-well plate, 10 wells, 5×10). 4 Cells were seeded in 500 μL of DMEM containing 2% FBS per well and cultured until 90% confluence was achieved after 48 hours. After 48 hours, a single plaque was collected using a 200 μL sterile tip and transferred to a 24-well plate for continued culture. The formation of fluorescent plaques was observed over time until the cells were fully confluent. The supernatant was removed, 200 μL of DMEM medium without FBS was added, and after repeated pipetting, the virus in the supernatant was recovered by centrifugation. The steps were the same as described above. Thymidine kinase (TK) primers were used to identify whether the selected recombinant viruses contained wild-type viruses, and the procedure was repeated until the solution did not contain wild-type viruses, as shown in Figure 14. As a result, the selected recombinant viruses did not contain wild-type viruses.

[0047] (4) In vitro amplification and purification of recombinant oncolytic virus VV-H Prepare Hela-S3 (cervical cancer) cells in multiple petri dishes, and after trypsin treatment, approximately 10 8The number of cells was counted. The recombinant virus (MOI=0.1) and cell mixture was seeded into 20 15cm petri dishes, and viral amplification was performed. Fluorescence expression was observed during the amplification process. When the fluorescence intensity reached its maximum, the cell and virus solution was transferred to a 50mL centrifuge tube and centrifuged at 2000g for 10min. The supernatant was discarded, and the cells were resuspended with 10mM Tris-HCl. The cell suspension was frozen and thawed three times at -80 / 37°C, centrifuged at 2000rpm for 5min, and the supernatant was collected. After adding 1 / 3 volume of trypsin to the supernatant and mixing, it was incubated in a 37°C water bath for 30min. Preparation of 36% sucrose: 13mL of 36% sucrose was added to an ultracentrifuge tube, and an equal volume of virus solution was gently overlaid on top of the sucrose solution. 32900g was centrifuged at 4°C for 2h using an ultracentrifuge. The supernatant was carefully discarded, and each tube was resuspended in 5 mL of PBS, with additional PBS added to supplement. The 32900 g was again ultracentrifuged at 4°C for 1 hour. The supernatant was discarded, and the virus pellet was resuspended in 1 mL of PBS in each tube. The virus solution was dispensed and stored at -80°C to obtain recombinant vaccinia virus.

[0048] Recombinant vaccinia virus titer measurement (TCID) 50 ) Hela-S3 cells were placed in a 96-well plate. 4 Seeds were seeded in cells / well. 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 A viral gradient was set, and 6 wells were repeated for each viral gradient, with 3 independent experiments performed for each virus. Fluorescent plaques were observed in each well, and the formula T = 0.7 × 10 × 10 was used. (1+S(d-0.5)) The viral titer was calculated using the following method: S = Log dilution - 1, and d = sum of positive rates.

[0049] Detection of HBsAg labeling ability of VV-H using an ELISA kit Hela-S3 tumor cells were infected with viruses having three MOI gradients of 0.01, 0.1, and 1. Cells were centrifuged and collected at 24, 48, and 72 hours post-infection. The cells were frozen and thawed three times at -80°C / 37°C, and the supernatant was collected after centrifugation at 2000 rpm for 10 minutes. HBsAg content was measured using an ELISA kit, and absorbance at OD450 nm was measured using a plate reader. Expression results are shown in Figure 15.

[0050] Example 8 Evaluation of the tumor therapeutic effect of VV-H by activating its specific memory T cells. The therapeutic effects were evaluated using four types of tumors: B16F10 melanoma (rapid growth rate, high malignancy), CT26 colorectal cancer (immunocytic tumor with strong tumor heterogeneity), 4T1 breast cancer (low mutational burden, spontaneous metastasis), and B16-OVA. Specific treatment methods are shown in Figure 16. Tumor cells were subcutaneously inoculated into a control group and C57BL / 6 or Balb / c mice 30 days post-immunization to construct B16F10, CT26, and 4T1 tumor models, respectively. Tumor volume was 50-100 mm². 3 If the tumors grew, these tumor-bearing mice were randomly divided into 5 groups. On day 0 (the day of group division), 3, and 5, 50 μL of each virus solution was administered into the tumor. 7 PFU (Psycholytic Fury) was injected into the mice. The results are shown in Figures 17-20 (Figure 17: B16F10 subcutaneous tumor model, Figure 18: B16-OVA subcutaneous tumor model, Figure 19: CT26 subcutaneous tumor model, Figure 20: 4T1 subcutaneous tumor model). VV-H+mRNA-H significantly suppressed tumors compared to the control group, demonstrating that VV-H+mRNA-H can effectively activate memory T cells and kill tumors by labeling them with the novel HBsAg antigen. Furthermore, there were no significant changes in the body weight of the mice in each group, indicating that this treatment has good safety.

[0051] In addition, in the 4T1 tumor model, the treated tumor was resected 30 days after the final treatment, and the mouse's body weight change was monitored. At the end of treatment, the survival curve and lung metastasis status were statistically analyzed, and as shown in Figure 20, this treatment policy was able to effectively delay the ability of the tumor to metastasize distally.

[0052] Example 9 Evaluation of the therapeutic effect of melanoma on HBsAg memory T cells activated by VV-H. After treatment of the B16F10 melanoma model and the B16-OVA subcutaneous tumor model in Example 7, the treated tumor and spleen were dissected, and single-cell suspensions were prepared. Subsequently, the tumor single-cell suspensions were stained on ice for 30 minutes with a mixture of CD45, CD3, CD4, and CD8 antibodies and a Live / Dead dye, and the T cell content was measured by flow cytometry.

[0053] As shown in Figure 17, in the B16F10 melanoma model, tumors in mice in the VV-H+mRNA-H group showed a higher CD8 count compared to the mRNA-H vaccine group. + T is at least 10 times, CD4 + The study showed that T cells increased at least fourfold, and that VV-H+mRNA-H efficiently degrades tumors, releases antigens, and further activates the anti-tumor immune response.

[0054] Similarly, as shown in Figure 18, in the B16-OVA subcutaneous tumor model, CD4 in the tumors of mice in the VV-H+mRNA-H group + CD8 + T levels increased significantly compared to other treatment groups, indicating that the VV-H+mRNA-H group achieved more effective immune infiltration.

[0055] Example 10 Evaluation of the ability of VV-H to induce HBsAg expression in in vivo tumors by immunofluorescence staining of sectioned tissue. A B16F10 tumor model was constructed by subcutaneously inoculating B16F10 cells into a control group and immunized C57BL / 6 mice. The tumor volume was 150 mm². 3 When the tumors grew to this stage, they were randomly divided into three groups (n=5): 1. VV-C, 2. VV-H, and 3. mRNA-H+VV-H. After dividing into groups, 50 μL of virus (10) was administered into each tumor. 7PFU (mouse) was injected, and the tumor was dissected on days 1, 3, and 7 after administration. Sections were prepared, and VV-H (mCherry, red) and the novel HBsAg antigen (FITC-HBsAg antibody, green) were observed.

[0056] As shown in Figure 21, a strong HBsAg signal was detected in the tumor after VV-H administration, demonstrating that VV-H can effectively label HBsAg.

[0057] Example 11 Safety evaluation of treatment with VV-H + mRNA-H During the above treatment, the mice's body weight was monitored daily to provide a preliminary assessment of safety. In addition, following the same treatment as in Example 8, major blood indicators such as red blood cells and white blood cells, as well as serum biochemical indicators such as alanine aminotransferase (ALT) and blood urea nitrogen (BUN), were measured on Day 1 and Day 14 after treatment. After the completion of treatment, the mice were euthanized, and the heart, liver, spleen, lungs, and kidneys were isolated and examined pathologically to further assess safety.

[0058] As shown in Figure 22, no significant changes were observed in serum biochemical indicators or major organ HE staining in VV-H+mRNA-H group mice, and preliminary confirmation of good safety was obtained.

Claims

1. A novel tumor antigen vaccine system that specifically labels heterologous proteins, A tumor antigen vaccine system that specifically labels heterologous proteins, characterized in that it consists of a heterologous protein mRNA vaccine and a recombinant oncolytic virus, wherein the heterologous protein mRNA vaccine induces the generation of specific memory T cells against a precisely labeled novel antigen, and the recombinant oncolytic virus is used to precisely label a novel antigen that is highly immunogenic against tumor cells.

2. The vaccine system according to claim 1, characterized in that the heterologous protein is one or more of the following: hepatitis B virus-derived protein, human papillomavirus-derived protein, EBV-derived protein, influenza virus-derived protein, tetanus toxoid protein, Staphylococcus aureus-derived protein, or flagellin from Salmonella typhi.

3. The vaccine system according to claim 1, characterized in that the oncolytic virus is one or more specific oncolytic viruses such as vaccinia virus, herpes simplex virus, or adenovirus.

4. The vaccine system according to claim 1, characterized in that the heterologous protein mRNA vaccine is produced by constructing, synthesizing, and amplifying an mRNA vaccine recombinant plasmid, and then encapsulating the mRNA vaccine recombinant plasmid with a lipid nanomaterial.

5. The vaccine system according to claim 1, characterized in that the recombinant oncolytic virus is produced by constructing an oncolytic virus that expresses a new antigen, and then performing plaque purification and amplification purification.

6. Use in a vaccine-based tumor immunotherapy as described in claim 1.

7. The use according to claim 6, characterized in that it is used to enhance tumor immunotherapy.

8. The use according to claim 6, characterized in that the order of administration is to administer a heterologous protein mRNA vaccine first, followed by the administration of recombinant oncolytic virus.

9. The use according to claim 8, characterized in that, in the administration described above, the heterologous protein mRNA vaccine induces immune memory by intramuscular injection, intranasal administration, or intravenous administration, and the recombinant oncolytic virus specifically labels tumor cells with a highly immunogenic neo-tumor antigen by intratumoral administration.

10. The use according to claim 6, characterized in that the tumor is a solid tumor or a metastatic tumor associated with a solid tumor, and includes melanoma, colorectal cancer, breast cancer, liver cancer, glioma, bile duct cancer, or pancreatic cancer.

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