Tumor neoantigen vaccine system specifically marking heterologous protein, and use thereof

Abstract

The present invention provides a tumor neoantigen vaccine system specifically marking a heterologous protein, and a use thereof. The vaccine system is composed of a heterologous protein mRNA vaccine and a recombinant oncolytic virus; the heterologous protein mRNA vaccine is used to induce an organism to generate heterologous protein-specific memory T cells, and the recombinant oncolytic virus is used to promote a tumor to express microorganism-derived tumor neoantigens. When pre-immunized memory T cells detect these marked specific neoantigens, the tumor expressing the neoantigens is quickly activated and killed, the release of tumor autoantigens is promoted, dendritic cells phagocytize antigens to further initiate an anti-tumor response, and a systemic anti-tumor immune effect is activated by means of antigenic epitope spreading.

Description

A tumor neoantigen vaccine system specifically labeling heterologous proteins and its application Technical Field

[0001] The present invention relates to an anti-tumor vaccine system, in particular to a tumor neoantigen vaccine system that specifically labels heterologous proteins, and also to the application of the above vaccine system. Background Art

[0002] Immunotherapy, a milestone in the history of cancer treatment, has greatly changed the landscape of clinical cancer treatment. Due to its unique tumor targeting, tumor neoantigen vaccines have become a research hotspot in immunotherapy. Neoantigens, also known as tumor-specific antigens, are expressed only in tumors and not in normal tissues. By activating the patient's immune system, they can produce tumor-specific T cells to inhibit tumor growth. Although compared with conventional tumor-associated antigens, tumor neoantigens have advantages such as high tumor specificity, the results of multiple clinical trials at home and abroad have shown that tumor neoantigen vaccines have poor response rates for the treatment of various solid tumors. For example, the personalized tumor neoantigen vaccine RO7198457 developed by BioNTech has an objective response rate of only 8% in the treatment of various solid tumors including melanoma, breast cancer, and colorectal cancer. Existing tumor neoantigens still have the following problems: low immunogenicity and extremely differential expression, and TIME inhibition of tumor neoantigen vaccine treatment response.

[0003] Oncolytic viruses are a new type of therapy for tumor treatment. 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 often lower than that of viral vector-derived proteins, the body can produce a strong antiviral response, inhibiting viral replication and oncolysis, but failing to produce a strong tumor treatment effect. Although oncolytic viruses that express immune checkpoint inhibitors such as PD-1, DC cell-activating factors such as GM-CSF, and cytokines such as IL-2 can enhance the anti-tumor immune effect of oncolytic viruses to a certain extent, they still rely on the de novo initiation of tumor-derived antigens. Therefore, they are still limited by the host immune system's inhibitory effect on the virus and are unable to produce a significant tumor treatment effect.

[0004] Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a highly efficient tumor neoantigen vaccine system that specifically labels heterologous proteins, utilize memory T cells generated by mRNA vaccines to recognize and kill tumor cells precisely labeled with neoantigens derived from heterologous proteins, thereby activating systemic anti-tumor immunotherapy effects, and provide applications of the above-mentioned vaccine system.

[0006] Technical solution: The tumor neoantigen vaccine system of the present invention specifically marks heterologous proteins. The vaccine system is composed of a heterologous protein mRNA vaccine and a recombinant oncolytic virus. The heterologous protein mRNA vaccine produces specific memory T cells of precisely marked neoantigens, and the recombinant oncolytic virus precisely marks highly immunogenic neoantigens to tumor cells.

[0007] Preferably, the heterologous protein is one or more of hepatitis B virus-derived protein (HBsAg), human papillomavirus-derived protein (E6, E7), EBV-derived protein (PP65), influenza virus-derived protein (MP), tetanus toxoid protein (TT), Staphylococcus aureus-derived protein (C2) or flagellin of Salmonella typhi.

[0008] Preferably, the oncolytic virus is one or more of vaccinia virus, herpes simplex virus or adenovirus.

[0009] Preferably, the heterologous protein mRNA vaccine is prepared by constructing, synthesizing and amplifying an mRNA vaccine recombinant plasmid, and then encapsulating the mRNA vaccine recombinant plasmid with lipid nanomaterials (LNP).

[0010] Preferably, the recombinant oncolytic virus is prepared by constructing a recombinant oncolytic virus expressing a new antigen, and performing plaque purification and amplification purification.

[0011] Application of the above vaccine system in the preparation of tumor immunotherapy drugs.

[0012] Preferably, it is used to enhance tumor immunotherapy; a heterologous protein mRNA vaccine is used to induce the body to produce heterologous protein-specific memory T cells, and a recombinant oncolytic virus is used to induce the tumor to express microbial-derived tumor neoantigens. When the pre-immunized memory T cells detect specific neoantigens, they are rapidly activated and kill the tumor expressing the neoantigen, promoting the release of tumor antigens. Dendritic cells (DCs) phagocytize the antigens to further initiate anti-tumor responses and activate systemic anti-tumor immune effects.

[0013] Preferably, the order of administration is to first administer the heterologous protein mRNA vaccine and then administer the recombinant oncolytic virus, or to administer the heterologous protein mRNA vaccine and the recombinant oncolytic virus at the same time.

[0014] Preferably, the heterologous protein mRNA vaccine can produce immune memory in the body through intramuscular injection, intranasal or intravenous administration, and the recombinant oncolytic virus can accurately mark highly immunogenic tumor neoantigens to tumor cells through intratumoral or intravenous administration.

[0015] Preferably, the tumor is a solid tumor or a solid tumor with metastatic disease, including melanoma, colorectal cancer, breast cancer, liver cancer, glioma, bile duct cancer or pancreatic cancer.

[0016] Principle of the invention: The present invention addresses the problems of poor immunogenicity of neoantigens, tumor heterogeneity (temporal and spatial differences in neoantigen expression within tumors) and TIME limitations that lead to poor responses to neoantigen vaccine treatment, and constructs a tumor neoantigen vaccine system consisting of a heterologous protein mRNA vaccine and a recombinant oncolytic virus that specifically labels heterologous proteins. On the one hand, by independently constructing tumor neoantigens derived from heterologous proteins with strong immunogenicity, the problems of low immunogenicity and expression heterogeneity of neoantigens derived from existing tumor gene mutations are solved; on the other hand, the mRNA vaccine of heterologous proteins is used to stimulate the body to produce neoantigen-specific memory T cells to break through the TIME limitation, thereby activating the anti-tumor immune effect in a two-pronged manner. It is hoped that by independently constructing highly immunogenic tumor neoantigens, inducing neoantigen memory T cells to break through TIME, and improving the response rate of neoantigen vaccine treatment, the following three problems of the prior art will be solved:

[0017] (1) Solve the problem of low immunogenicity and differential expression of existing tumor neoantigens: Use gene-defective oncolytic viruses to specifically mark tumor cells with new antigens derived from heterologous proteins that are highly safe and immunogenic. The low immunogenicity and differential expression of tumor mutation-derived neoantigens are key bottlenecks that lead to poor response rates in solid tumor treatment with neoantigen vaccines and need to be solved urgently. This project utilizes the characteristic that gene-defective oncolytic viruses only replicate in tumors to specifically mark tumor cells with new antigens derived from heterologous proteins that are highly safe and immunogenic, in order to solve the problem of low immunogenicity and differential expression of existing mutation-derived neoantigens. More importantly, the heterologous protein constructed by the present invention has known strong immunogenic T cell epitopes, and there is no need for time-consuming and costly processes such as neoantigen sequencing and screening, and can be quickly used for tumor treatment;

[0018] (2) Solve the problem of TIME's inhibition of tumor neoantigen vaccine treatment response: Use mRNA vaccines to induce the body to produce neoantigen-specific memory T cells (not limited by TIME) and specifically labeled neoantigens to quickly and efficiently kill tumors and activate anti-tumor immune effects. Even if tumor mutation-derived neoantigen-specific T cells are generated, TIME can still make these neoantigen T cells unable to recognize and kill tumors by exhausting neoantigen-specific T cells. In contrast, immune memory T cells, especially heterologous protein-specific memory T cells, have a low activation threshold when recognizing antigens and are not limited by TIME. The present invention utilizes the characteristics of heterologous protein-specific memory T cells that are not limited by TIME and specifically labeled heterologous protein-derived neoantigens to quickly and efficiently kill tumors and release tumor self-antigens (epitope expansion), further activating anti-tumor immune effects and improving the response rate of neoantigen vaccine treatment;

[0019] (3) Solve the scientific problem of poor anti-tumor immune effect activated by oncolytic viruses: In theory, oncolytic viruses can eliminate tumors by lysing tumor cells, releasing tumor antigens, and activating anti-tumor immune responses. However, since the immunogenicity of tumor antigens is often lower than that of viruses, the body produces a stronger anti-viral immune response, which further limits the oncolytic effect of the virus, lysing tumors and releasing less antigens, resulting in poor anti-tumor immune effects. Oncolytic viruses expressing PD-L1, GM-CSF, new antigens, etc. can enhance anti-tumor immune responses to a certain extent, but still rely on the immune initiation of tumor antigens. Therefore, it is still limited by the host immune inhibition of the virus. To solve the above problems, the applicant, on the one hand, improves tumor immunogenicity by delivering tumor new antigens derived from heterologous proteins; on the other hand, uses mRNA-H vaccines to pre-induce the body to produce a large number of new antigen memory T cells. These heterologous protein-specific memory T cells can quickly and efficiently kill tumors expressing new antigens, release tumor antigens, and activate anti-tumor immune effects. Therefore, the present invention can also solve the problem of poor anti-tumor immune effects of oncolytic viruses due to host immune restrictions.

[0020] Since most microbial-derived proteins, such as human papillomavirus proteins E6 and E7, EBV virus-derived proteins PP65, influenza virus-derived proteins, tetanus toxoid proteins, Staphylococcus aureus-derived proteins, or flagellin from Salmonella typhi, are also heterologous microbial proteins with known strong immunogenic epitopes, they are more immunogenic than neoantigens derived from single tumor gene mutations. Therefore, the mRNA vaccines corresponding to these proteins (generating memory T cells derived from the heterologous protein-derived neoantigens, breaking through the TIME limit) are combined with oncolytic viruses to accurately mark the neoantigens to tumor cells (solving the problems of low immunogenicity and differential expression of tumor-derived mutant antigens), which can achieve therapeutic effects similar to those of the above-mentioned labeled HBsAg-derived neoantigens. Similar to vaccinia viruses, oncolytic viruses can specifically infect tumor cells without infecting other normal cells around them. Therefore, the use of oncolytic viruses such as herpes simplex virus or adenovirus as delivery vehicles for precisely marking heterologous protein-derived neoantigens can also have similar effects to oncolytic vaccinia viruses, because they can accurately mark tumor cells with heterologous protein-derived neoantigens.

[0021] The vaccine system of the present invention uses oncolytic viruses to accurately label tumor cells with highly immunogenic heterologous proteins, constructs highly immunogenic tumor neoantigens, solves the problems of low immunogenicity and differential expression of existing tumor neoantigens, and can also use mRNA vaccines to pre-stimulate the body to produce autonomously labeled neoantigen-specific T cells (not limited by TIME). By utilizing the characteristics of precisely labeled highly immunogenic neoantigens and their memory T cells not being limited by TIME, solid tumors are killed quickly and efficiently, and the anti-tumor immune effect is activated in a two-pronged manner, thereby improving the therapeutic response rate of tumor vaccines. In addition, the memory T cells produced by the mRNA vaccine body efficiently solve the problem that oncolytic viruses cannot activate strong anti-tumor immune effects due to host immune restrictions. Before the body's immune system recognizes and eliminates the tumor, it quickly identifies, kills tumor cells and activates anti-tumor immune effects, and the recombinant oncolytic virus is a genetically defective vaccinia virus that only replicates in tumors and not in normal tissues, so it can achieve precise delivery of heterologous proteins.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] (1) The heterologous protein mRNA vaccine of the present invention can stimulate the body to produce a stronger memory T cell response. Compared with the recombinant protein vaccine on the market, the spleen cells of mice in the mRNA-H group, i.e., the heterologous protein HBsAg mRNA vaccine group, were stimulated by antigen restimulation and IFN-γ expression was higher than that in mice in the mRNA-H group. + Cells increased 150-fold, IFN-γ + CD8 + The number of T cells increased by at least 25 times, effectively overcoming the tumor's immunosuppressive microenvironment and improving the response rate of neoantigen vaccine treatment. The extremely high response rate combined with the recombinant oncolytic virus achieved a more precise and efficient treatment effect;

[0024] (2) In the mouse B16F10 melanoma experiment, the vaccine system of the present invention significantly inhibited the tumor growth in the treatment group; and the flow cytometry results showed that the CD8 + T cells and CD4 + T cells increased by at least 10-fold and 4-fold, respectively, and the effector memory T cells (T EM ) also reached a significant increase;

[0025] (3) In three different levels of solid tumors, namely B16F10-OVA melanoma, 4T1 breast cancer (low immunogenicity, high malignancy, and easy to spontaneously metastasize), and CT26 (immune cold tumor), the recombinant oncolytic virus VV-H of the present invention successfully labeled new antigens derived from heterologous proteins and activated their specific memory T cells, resulting in synergistic enhancement and producing a significant difference in the treatment effect of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a background technology process flow chart and description;

[0027] Figure 2 is a flow chart of the application of the vaccine system of the present invention in tumor immunotherapy, wherein ab is the use of mRNA-H vaccine to immunize mice (muscular injection) to stimulate the body to produce HBsAg memory T cells, c is the use of VV-H (intratumoral injection) to specifically mark tumors with HBsAg neoantigens, d is the HBsAg neoantigen to activate its memory T cells, rapidly kill the tumor and release tumor autoantigens, e is through epitope expansion, and f is the generation of systemic anti-tumor immune effects;

[0028] Figure 3 is a map of the pUC-HBsAg recombinant plasmid;

[0029] Figure 4 shows the average particle size, surface potential, and average dispersion coefficient values ​​of LNP HBsAg mRNA;

[0030] Figure 5 shows the quantification of serum HBsAb after the first (a) and second (b) immunization with mRNA-H vaccine;

[0031] Figure 6 shows the characterization of HBsAg memory T cells in the spleen after HBsAg vaccine immunization; ab is the number of IFN-γ+ cells after ELISPOT quantification of HBsAg peptide library stimulation, and cd is the flow cytometry gating strategy and quantification of IFNγ+CD8+T cells after restimulation with HBsAg peptide library;

[0032] Figure 7 shows the efficacy of mRNA-H vaccine in preventing B16-HBsAg tumors; a is tumor volume monitoring (n=5); b is weight monitoring of tumor-bearing mice during treatment (n=5); c is the survival rate of mice after immunization with different vaccines (n=6). HepB-CHO is a commercially available recombinant HBsAg hepatitis B vaccine.

[0033] Figure 8 shows the immunogenicity evaluation of the new antigen HBsAg, including tumor volume and body weight monitoring of B16-HBsAg, B16-OVA, and B16F10 tumors (n=10);

[0034] Figure 9 shows the epitope expansion evaluation of the neoantigen HBsAg, and the number of IFN-γ+ cells after stimulation with a melanoma peptide library in B16-HBsAg tumor-bearing mice was quantified by Elispot;

[0035] Figure 10 shows the oncolytic virus plasmid map and gene construction process, where A is the vaccinia virus plasmid map and B is the gene construction process;

[0036] FIG11 is a red fluorescence image of Hela-s3 cells undergoing recombinant virus screening and culture;

[0037] Figure 12 shows PCR identification of the expression of the target gene of the recombinant oncolytic vaccinia virus using HBsAg and GM-CSF gene primers. The left image shows HBsAg and the right image shows GM-CSF.

[0038] FIG13 is a bar graph showing the ability of VV-H to induce B16F10 tumor cells to express HBsAg and GM-CSF as detected by ELISA;

[0039] FIG14 shows the expression of the TK gene identified by recombinant oncolytic vaccinia virus gDNA after plaque purification;

[0040] Figure 15 shows the HBsAg content in Hela-s3 tumor cells at different MOIs by ELISA after 24h, 48h, and 72h of VV-H infection;

[0041] FIG16 is a treatment plan for activating anti-tumor immune effects by the vaccine system of the present invention, wherein sc is subcutaneous injection and it is intratumoral injection;

[0042] Figure 17 shows the evaluation of the therapeutic effect of VV-H on melanoma (B16F10) produced by HBsAg memory T cells; a is tumor volume monitoring, b is weight detection of tumor-bearing mice, ce are 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 strategy;

[0043] FIG18 is an evaluation of the therapeutic effect of B16-OVA produced by VV-H activated HBsAg memory T cells; wherein a-c are tumor volume and body weight monitoring, d is the tumor-bearing status of mice at the end of treatment, e is the tumor weight of tumor-bearing mice, and fg are quantitative analysis of CD3+CD8+ and CD3+CD4+ T cells in the tumor;

[0044] Figure 19 shows the evaluation of the therapeutic effect of VV-H activated HBsAg memory T cells combined with the immune checkpoint inhibitor PD-L1 on colorectal cancer (CT26); a is tumor volume monitoring, and b is weight detection of tumor-bearing mice;

[0045] Figure 20 shows the evaluation of the therapeutic effect of VV-H on breast cancer (4T1) produced by HBsAg memory T cells; a is the tumor volume monitoring, b is the mouse survival rate statistics, and c is the mouse recurrence rate statistics;

[0046] FIG21 is an immunofluorescence image of VV-H-induced tumor expression of HBsAg in vivo; after intratumoral injection of VV-H, the tumor was removed and subjected to immunofluorescence staining for HBsAg (HBsAg, green) and VV (mcherry, the red fluorescence of the virus itself);

[0047] Figure 22 shows the safety evaluation of VV-H+mRNA-H after treatment; a is the detection of serum biochemical indicators after treatment, including alanine aminotransferase (ALT), total protein (TP), serum creatine kinase isoenzyme (CKI), and blood urea nitrogen (BUN); b is HE staining of major organs after treatment. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0049] Example 1

[0050] Preparation of heterologous protein mRNA vaccines

[0051] 1. Construction of mRNA-H:

[0052] (1) Construction, synthesis and amplification of mRNA vaccine recombinant plasmids

[0053] The HBsAg, T7 promoter, and UTR sequences were ligated into the vector to construct the pUC-HBsAg recombinant plasmid. The plasmid map is shown in Figure 3. This plasmid was synthesized and prepared by GeneWeiZ. An appropriate amount of E. coli culture containing the HBsAg plasmid was taken and cultured overnight at 37°C. Three monoclonal plaques were placed in a centrifuge tube containing 5 mL of culture medium and incubated at 37°C, 220 rpm / min, for 14-18 hours. The pHBsAg plasmid was extracted and quantified using a kit.

[0054] (2) HBsAg plasmid PCR, purification and identification

[0055] Add the PCR reaction system and run the PCR program. Prepare a 1% agarose gel, mix the PCR products, and add 3 volumes of Buffer DC to further mix. Transfer the gel to an adsorption column. Centrifuge at 12,000 rpm for 1 minute at room temperature and discard the filtrate. Add Buffer WB to the adsorption column and let it sit for 2 minutes. Centrifuge at 12,000 rpm for 30 seconds at room temperature and discard the filtrate. Add sterile water to the center of the adsorption column and centrifuge to collect the sample.

[0056] (3) In vitro transcription (IVT) and capping

[0057] Add the IVT reaction system, mix well, and run in a 37°C PCR instrument for 2 hours. Add DNase I, add sterile water to the PCR product to 50 μL, pipette to mix, and PCR for 15 minutes at 37°C to remove the DNA template. Add RNA cleanup binding buffer to 100 μL of sample, then add ethanol and pipette to mix. Transfer the liquid to the purification column, centrifuge at 12,000 rpm at room temperature for 1 minute, and discard the filtrate. Add RNA cleanup wash buffer and centrifuge at 12,000 rpm at room temperature for 1 minute. Repeat the previous step and transfer the column to an RNase-free centrifuge tube. Add L sterile water, let it stand for 5 minutes, and then collect by centrifugation. Quantitatively dilute for agarose gradient electrophoresis.

[0058] 2. Preparation of heterologous protein mRNA vaccines

[0059] The lipid components were mixed in a molar ratio of Lipid / DSPC / cholesterol / PEG-DMG = 50 / 10 / 38.5 / 1.5, and then the Lipid solutions were mixed using anhydrous ethanol to prepare a 2.85 mg / mL Lipid mixture. The mRNA-containing aqueous solution was prepared by diluting 100 μg of mRNA in 2 mL of D 10 mM citric acid-sodium citrate buffer to obtain the mRNA dilution solution. The mRNA-containing aqueous phase and the lipid-containing alcohol phase were passed through a microfluidic device at a flow rate ratio of 3:1 to complete the preparation of mRNA LNPs. After ultrafiltration and centrifugation, the mRNA LNP particle size and zeta potential were measured.

[0060] As shown in Figure 4, the physical parameters of the heterologous protein mRNA vaccine mRNA-H are suitable, which is conducive to entering the cells to express HBsAg; the plasmid nucleotide sequence of HBsAg mRNA is SEQ ID NO.1.

[0061] Example 2

[0062] Detection of anti-HBsAg antibody (HBsAb) in serum after mRNA-H immunization

[0063] The immune efficacy of the prepared mRNA-H vaccine and the commercially available HBsAg hepatitis B vaccine (HepB-CHO) was preliminarily evaluated by serum HBsAb detection. Six-week-old C57BL / 6 mice were randomly divided into three groups: 1. Saline, 2. mRNA-H vaccine, and 3. HepB-CHO. Mice were immunized twice via intramuscular injection on days 0 and 14 after grouping. Serum HBsAb levels were measured 7 days after each immunization.

[0064] As shown in Figure 5 , 7 days after the second immunization, the serum HBsAb level in the mRNA-H group was much higher than that in the blank group (HBsAb ≥ 10 mIU / mL, which can prevent hepatitis B).

[0065] Example 3

[0066] Detection of the ability of mRNA-H vaccine to induce the formation of splenic memory T cells

[0067] ELISPOT assay: Immunize mice using the method described in Example 3. 30 days after the last immunization, remove the spleen under sterile conditions and prepare a single-cell suspension. Centrifuge at 350g for 5 minutes. Discard the supernatant, add 1 mL of erythrocyte lysis buffer, and lyse for 10 minutes. Then, add 3 mL of buffer to terminate lysis and collect the cells by centrifugation at 350g for 5 minutes. Resuspend the cells in 4 mL of 1640 medium, count, and adjust the volume to 2 × 10^6 / mL. Activate the pre-coated plates by adding 200 μL / well of RPMI-1640 medium (5% FBS), let it stand for 10 minutes, and then remove the plate. ELISPOT assays were performed as follows: negative control: 100 μL cells + 5 μL PBS; HBsAg protein group: 100 μL cells + 10 μL HBsAg epitope peptide library; positive group: 100 μL cells + 10 μL positive stimulant. After addition, cover the plate and incubate for 16-24 hours. After incubation, the spots were detected and statistically analyzed. In addition, flow cytometry was used to verify the ability of spleen memory T cells to secrete IFN-γ after stimulation with the HBsAg epitope antigen library.

[0068] As shown in Figure 6, compared with the recombinant HBsAg vaccine group, the HBsAg-specific memory T cells produced by the mRNA-H vaccine increased by at least 150 times, and the number of IFN-γ+CD8+T cells increased by at least 25 times.

[0069] Example 4

[0070] Evaluation of the efficacy of mRNA-H-induced memory T cells in preventing B16-HBsAg tumors

[0071] Six-week-old C57BL / 6 female mice were randomly divided into three groups: 1. Blank, 2. mRNA-H, and 3. HepB-CHO. Mice were immunized according to the method described in Example 3. Furthermore, lentiviral vectors expressing HBsAg in B16F10 tumors (B16-HBsAg) were constructed. Thirty days after the last immunization, B16-HBsAg was inoculated. Mice were observed and tumor volume was monitored. Furthermore, treatments were repeated to analyze survival data.

[0072] As shown in Figure 7, after treatment, all mice in the mRNA-H group were tumor-free, while the average tumor volume in the control group (Saline) and the HepB-cho-immunized mice exceeded 1000 mm³. Furthermore, there was no significant change in the body weight of the mRNA-H-treated mice. While all mice in the control group died from excessive tumor burden on day 26, all mice in the mRNA vaccine-immunized group were still alive and tumor-free on day 33.

[0073] The B16-HBsAg tumor model was used to verify the therapeutic effect of memory T cells produced by the mRNA-H vaccine on B16-HBsAg tumors. The results showed that 100% of mice immunized with the mRNA-H vaccine prevented the growth of B16-HBsAg tumors, while the control group had 0%. In addition, there was no significant change in the body weight of mice in the mRNA vaccine group, indicating good safety. The results showed that heterologous protein (HBsAg) memory T cells can completely eliminate B16-HBsAg tumors, providing a strong guarantee for the subsequent killing of tumors expressing new antigens derived from heterologous proteins.

[0074] Example 5

[0075] Evaluation of the immunogenicity of the new antigen HBsAg

[0076] Six-week-old C57BL / 6 female mice were divided into three groups: 1. B16-HBsAg, 2. B16-OVA, and 3. B16-Neo. They were immunized twice with mRNA-H, mRNA-OVA, and mRNA-Neo, respectively. One week after the final immunization, mice were inoculated with B16-HBsAg, B16-OVA, and B16F10 cells. The mice were observed and tumor volumes were measured.

[0077] As shown in Figure 8, the B16-HBsAg group significantly inhibited the tumor, indicating that mRNA-H can effectively activate memory T cells to kill tumors by labeling HBsAg neoantigens. It also shows that heterologous proteins derived from microorganisms have higher immunogenicity than neoantigens and other highly immunogenic proteins, and can be quickly recognized by memory cells and kill tumors.

[0078] Example 6

[0079] Evaluation of epitope expansion of the new antigen HBsAg

[0080] According to the method in Example 5, when the average tumor volume of the B16-Neo group reached 2000 mm 3Spleens from mice in the B16-HBsAg group were removed under sterile conditions and prepared into a single-cell suspension. The suspension was centrifuged at 350 g for 5 minutes. The supernatant was discarded, and 1 mL of red blood cell lysis buffer was added and lysed for 10 minutes. Lysis was then terminated by adding 3 mL of buffer, and the cells were collected by centrifugation at 350 g for 5 minutes. Cells were resuspended in 4 mL of 1640 medium, counted, and adjusted to 2 × 10^6 / mL. ELISPOT assays were performed as follows: negative control, 100 μL of cells + 100 μL of PBS; HBsAg protein group, 100 μL of cells + 100 μL of HBsAg epitope peptide library; M30 group, 100 μL of cells + 100 μL of M30 peptide; M27 group, 100 μL of cells + 100 μL of M27 peptide. After addition, the plates were covered and incubated for 16-24 hours. After incubation, spots were detected and statistically analyzed.

[0081] As shown in Figure 9, tumors labeled with HBsAg neoantigens induced and amplified immune responses to secondary antigens under the continuous killing of memory T cells, effectively achieving epitope expansion and contributing to the overall treatment outcome.

[0082] Example 7

[0083] Construction and characterization of recombinant oncolytic viruses expressing neoantigens:

[0084] (1) Construction and synthesis of HBsAg shuttle plasmid

[0085] The flanking sequences of TK (VACWR094) VACWR093 (TK-R) and VACWR095 (TK-L) were used as homology arms; mCherry (fluorescent group) and xanthine guanine phosphotransferase (gpt) were used as screening genes; the Pse / l promoter was used to induce HBsAg expression, and the vector resistance was AmpR (ampicin). The pVV-mcherry-HBsAg plasmid was constructed. In addition, an empty vaccinia virus VV-C and a vaccinia virus VV-G expressing GM-CSF (granulocyte macrophage colony stimulating factor) were constructed (the same position as HBsAg in the plasmid in the figure) and handed over to GenScript for synthesis. The plasmid map is shown in Figure 10. The nucleotide sequence of the recombinant viral plasmid is SEQ ID NO.2.

[0086] (2) Construction and screening of recombinant oncolytic viruses (VV-H) expressing HBsAg

[0087] 293T cells were cultured, trypsinized, counted, and plated at 5x10^5 cells / well in a 6-well plate. The cells were incubated at 37°C and 5% CO2. When the cells reached 90% confluence, wild-type virus (VV) was added at a multiplicity of infection (MOI) of 1. Two hours after infection, the DMEM medium was replaced and 1 μg of plasmid was transfected / well. 24-72 hours after transfection, red fluorescence was observed and photographed. The medium was discarded, and 500 μL of DMED medium without FBS was added. The cells were pipetted and collected into a centrifuge tube. After three freeze-thaw cycles at -80°C / 37°C, the tubes were centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected. Hela-S3 cells were plated in a 6-well plate. When the confluence reached 90%, 10 and 100 μL of virus were added to three wells, respectively. Two hours later, the medium was removed, and selection culture was performed using selection medium containing xanthine, hypoxanthine, and mycophenolic acid. Observe mCherry fluorescence within 24-72 hours after adding the screening medium. Fluorescence images are shown in Figure 11. After 72 hours, carefully discard the screening medium, add 500 μL of DMED medium, centrifuge, collect the supernatant, and proceed as above. Perform three to four rounds of screening on the recombinant virus. The screening results are shown in Figure 9.

[0088] 10 and 100 μL of the selected virus were taken and used to infect cells in two 6-well plates, respectively. DNA was extracted from these cells, and PCR was performed using target gene primers to identify the presence of recombinant virus. The results are shown in Figure 12. ELISA kits were also used to identify the target proteins in the cell lysates. As shown in Figure 13, VV-H and VV-G promoted the expression of HBsAg and GM-CSF in tumor cells, respectively. After correct identification at the genomic DNA and protein levels, the recombinant virus was amplified in 1x screening medium.

[0089] (3) Plaque purification of HBsAg recombinant oncolytic virus (VV-H)

[0090] HELA-S3 cells were seeded and infected with the virus as described above. Different volumes of virus solution were added to 1x selection medium for plaque purification to remove wild-type virus. After 24 hours, fluorescence was observed. 2x selection medium (1 mL) was replaced in a well plate containing an appropriate number of plaques. 1 mL of 2% low-melting-point agarose was added and the cells were incubated at 37°C in a 5% CO2 incubator. Based on the fluorescence observed after 24 hours, HELA-S3 cells were plated in 24-well plates and allowed to reach 90% confluency at 48 hours (24-well plate, 10 wells, 5 x 104 cells / well, 500 μL of DMEM with 2% FBS). At 48 hours, a single plaque was picked using a 200 μL sterile pipette tip and transferred to the 24-well plate for further incubation. The formation of fluorescent plaques was continuously observed until the cells were fully confluent. The supernatant was removed and 200 μL of DMED medium without FBS was added. The supernatant was then pipetted repeatedly and the virus was collected by centrifugation, and the procedure was repeated as described above. Thymidine kinase (TK) primers were used to identify whether the selected recombinant viruses contained wild-type viruses, until the solution was free of wild-type viruses. As shown in FIG14 , the selected recombinant viruses no longer contained wild-type viruses.

[0091] (4) In vitro amplification and purification of recombinant oncolytic virus VV-H

[0092] Prepare several dishes of Hela-S3 (cervical cancer) cells, digest them, and count approximately 10^8 cells. Plate the recombinant virus (MOI = 0.1) and cell mixture onto 20 15cm culture dishes for recombinant virus amplification, observing fluorescence expression during amplification. When fluorescence intensity reaches its peak, transfer the cells and virus solution to a 50mL centrifuge tube and centrifuge at 2000g for 10 minutes. Discard the supernatant and resuspend the cells in 10mM Tris-HCl. Repeat freeze-thaw the cell suspension three times at -80°C / 37°C, centrifuge at 2000rpm for 5 minutes, and collect the supernatant. Add 1 / 3 volume of trypsin to the supernatant, mix thoroughly, and incubate in a 37°C water bath for 30 minutes. Layer 36% sucrose: Add 13mL of 36% sucrose to an ultracentrifuge tube and gently add an equal volume of virus solution on top of the sucrose solution. Centrifuge at 32900g for 2 hours at 4°C in an ultracentrifuge. Gently discard the supernatant and resuspend the pellet in 5 mL of PBS per ultracentrifuge tube. Fill to the top with PBS and ultracentrifuge again at 32,900 g for 1 hour at 4°C. Discard the supernatant and resuspend the pellet in 1 mL of PBS per ultracentrifuge tube. Aliquot the virus solution and store at -80°C to obtain recombinant vaccinia virus.

[0093] Titer determination of recombinant vaccinia virus (TCID50)

[0094] Hela-S3 cells were plated at a density of 10⁻¹ cells per well in a 96-well plate. Virus titers were set up at 10⁻³, 10⁻4, 10⁻5, 10⁻6, 10⁻7, 10⁻8, 10⁻9, and 10⁻10, with six replicates per viral gradient and three replicates per virus. Fluorescent plaques were observed in each well, and viral titer was calculated using the formula T = 0.7 × 10 × 10^(1 + S(d-0.5)). Where S = Log dilution - 1, and d = sum of positive ratios.

[0095] ELISA kit to detect VV-H labeled HBsAg

[0096] Hela-S3 tumor cells were infected with the virus at three MOIs of 0.01, 0.1, and 1. Cells were harvested by centrifugation at 24, 48, and 72 hours post-infection. The cells were frozen and thawed three times at -80°C / 37°C, centrifuged at 2000 rpm for 10 minutes, and the supernatant collected. HBsAg levels were measured using an ELISA kit and the absorbance at 450 nm was measured using a microplate reader. The expression results are shown in Figure 15.

[0097] Example 8

[0098] Evaluate the tumor therapeutic effect of VV-H activated specific memory T cells

[0099] The therapeutic effect was evaluated using four tumor types: B16F10 melanoma (fast growth rate and high malignancy), CT26 colorectal cancer (strong tumor heterogeneity and immune-cold tumor), 4T1 breast cancer (low mutation load and spontaneous metastasis), and B16-OVA. The specific treatment method is shown in Figure 16: Using the control group and C57BL / 6 or Balb / c mice 30 days after immunization, tumor cells were subcutaneously inoculated to construct B16F10, CT26, and 4T1 tumor models, respectively. When the tumor volume was 50-100 mm3, these tumor-bearing mice were randomly divided into 5 groups. On Day 0 (grouping day), 3, and 5 days, 50 μL of each virus solution (10^7 PFU / mouse) was injected intratumorally. The results are shown in Figures 15-17 (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). Compared with the control group, VV-H + mRNA-H significantly inhibited tumor growth, indicating that VV-H + mRNA-H can effectively activate memory T cells to kill tumors by labeling HBsAg neoantigens. In addition, there was no significant change in the body weight of mice in each group, indicating that the treatment has a good safety profile.

[0100] In the 4T1 tumor model, 30 days after the last treatment, the treated side of the tumor was resected and the mice's weight was monitored. At the end of treatment, survival curves and lung metastasis were calculated, as shown in Figure 20. This treatment strategy effectively slowed the tumor's ability to metastasize to distant sites.

[0101] Example 9

[0102] Evaluation of the therapeutic effect of melanoma generated by VV-H activated HBsAg memory T cells

[0103] After treatment of the B16F10 melanoma model and B16-OVA subcutaneous tumor model described in Example 7, the treated tumors and spleens were dissected and single-cell suspensions prepared. Subsequently, the tumor single-cell suspensions were stained with a mixture of CD45, CD3, CD4, and CD8 antibodies and a Live / Dead dye on ice for 30 minutes, and the effector T cell counts were measured by flow cytometry.

[0104] As shown in Figure 17, in the B16F10 melanoma model, compared with the mRNA-H vaccine group, the CD8+T in the tumors of mice in the VV-H+mRNA-H group increased by at least 10 times, and the CD4+T cells increased by at least 4 times, indicating that VV-H+mRNA-H further activates the anti-tumor immune response by efficiently lysing tumors and releasing antigens.

[0105] Also as shown in Figure 18, in the B16-OVA subcutaneous tumor model, compared with other treatment groups, the CD4+ and CD8+ T cells in the tumors of mice in the VV-H+mRNA-H group were significantly increased, indicating that the VV-H+mRNA-H group achieved more effective immune infiltration.

[0106] Example 10

[0107] Immunofluorescence staining to evaluate the ability of VV-H to induce HBsAg expression in tumors in vivo

[0108] B16F10 tumor models were established using control and immunized C57BL / 6 mice subcutaneously inoculated with B16F10 cells. When tumors reached 150 mm³, mice were randomly divided into three groups (n=5): 1. VV-C, 2. VV-H, and 3. mRNA-H + VV-H. After grouping, 50 μL of virus (10^7 PFU / mouse) was injected intratumorally. Tumors were dissected on days 1, 3, and 7 after administration, and sections were prepared to visualize VV-H (mCherry, red) and HBsAg neoantigens (FITC-HBsAg antibody, green).

[0109] As shown in Figure 21, after VV-H administration, a strong HBsAg signal was detected in the tumor, indicating that VV-H can effectively label HBsAg.

[0110] Example 10

[0111] Safety evaluation of VV-H+mRNA-H treatment

[0112] During the above treatment, the body weight of the mice was monitored daily for preliminary safety assessment. In addition, following the same treatment as in Example 8, major blood indicators such as red blood cell and white blood cell levels, 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. Following completion of treatment, the mice were euthanized, and the heart, liver, spleen, lung, and kidney were isolated for pathological examination to further assess safety.

[0113] As shown in Figure 22, no significant changes were found in the serum biochemical indicators and HE staining of major organs of mice in the VV-H+mRNA-H group, which preliminarily confirmed its good safety.

Claims

1. A tumor neoantigen vaccine system that specifically labels heterologous proteins, characterized in that: The vaccine system consists of a heterologous protein mRNA vaccine and a recombinant oncolytic virus. The heterologous protein mRNA vaccine produces specific memory T cells of precisely labeled new antigens, and the recombinant oncolytic virus is used to accurately label tumor cells with highly immunogenic new antigens.

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

3. The vaccine system according to claim 1, characterized in that The oncolytic virus is one or more of 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 prepared by constructing, synthesizing and amplifying the mRNA vaccine recombinant plasmid, and then encapsulating the mRNA vaccine recombinant plasmid with lipid nanomaterials.

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

6. Use of the vaccine system according to claim 1 in tumor immunotherapy.

7. The use according to claim 6, characterized in that: Used to enhance tumor immunotherapy.

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

9. The use according to claim 8, characterized in that: In the administration, the heterologous protein mRNA vaccine produces immune memory through intramuscular injection, intranasal administration or intravenous administration, and the recombinant oncolytic virus accurately marks highly immunogenic tumor neoantigens to tumor cells through intratumoral administration.

10. The use according to claim 6, characterized in that: The tumor is a solid tumor or a solid tumor with metastasis, including melanoma, colorectal cancer, breast cancer, liver cancer, glioma, bile duct cancer or pancreatic cancer.

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