Cancer immunotherapy by mRNA delivery

Abstract

The present invention relates to cancer immunotherapy. A method for increasing the antigenicity or immunogenicity of a tumor in a subject is provided. In one embodiment, the method comprises delivering lipid nanoparticles carrying mRNA sequences encoding one or more pathogen antigen proteins to the tumor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 439,557, filed January 17, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to cancer immunotherapy, and in particular to immunotherapeutic agents. [Background technology]

[0003] Over the past few decades, cancer immunotherapy has shown promise for improving clinical cancer therapy compared with conventional cancer treatment strategies. However, existing cancer immunotherapies fail to benefit all cancer patients with different cancer types and stages. Effective cancer immunotherapy always relies on the induction of a cancer-immune cycle [1]. In this cycle, tumor antigens are released by antitumor responses and captured by professional antigen-presenting cells (APCs). APCs then present the antigens on MHC molecules to stimulate and activate tumor-specific T cells. Ultimately, these activated tumor-specific T cells are recruited to tumors, where they recognize and kill their target cancer cells. The elimination of cancer cells releases additional tumor-associated antigens, inducing a broader and more potent antitumor response in subsequent cycles [1]. Nevertheless, tumors continually evolve to escape immune surveillance or subvert the cancer-immune cycle, ultimately leading to immunotherapy failure or tumor recurrence [2,3].

[0004] The three most important factors for improving therapeutic efficacy in this cancer-immunity cycle are tumor antigenicity, tumor immunogenicity, and the tumor microenvironment [3], which are important for easy tumor recognition, induction of strong tumor-specific immune responses, and effective activation of intratumoral immune cells.

[0005] One strategy for inducing an effective cancer immune cycle is to introduce pathogens into tumors to increase tumor immunogenicity and reverse the suppressive tumor microenvironment. For example, one study showed that intratumoral administration of an unadjuvanted seasonal influenza vaccine converted a "cold" tumor microenvironment into a "hot" one, resulting in reduced tumor growth [4]. Another promising cancer treatment approach is oncolytic viruses, which can selectively infect cancer tissue without infecting normal tissues and can be engineered to deliver or redirect drugs to specific targets. In addition, the immunogenicity of oncolytic viruses also helps to reverse the suppressive tumor microenvironment [5,6]. Furthermore, bacterial-based cancer therapies are also becoming attractive, and genetically engineered tumor-targeting attenuated bacteria have shown potent tumoricidal effects and significantly increased survival rates. It has previously been shown that in vivo delivery of engineered tumor-targeting Salmonella can effectively suppress tumor growth [7,8] and tumor metastasis [9].

[0006] On the other hand, to make tumors more recognizable by the immune system, researchers have introduced foreign antigens into tumors, increasing their antigenicity and immunogenicity. One strategy is to directly introduce foreign MHC into tumor cells to increase their antigenicity and immunogenicity and generate a systemic antitumor immune response [10,11]. Furthermore, researchers have transduced the MHC of tumor cells with influenza virus-derived peptides, which are used as vaccines

[12] , to help induce a stronger antitumor response.

[0007] However, not only pathogen-based or tumor antigen-based cancer immunotherapy, but also widely used clinical strategies such as immune checkpoint blockade [13-16] and CAR-T therapy [17-21] only focus on one aspect of tumor eradication, namely, either tumor microenvironment transformation or tumor recognition, and when used alone, they have limited therapeutic efficacy. One strategy is to express foreign antigens with sufficient immunogenicity and antigenicity to not only control the tumor microenvironment but also increase tumor recognition, thereby rapidly inducing potent tumor elimination.

[0008] BNT162b2 (Comirnaty) is an mRNA COVID-19 vaccine developed by BioNTech. BNT162b2 is composed of spike protein mRNA and lipid nanoparticles

[22] . Studies have shown that administration of BNT162b2 can effectively prevent SARS-CoV-2 infection in humans and mice by eliciting strong anti-spike protein humoral and cellular immune responses [23-25]. Due to the COVID-19 pandemic, more than 69% of the world's population has currently received at least one dose of a COVID-19 vaccine

[26] . This vaccine induces a memory immune response targeting the SARS-CoV-2 spike protein, preventing infection or severe symptoms. Therefore, it is an ideal candidate for demonstrating the general cancer treatment strategy of the present invention.

[0009] In addition to the BNT162b2 mRNA vaccine encoding the SARS-CoV-2 spike protein, lipid nanoparticle-encapsulated mRNA vaccines encoding the HKU1 coronavirus spike protein or the Staphylococcal enterotoxin A (SEA) superantigen have also been developed for cancer treatment in the present invention.

[0010] The present invention demonstrates that lipid nanoparticle-encapsulated mRNA expresses spike protein in tumor cells and induces a potent anti-spike protein T cell immune response, thereby offering significant therapeutic potential for various types of cancer. Furthermore, this initial antitumor attack may induce the release of spike protein-containing tumor exosomes and potent tumor antigen dissemination by dead tumor cells. Therefore, the present invention provides a powerful cancer treatment strategy by repurposing existing mRNA vaccines, such as the COVID-19 mRNA vaccine, for cancer treatment with potent therapeutic efficacy and the potential for rapid clinical translation across multiple cancers. Furthermore, the present invention also demonstrates that antigens derived from other pathogens have significant therapeutic potential. Summary of the Invention

[0011] The present invention provides a method for increasing the antigenicity or immunogenicity of a tumor in a subject. In one embodiment, the method comprises delivering lipid nanoparticles carrying mRNA sequences encoding one or more pathogen antigen proteins to the tumor.

[0012] The present invention also provides a method of treating a subject having one or more tumors, the method comprising increasing the antigenicity or immunogenicity of at least one of the one or more tumors using a method of the present invention.

[0013] The present invention further provides intratumoral administration of lipid nanoparticles loaded with mRNA encoding one or more pathogen antigen proteins for treating tumors in a subject.

[0014] The present invention also provides a kit for treating a tumor in a subject, comprising intratumoral administration of the present invention.

[0015] The present invention also provides combination therapies of the present invention with immune checkpoint inhibitors. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 illustrates an embodiment of the therapeutic strategy of the present invention. [Figure 2] FIG. 2 shows the plasmid genetic map for the construction of the B16F10-OVA cell line. [Figure 3A] Figure 3A shows the experimental design of BNT162b2 cancer treatment. BNT162b2 intramuscular vaccination was performed before cancer cell transplantation. n=5 per group. [Figure 3B] FIG. 3B shows tumor growth curves for the BNT162b2 intratumoral treatment group versus the PBS intratumoral control group for the experiment described in FIG. 3A. [Figure 3C] FIG. 3C shows the tumor growth curve of the PBS intratumoral administration control group for the experiment described in FIG. 3A. [Figure 3D] FIG. 3D shows tumor growth curves for the BNT162b2 intratumoral administration treatment group for the experimental design described in FIG. 3A. [Figure 3E] FIG. 3E shows comparative photographs of tumors from the treatment and control groups of the experiment described in FIG. 3A. [Figure 4A] Figures 4A-4V show that intratumoral administration of BNT162b2 reduces tumor growth or tumor lung metastasis in vaccinated mice. BNT162b2 intramuscular vaccination was performed after cancer cell transplantation. Figures 4A, 4F, 4K, and 4R show the experimental design of BNT162b2 cancer therapy. BNT162b2 vaccination was performed after cancer cell transplantation. n = 5 or 6 per group. [Figure 4B] FIG. 4B shows B16F10 tumor growth curves for the BNT162b2 intratumoral treatment group versus the PBS intratumoral control group for the experiment described in FIG. 4A. [Figure 4C] FIG. 4C shows the B16F10 tumor growth curve for the PBS intratumoral administration control group for the experiment described in FIG. 4A. [Figure 4D] FIG. 4D shows the B16F10 tumor growth curves for the BNT162b2 intratumoral administration treatment group for the experimental design described in FIG. 4A. [Figure 4E] FIG. 4E shows comparative photographs of B16F10 tumors from the treatment and control groups of the experiment described in FIG. 4A. [Figure 4F]Figures 4A, 4F, 4K, and 4R show the experimental design of BNT162b2 cancer therapy. BNT162b2 vaccination was performed after cancer cell transplantation. n=5 or 6 per group. [Figure 4G] FIG. 4G shows MB49 tumor growth curves for the BNT162b2 intratumoral treatment group versus the PBS intratumoral control group from the experiment described in FIG. 4F. [Figure 4H] FIG. 4H shows the MB49 tumor growth curve for the PBS intratumoral administration control group from the experiment described in FIG. 4F. [Figure 4I] FIG. 4I shows the MB49 tumor growth curves for the BNT162b2 intratumoral administration treatment group from the experiment described in FIG. 4F. [Figure 4J] FIG. 4J shows comparative photographs of MB49 tumors from the treatment group versus the control group of the experimental design described in FIG. 4F. [Figure 4K] Figure 4K shows the experimental design of BNT162b2 cancer therapy. BNT162b2 vaccination was performed after 4T1 cancer cell transplantation. n=6 per group. [Figure 4L] FIG. 4L shows 4T1 tumor growth curves for the BNT162b2 intratumoral treatment group versus the PBS intratumoral control group from the experiment described in FIG. 4K. [Figure 4M] FIG. 4M shows the 4T1 tumor growth curve for the PBS intratumoral administration control group of the experiment described in FIG. 4K. [Figure 4N] FIG. 4N shows 4T1 tumor growth curves for the BNT162b2 intratumoral administration treatment group from the experiment described in FIG. 4K. [Figure 4O] FIG. 4O shows comparative photographs of 4T1 tumors from the treatment group versus the control group of the experimental design described in FIG. 4K. [Figure 4P] FIG. 4P shows comparative photographs of 4T1 tumor lung metastases in the treatment and control groups at the endpoint of the experiment described in FIG. 4K. [Figure 4Q] FIG. 4Q shows the number of lung metastases per mouse for each group shown in FIG. 4P (n=6 per group). [Figure 4R] Figure 4R shows the CT26 subcutaneous model of the present invention. Tumor growth was monitored and measured every two days. Intramuscular vaccination with BNT162b2 was performed after cancer cell implantation. [Figure 4S] Figures 4S-4V show that BNT162b2 treatment can also effectively inhibit tumor growth of CT26 colon cancer. [Figure 4T] Figures 4S-4V show that BNT162b2 treatment can also effectively inhibit tumor growth of CT26 colon cancer. [Figure 4U] Figures 4S-4V show that BNT162b2 treatment can also effectively inhibit tumor growth of CT26 colon cancer. [Figure 4V] Figures 4S-4V show that BNT162b2 treatment can also effectively inhibit tumor growth of CT26 colon cancer. [Figure 5A] Figures 5A-5F and 5H-5V show that intratumoral BNT162b2 vaccine injection induced a potent antitumor T cell immune response. Figures 5G-1, 5G-2, 5G-3, and 5G-4 demonstrate the recruitment of immune cells in tumors. Figures 5A and 5K show the experimental design. [Figure 5B] Figures 5B, 5C, 5G-1, 5G-2, 5G-3, and 5G-4 show the detection of tumor-infiltrating immune cells by flow cytometry or immunofluorescence staining. [Figure 5C] Figures 5B, 5C, 5G-1, 5G-2, 5G-3, and 5G-4 show the detection of tumor-infiltrating immune cells by flow cytometry or immunofluorescence staining. [Figure 5D] Figures 5D and 5E show detection of spike protein expression on tumor cell surfaces and within tumors by immunofluorescence staining of tumor sections. BNT im-BNT it: Tumor sections from mice intramuscularly and intratumorally administered BNT162b2. BNT im-PBS it: Tumor sections from mice intramuscularly vaccinated with BNT and intratumorally administered PBS. Spikes on the tumor cell surface, intratumor spikes, and DAPI are indicated in the figures. [Figure 5E]Figures 5D and 5E show detection of spike protein expression on tumor cell surfaces and within tumors by immunofluorescence staining of tumor sections. BNT im-BNT it: Tumor sections from mice intramuscularly and intratumorally administered BNT162b2. BNT im-PBS it: Tumor sections from mice intramuscularly vaccinated with BNT and intratumorally administered PBS. Spikes on the tumor cell surface, intratumor spikes, and DAPI are indicated in the figures. [Figure 5F] Figures 5F, 5G-1, 5G-2, 5G-3, and 5G-4 show a comparison of tumor-infiltrating T cells, B cells, NK cells, antigen-presenting cells, macrophages, and neutrophils in the BNT162b2 intratumoral administration group and the PBS intratumoral administration control group. Figures 5G-2 and 5G-4 show the results of intratumoral immune cell staining by flow cytometry. [Figure 5G-1] Figures 5F, 5G-1, 5G-2, 5G-3, and 5G-4 show a comparison of tumor-infiltrating T cells, B cells, NK cells, antigen-presenting cells, macrophages, and neutrophils in the BNT162b2 intratumoral administration group and the PBS intratumoral administration control group. Figures 5G-2 and 5G-4 show the results of intratumoral immune cell staining by flow cytometry. [Figure 5G-2] Figures 5F, 5G-1, 5G-2, 5G-3, and 5G-4 show a comparison of tumor-infiltrating T cells, B cells, NK cells, antigen-presenting cells, macrophages, and neutrophils in the BNT162b2 intratumoral administration group and the PBS intratumoral administration control group. Figures 5G-2 and 5G-4 show the results of intratumoral immune cell staining by flow cytometry. [Figure 5G-3] Figures 5F, 5G-1, 5G-2, 5G-3, and 5G-4 show a comparison of tumor-infiltrating T cells, B cells, NK cells, antigen-presenting cells, macrophages, and neutrophils in the BNT162b2 intratumoral administration group and the PBS intratumoral administration control group. Figures 5G-2 and 5G-4 show the results of intratumoral immune cell staining by flow cytometry. [Figure 5G-4]Figures 5F, 5G-1, 5G-2, 5G-3, and 5G-4 show a comparison of tumor-infiltrating T cells, B cells, NK cells, antigen-presenting cells, macrophages, and neutrophils in the BNT162b2 intratumoral administration group and the PBS intratumoral administration control group. Figures 5G-2 and 5G-4 show the results of intratumoral immune cell staining by flow cytometry. [Figure 5H] Figure 5H shows anti-spike protein IgG antibody titers after intratumoral BNT162b2 treatment. [Figure 5I] FIG. 5I shows a representative IFN-γ ELISpot showing tumor antigen-specific T cell responses after intratumoral BNT treatment. [Figure 5J] Figure 5J shows a bar graph summarizing ELISpot data comparing tumor antigen-specific T cell responses in the BNT162b2 intratumoral treatment group and the PBS intratumoral control group. Tumor cell line lysate was used as the tumor antigen stimulus. X-PBS it: splenocytes from mice treated with intratumoral PBS without intramuscular vaccination. BNT im-BNT it: splenocytes from mice treated with intramuscular and intratumoral BNT162b2. BNT im-PBS it: splenocytes from mice treated with intramuscular BNT162b2 and intratumoral PBS. [Figure 5K] Figures 5A-5F and 5H-5V show that intratumoral BNT162b2 vaccine injection induced a potent antitumor T cell immune response. Figures 5G-1, 5G-2, 5G-3, and 5G-4 demonstrate the recruitment of immune cells in tumors. Figures 5A and 5K show the experimental design. [Figure 5L] FIG. 5L shows tumor growth curves for the BNT162b2 intratumoral treatment group versus the PBS intratumoral control group for the experiment described in FIG. 5K. [Figure 5M] FIG. 5M shows comparative photographs of tumors from the treatment and control groups of the experiment described in FIG. 5K. [Figure 5N] FIG. 5N shows tumor growth curves for the BNT162b2 intratumoral administration treatment group and the PBS intratumoral administration control group in μMT mice for the experiment described in FIG. 5K. [Figure 5O]FIG. 5O shows tumor growth curves for the BNT162b2 intratumoral administration treatment group and the PBS intratumoral administration control group in Rag1 − / − mice for the experiment described in FIG. 5K. [Figure 5P] FIG. 5P shows the experimental design for detecting memory T cell activation after intratumoral BNT162b2 treatment. [Figure 5Q] FIG. 5Q shows CD69+ activated T cell detection at the time points described in FIG. 5P. [Figure 5R] FIG. 5R shows IFN-γ+ activated T cell detection at the time points indicated in FIG. 5P. [Figure 5S] Figure 5S shows a schematic diagram of the RNA-seq experimental design. [Figure 5T] Figure 5T shows a Venn diagram comparing differential gene expression between the intratumoral BNT162b2 treatment group (BNT im-BNT it group) and two control groups (BNT im-PBS it group and PBS it only group) at different time points. [Figure 5U] Figure 5U shows the enriched GO terms of genes that were significantly upregulated after intratumoral BNT162b2 administration. [Figure 5V] Figure 5V shows the correlation between APC markers and T cell activation markers. Statistical differences were analyzed using the Wilcoxon test. [Figure 6A] 6A to 6D show tumor microenvironment tests after intratumoral administration of BNT162b2. Figures 6A and 6B show the phenotype of intratumoral macrophages in tumors after BNT162b2 or PBS treatment. [Figure 6B] 6A to 6D show tumor microenvironment tests after intratumoral administration of BNT162b2. Figures 6A and 6B show the phenotype of intratumoral macrophages in tumors after BNT162b2 or PBS treatment. [Figure 6C-1] Figures 6C-1 and 6C-2 show cytokine profiles in tumors or blood of mice after treatment with BNT162b2 or PBS. [Figure 6C-2]Figures 6C-1 and 6C-2 show cytokine profiles in tumors or blood of mice after treatment with BNT162b2 or PBS. [Figure 6D] FIG. 6D shows MHC-I and MHC-II expression in tumors from the BNT162b2-treated and control groups. [Figure 7A] 7A-7N show mechanistic studies of antigen spreading induced by BNT162b2 cancer therapy. Figure 7A shows the experimental design for the study of exosomes induced by BNT162b2 therapy. [Figure 7B] FIG. 7B shows flow cytometry intracellular staining comparing CD63+ exosome secretion levels after BNT162b2 treatment and DMSO treatment. [Figure 7C] Figure 7C shows immunofluorescence staining of intracellular CD63 to detect exosome secretion levels in tumors after intratumoral administration of BNT162b2 and PBS. [Figure 7D] FIG. 7D shows the experimental design for the study of exosomes of B16F10-OVA after BNT162b2 transfection. [Figure 7E] FIG. 7E shows a transmission electron microscope (TEM) image of isolated B16F10-OVA-derived exosomes. [Figure 7F] FIG. 7F shows the identification of exosomes derived from B16F10-OVA by Western blot. [Figure 7G] FIG. 7G shows the experimental design to investigate the potential of antigen spreading induced by tumor cell-derived exosomes. [Figure 7H] FIG. 7H shows an ELISpot assay detecting antigen spreading induced by tumor cell-derived exosomes. [Figure 7I] FIG. 7I shows the experimental design for the study of heat shock protein secretion induced by BNT162b2 transfection in tumor cells. [Figure 7J]FIG. 7J shows a statistical table of calreticulin and HSP70 heat shock protein secretion levels after BNT162b2 transfection in tumor cells. [Figure 7K] FIG. 7K shows the experimental design for antigen spread and distant therapeutic effect detection in a bilateral tumor model. [Figure 7L] FIG. 7L shows tumor photographs and tumor growth curves from the experiment described in FIG. 7K. [Figure 7M] FIG. 7M shows the experimental design of the potential for antigen spreading induced by dead tumor cells. [Figure 7N] FIG. 7N shows an ELISpot assay to detect the immunogenicity of dead tumor cells for antigen spreading. [Figure 8A] Figures 8A-8N show the role of PD-L1 expression in BNT162b2 cancer therapy. Figure 8A shows the experimental design of BNT162b2 cancer therapy, where samples are collected at the endpoint for immunodetection. [Figure 8B] Figure 8B shows PD-L1 expression detection in tumors by immunofluorescence staining. [Figure 8C] Figure 8C shows detection of intratumoral PD-L1 expression on tumor cells and CD45+ leukocytes by flow cytometry. [Figure 8D] Figure 8D shows transcriptome analysis of PD-L1 expression in tumors at different time points during BNT162b2 cancer treatment. [Figure 8E] Figure 8E shows transcriptome analysis of the correlation between PD-L1 expression and cell type. [Figure 8F] The heat map in Figure 8F shows the fold change in neutrophil and T cell activation genes after intratumoral administration of BNT162b2. [Figure 8G] Figure 8G shows the percentage of PD-L1+ macrophages and PD-L1+ neutrophils among PD-L1+ leukocytes. [Figure 8H]Figure 8H shows colocalized immunofluorescence staining of PD-L1 and the neutrophil marker Gr-1 in tumors from the BNT162b2-treated and control groups. The percentage of intratumoral PD-L1+ neutrophils among total live cells and the percentage of intratumoral PD-L1+ neutrophils among total neutrophils. [Figure 8I] FIG. 8I shows the experimental design for testing the therapeutic efficacy of exosomes derived from BNT162b2-transfected tumor cells. [Figure 8J] Figure 8J shows tumor photographs of the therapeutic effect of exosomes derived from BNT162b2-transfected tumor cells. [Figure 8K] FIG. 8K shows the tumor growth curve of the therapeutic effect of exosomes derived from BNT162b2-transfected tumor cells in the experiment described in I. [Figure 8L] FIG. 8L shows the experimental design for testing the preventive effect of exosomes derived from BNT162b2-transfected tumor cells. [Figure 8M] Figure 8M shows tumor photographs demonstrating the preventive effect of exosomes derived from BNT162b2-transfected tumor cells. [Figure 8N] Figure 8N shows tumor growth curves of the preventive effect of exosomes derived from BNT162b2-transfected tumor cells in the experiment described in L. [Figure 9A] 9A-9F show the combination therapy of BNT162b2 and anti-PD-L1 treatment, and the therapeutic effect of BNT162b2 in advanced cancer. Figure 9A shows the experimental design of the combination therapy of BNT162b2 and anti-PD-L1. [Figure 9B] Figure 9B shows tumor photographs of the therapeutic effect of the combination therapy of BNT162b2 and anti-PD-L1. [Figure 9C] Figure 9C shows tumor growth curves of the therapeutic effect of the combination therapy of BNT162b2 and anti-PD-L1 in the experiment described in Figure 9A. [Figure 9D] FIG. 9D shows the experimental design of the therapeutic effect of BNT162b2 in an advanced tumor model. [Figure 9E] FIG. 9E shows tumor photographs of the therapeutic effect of BNT162b2 and combination therapy in an advanced tumor model. [Figure 9F] FIG. 9F shows the tumor volumes of different groups of mice at the endpoint in the experimental design described in FIG. 9D. [Figure 10A] FIG. 10A shows IFN-γ ELISpot showing tumor antigen-specific T cell responses after intratumoral BNT treatment in an MB49 tumor model. [Figure 10B] Figure 10B shows a bar graph summarizing ELISpot data in the MB49 tumor model comparing tumor antigen-specific T cell responses in the BNT162b2 intratumoral treatment group and the PBS intratumoral control group. An MB49 tumor neoantigen peptide pool was used as the tumor antigen stimulator. BNT im-BNT it: splenocytes from mice intramuscularly and intratumorally administered BNT162b2. BNT im-PBS it: splenocytes from mice intramuscularly vaccinated with BNT162b2 and intratumorally administered PBS. [Figure 11A] Figure 11A shows the experimental design of the therapeutic effect of HKU1 CoV spike protein-encoding mRNA vaccine in a subcutaneous melanoma model. [Figure 11B] Figure 11B shows tumor growth curves for the treatment group administered intratumorally with HKU1 CoV spike protein-encoding mRNA vaccine versus the control group administered intratumorally with PBS for the experiment described in Figure 11A. [Figure 11C] FIG. 11C shows the tumor growth curve of the PBS intratumoral administration control group for the experiment described in FIG. 11A. [Figure 11D] Figure 11D shows the tumor growth curves of the treatment group administered intratumorally with an mRNA vaccine encoding the HKU1 CoV spike protein for the experimental design described in Figure 11A. [Figure 11E] FIG. 11E shows comparative photographs of tumors from the treatment and control groups of the experiment described in FIG. 11A. [Figure 12A] FIG. 12A shows the experimental design of the therapeutic effect of an SEA protein-encoding mRNA vaccine in a subcutaneous melanoma model. [Figure 12B]Figure 12B shows tumor growth curves for the experiment described in Figure 12A for the treatment group receiving intramuscular administration of an SEA protein-encoding mRNA vaccine followed by intratumoral administration of an SEA protein-encoding mRNA vaccine, versus the control group receiving intramuscular administration of an SEA protein-encoding mRNA vaccine followed by intratumoral administration of PBS, versus the untreated group, versus the group receiving intratumoral administration of SEA only. [Figure 12C] FIG. 12C shows the tumor growth curve of the control group administered intramuscularly with SEA protein-encoding mRNA vaccine followed by intratumoral administration of PBS for the experiment described in FIG. 12A. [Figure 12D] FIG. 12D shows tumor growth curves for the treatment group in which an SEA protein-encoding mRNA vaccine was administered intramuscularly followed by intratumoral administration of an SEA protein-encoding mRNA vaccine, for the experimental design described in FIG. 12A. [Figure 12E] FIG. 12E shows the untreated control group for the experiment described in FIG. 12A. [Figure 12F] FIG. 12F shows the tumor growth curves of the treatment group that received only intratumoral administration of the SEA protein-encoding mRNA vaccine for the experimental design described in FIG. 12A. [Figure 12G] FIG. 12G shows comparative photographs of tumors from the treatment and control groups of the experiment described in FIG. 12A. DETAILED DESCRIPTION OF THE INVENTION

[0017] Vaccine-based immunotherapy for cancer faces multiple obstacles: weak tumor antigens, an immunosuppressive tumor microenvironment, and insufficient recruitment of immune cells. Immune evasion by cancer cells after anti-tumor immunotherapy further reduces therapeutic efficacy. To overcome these obstacles, the present invention provides a strategy for cancer treatment by increasing the antigenicity or immunogenicity of tumors in a subject based on the subject's immune response to the vaccine. As an example, a COVID-19 mRNA vaccine was used in this study for the purpose of cancer treatment. However, those skilled in the art will readily understand that other pathogen-derived antigens have similar cancer therapeutic potential as BNT162b2 in this study. Intratumoral administration of a COVID-19 mRNA vaccine reactivates anti-spike memory immune responses and directs them to tumors expressing the spike protein. Importantly, intratumoral vaccination with a COVID-19 mRNA vaccine potently inhibits the growth of tested cancers and extends the lifespan of tumor-bearing mice. Careful analysis indicates that COVID-19 mRNA vaccines can induce potent T cell responses against tumor-specific antigens other than the spike protein, recruit immune cells to tumors, and modify the tumor immune microenvironment. Due to the SARS-CoV-2 pandemic, a large proportion of the population has been vaccinated against COVID-19. This COVID-19 vaccine cancer therapy offers great potential for cancer treatment of various cancer types and may be rapidly translated into clinical use. Furthermore, the versatility of this cancer immunotherapy strategy was verified by using HKU1 CoV spike protein-encoding mRNA vaccines and SEA protein-encoding mRNA vaccines. The results demonstrate that this cancer immunotherapy strategy can be effectively applied using different mRNAs suitable for increasing tumor antigenicity or immunogenicity.

[0018] In the present invention, a new cancer treatment is developed. This method utilizes the BNT162b2 vaccine not only to induce a potent antitumor immune response but also to convert the tumor-suppressive microenvironment from "cold" to "hot." To achieve this goal, it is hypothesized that if spike proteins or other pathogen-derived antigenic proteins are expressed in tumors as tumor neoantigens by intratumoral administration of BNT162b2 or other antigenic protein-encoding mRNA vaccines, the tumors may be more easily recognized by the immune system of individuals intramuscularly vaccinated with BNT162b2 or other antigenic protein-encoding mRNA vaccines. On the other hand, the expression of spike proteins or other antigenic proteins in tumors triggers the rapid activation of anti-spike or anti-pathogen antigen memory immune responses. Additionally, intratumoral administration of BNT162b2 or other antigenic protein-encoding mRNA vaccines also helps convert the suppressive tumor microenvironment (Figure 1).

[0019] The present invention provides a method for increasing the antigenicity or immunogenicity of a tumor in a subject. In one embodiment, the method comprises delivering lipid nanoparticles carrying mRNA sequences encoding one or more pathogen antigen proteins to the tumor.

[0020] In one embodiment, the subject is a human, animal, or any organism in need of increasing the antigenicity or immunogenicity of one or more tumors.

[0021] In one embodiment, the one or more pathogen antigenic proteins are antigens targeted in a vaccine previously administered to the subject or a vaccine to be administered to the subject.

[0022] In one embodiment, the one or more pathogen antigenic proteins are selected from the group consisting of coronavirus proteins, human papillomavirus proteins, respiratory syncytial virus proteins, human immunodeficiency virus proteins, hepatitis virus proteins, and influenza virus proteins.

[0023] In one embodiment, the lipid nanoparticle vector is delivered by intratumoral administration.

[0024] In one embodiment, the intratumoral administration is an mRNA vaccine.

[0025] In one embodiment, the mRNA vaccine is selected from the group consisting of BioNTech BNT162b2 (Comirnaty), BioNTech COVID-19 Omicron strain bivalent vaccine (Comirnaty Original / Omicron BA.4-5), and Moderna vaccine: mRNA-1273 (Spikevax).

[0026] In one embodiment, the mRNA vaccine is an mRNA vaccine against one or more antigens selected from the group consisting of a bacterial antigen, a bacterial superantigen, a viral antigen, and a viral superantigen.

[0027] In one embodiment, the bacterial antigen is CT529, CT511, CT461 (C. trachomatis), VirB9-1, VirB9-2, VirB10, conjugative transfer protein (CTP) (A. marginale), Erum0660, Erum2330, Erum2540, Erum2580, Erum5000 (E. ruminantum), OMP-19 (E. muris, E. chaffeensis), OmpA, OmpB, Adr2, YbgF, RP403, RP598 RP739, RP778, RP177, T4SS-associated protein C. Burnetii), CBU_1835 / protoporphyrinogen oxidase, CBU_1513 / protoporphyrinogen oxidase, CBU_1398 / SucB, CBU_0718, and CBU_0307 / outer membrane protein.

[0028] In one embodiment, the viral antigen is selected from the group consisting of: a) influenza virus hemagglutinin (HA) antigen, neuraminidase (NA) antigen, nucleoprotein (NP), nonstructural protein 1 (NSP1), nonstructural protein 2 / nuclear export protein (NS2 / NEP), polymerase basic protein 1 / 2 / 1-F2 (PB1 / 2 / 1-F2), matrix protein 1, and matrix protein 2 of influenza virus; b) hepatitis A virus antigen (HAV) or hepatitis B virus (HBV) antigen; c) human papillomavirus (HPV) antigen, including the major capsid (L1) protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, or 58; d) nucleoprotein (NP) of influenza virus; e) a rabies vaccine antigen comprising a protein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), or an RNA-dependent RNA polymerase; f) a varicella-zoster vaccine antigen comprising a varicella-zoster virus glycoprotein E; f) a smallpox vaccine antigen comprising a B5 antigenic region (pB5); g) a varicella-zoster virus vaccine antigen comprising a varicella-zoster virus glycoprotein E antigen; and h) a vaccine antigen against one or more of yellow fever, monkeypox, polio, mumps, rubella, or measles; and i) a respiratory syncytial virus antigen comprising seven structural proteins (G, F, M1, M2, P, L, N) and three non-structural proteins (NS1, NS2, SH).

[0029] In one embodiment, the bacterial or viral superantigen is selected from the group consisting of toxic shock syndrome toxin-1 (TSST-1), streptococcal pyrotoxin (Spe), staphylococcal enterotoxin (SE), ETEC enterotoxin, streptococcal superantigen (SSA), streptococcal mitogen-enhancing toxin (SMEZ) 1 and 2, and the human endogenous retroviruses HERV-K18.1, HERV-W, and MSRV.

[0030] In one embodiment, the mRNA vaccine is an mRNA vaccine against one or more antigens selected from the group consisting of coronavirus proteins, human papillomavirus proteins, respiratory syncytial virus, human immunodeficiency virus proteins, hepatitis virus, and influenza virus proteins.

[0031] In one embodiment, the coronavirus protein comprises one or more of a spike protein, envelope protein, membrane protein, nucleocapsid protein, accessory protein, or nonstructural protein of SARS-CoV-1, SARS-CoV-2, a common cold coronavirus, MERS, or coronavirus HuPn-2018.

[0032] In one embodiment, the common cold coronavirus comprises one or more of HCoV-OC43, HcoV-HKU1, HcoV-229E, or HcoV-NL63.

[0033] In one embodiment, the HIV protein comprises one or more of an HIV (human immunodeficiency virus) envelope protein, structural protein, protease, integrase, reverse transcriptase, viral protein u, viral infectivity factor, viral protein r, P6, negative regulatory factor, regulator of virion, or transactivator of transcription.

[0034] In one embodiment, the influenza virus proteins include one or more of influenza virus hemagglutinin (HA), neuraminidase (NP), nucleoprotein (NP), nonstructural protein 1 (NSP1), nonstructural protein 2 / nuclear export protein (NS2 / NEP), polymerase basic protein 1 / 2 / 1-F2 (PB1 / 2 / 1-F2), matrix protein 1, or matrix protein 2.

[0035] In one embodiment, the method further comprises vaccinating the subject with one or more pathogen antigenic proteins or variants thereof.

[0036] In one embodiment, the subject has previously been vaccinated against one or more pathogen antigenic proteins or variants thereof.

[0037] In one embodiment, the tumor is selected from the group consisting of melanoma, breast cancer, bladder cancer, colon cancer, gastric cancer, pancreatic cancer, blood cancer, lung cancer, and liver cancer.

[0038] The present invention also provides a method of treating a subject having one or more tumors. In one embodiment, the method comprises increasing the antigenicity or immunogenicity of at least one tumor of the one or more tumors using a method of the present invention.

[0039] In one embodiment, the at least one tumor is a primary tumor or a secondary tumor.

[0040] In one embodiment, the one or more tumors include a primary tumor and a secondary tumor.

[0041] In one embodiment, the method further comprises co-administering an immune checkpoint inhibitor to the subject.

[0042] In one embodiment, the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, or intravenously, hi another embodiment, the immune checkpoint inhibitor is administered by any route of administration appropriate for the immune checkpoint inhibitor.

[0043] In one embodiment, the immune checkpoint inhibitor is one or more selected from the group consisting of anti-PD-1, anti-CTLA4, and anti-PD-L1.

[0044] In one embodiment, the subject is a patient with advanced cancer. In another embodiment, the present invention also demonstrated therapeutic effects in advanced cancer.

[0045] The present invention further provides intratumoral administration for treating a tumor in a subject. In one embodiment, the intratumoral administration comprises lipid nanoparticles loaded with mRNA encoding one or more pathogen antigen proteins.

[0046] In one embodiment, the one or more pathogen antigenic proteins are antigens targeted in a vaccine previously administered to the subject or a vaccine to be administered to the subject.

[0047] In one embodiment, the tumor is selected from the group consisting of melanoma, breast cancer, bladder cancer, colon cancer, gastric cancer, pancreatic cancer, blood cancer, lung cancer, and liver cancer.

[0048] In one embodiment, the intratumoral administration is an mRNA vaccine against one or more antigens selected from the group consisting of bacterial antigens, bacterial superantigens, viral antigens, and viral superantigens.

[0049] In one embodiment, the one or more pathogen antigen proteins are selected from the group consisting of bacterial antigens, bacterial superantigens, viral antigens, and viral superantigens.

[0050] In one embodiment, the bacterial antigen is CT529, CT511, CT461 (C. trachomatis), VirB9-1, VirB9-2, VirB10, conjugative transfer protein (CTP) (A. marginale), Erum0660, Erum2330, Erum2540, Erum2580, Erum5000 (E. ruminantum), OMP-19 (E. muris, E. chaffeensis), OmpA, OmpB, Adr2, YbgF, RP403, RP598, RP739, RP778, RP177, T4SS-associated protein (C. Burnetii), CBU_1835 / protoporphyrinogen oxidase, CBU_1513 / protoporphyrinogen oxidase, CBU_1398 / SucB, CBU_0718, and CBU_0307 / outer membrane protein.

[0051] In one embodiment, the viral antigens are selected from the group consisting of: a) influenza virus hemagglutinin (HA) antigen or neuraminidase (NA) antigen; b) hepatitis A virus antigen (HAV) or hepatitis B virus (HBV) antigen; c) human papillomavirus (HPV) antigens including the major capsid (L1) protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, or 58; d) nucleoprotein (N), phosphoprotein (P), matrix protein (MTP), and / or IL-1. e) a rabies vaccine antigen comprising a protein (M), a glycoprotein (G), or an RNA-dependent RNA polymerase; f) a varicella-zoster vaccine antigen comprising a varicella-zoster virus glycoprotein E; f) a smallpox vaccine antigen comprising a B5 antigenic region (pB5); g) a varicella-zoster virus vaccine antigen comprising a varicella-zoster virus glycoprotein E antigen; and h) a vaccine antigen against one or more of yellow fever, polio, mumps, or measles.

[0052] In one embodiment, the bacterial or viral superantigen is selected from the group consisting of toxic shock syndrome toxin-1 (TSST-1), streptococcal pyrotoxin (Spe), staphylococcal enterotoxin (SE), ETEC enterotoxin, streptococcal superantigen (SSA), streptococcal mitogen-enhancing toxin (SMEZ) 1 and 2, and the human endogenous retroviruses HERV-K18.1, HERV-W, and MSRV.

[0053] In one embodiment, the one or more pathogen antigenic proteins are selected from the group consisting of a coronavirus protein, an HIV protein, and an influenza virus protein.

[0054] In one embodiment, the coronavirus protein comprises one or more of a spike protein, envelope protein, membrane protein, nucleocapsid protein, accessory protein, or nonstructural protein of SARS-CoV-1, SARS-CoV-2, a common cold coronavirus, MERS, or coronavirus HuPn-2018.

[0055] In one embodiment, the common cold coronavirus comprises one or more of HCoV-OC43, HcoV-HKU1, HcoV-229E, or HcoV-NL63.

[0056] In one embodiment, the HIV protein comprises one or more of an HIV (human immunodeficiency virus) envelope protein, structural protein, protease, integrase, reverse transcriptase, viral protein u, viral infectivity factor, viral protein r, P6, negative regulator, virion regulator, or transcriptional activator.

[0057] In one embodiment, the influenza virus proteins include one or more of influenza virus hemagglutinin (HA), neuraminidase (NP), nucleoprotein (NP), nonstructural protein 1 (NSP1), nonstructural protein 2 / nuclear export protein (NS2 / NEP), polymerase basic protein 1 / 2 / 1-F2 (PB1 / 2 / 1-F2), matrix protein 1, or matrix protein 2.

[0058] In one embodiment, the one or more pathogen antigenic proteins are antigens targeted in a vaccine.

[0059] In one embodiment, the vaccine is a Bacillus Calmette-Guerin vaccine (live attenuated Mycobacterium bovis). bovis), diphtheria-tetanus-pertussis vaccine (DTaP) (tetanus toxoid, attenuated diphtheria toxoid, and adsorbed purified acellular pertussis), MenACWY vaccine (MenACWY vaccine provides protection against four types of bacteria that can cause meningitis: groups A, C, W, and Y meningococcus), MenB vaccine, Hib / MenC vaccine (Haemophilus influenzae type b (Hib) and meningococcus C), smallpox vaccine (variola virus (often called smallpox virus)), monkeypox vaccine (Empoxvirus), polio vaccine, inactivated polio vaccine, human hepatitis A vaccine, human hepatitis B vaccine, measles-mumps-rubella vaccine (MMR), and varicella vaccine (Varicella / chickenpox).

[0060] In one embodiment, antigens targeted by the Bacillus Calmette-Guerin vaccine include one or more of the cell surface glycolipoproteins MPB63, MPB70, MPB83, the outer membrane channel protein CpnT, and other antigens.

[0061] In one embodiment, the antigens targeted by the diphtheria-tetanus-pertussis vaccine (DTaP) include one or more of the following: diphtheria toxin (consisting of two subunits linked by a disulfide bridge, known as toxin A and toxin B); tetanus toxoid (consisting of one light chain (approximately 50 kDa) and one heavy chain (approximately 100 kDa)); pertussis toxin (a hexamer consisting of five different subunits: S1, S2, S3, two S4, and S5).

[0062] In one embodiment, the antigens targeted by the MenACWY vaccine include one or more of the antigens in groups A, C, W, and Y meningococcus.

[0063] In one embodiment, the antigens targeted in the MenB vaccine include one or more of the antigens in group B meningococcus.

[0064] In one embodiment, the antigens targeted in the Hib / MenC vaccine include one or more of the antigens in Haemophilus influenzae type b (Hib) and meningitis type C.

[0065] In one embodiment, antigens targeted in a smallpox vaccine include one or more of virion membrane protein OPG144 precursor / OPG141 / OPG135 / OPG105 / OPG143 / OPG140, envelope protein H3 / OPG155, envelope phospholipase OPG057, superinfection exclusion protein (OPG040), chemokine-binding protein (OPG001 (B29R, C23L)), cell surface-binding protein OPG105, major core protein OPG136 precursor / OPG130 / VP8 / D2, scaffold protein D13, and all proteins in smallpox.

[0066] In one embodiment, antigens targeted in the monkeypox vaccine include one or more of OPG105, OPG077, DNA-dependent RNA polymerase 132 kDa polypeptide (RPO132), profilin (OPG171), p28, OPG101(TK), OPG188(B4R), Cu-Zn superoxide dismutase-like protein (A46R), virion membrane protein OPG140 / OPG139 / OPG135 / OPG144 precursor, core protein OPG142 / OPG138 / OPG130 / OPG129, and other antigens in monkeypox.

[0067] In one embodiment, the antigens targeted by the polio vaccine or inactivated polio vaccine include one or more of capsid proteins VP0 / VP1 / VP2 / VP3 / VP4, protease 2A, protein 2B, protein 2C, protein 3AB, protein 3A, protein 3CD, protease 3C, and other antigens.

[0068] In one embodiment, antigens targeted in a human hepatitis A vaccine include one or more of capsid proteins VP0 / VP1 / VP2 / VP3 / VP4, protein VP1-2A, protein 2B, protein 2BC, protein 2C, protein 3ABC, protein 3A, protein 3CD, protease 3C viral protein genome-linked, and other antigens.

[0069] In one embodiment, the antigens targeted in the human hepatitis B vaccine include one or more of external core antigen C, large envelope protein S, capsid protein C, protein P, protein X, and other antigens.

[0070] In one embodiment, the antigens targeted by the measles-mumps-rubella (MMR) vaccine include one or more measles antigens selected from the group consisting of nonstructural protein V, nucleoprotein N, hemagglutinin glycoprotein H, fusion glycoprotein F0, RNA-dependent RNA polymerase L, matrix protein M, phosphoprotein P / V, and other antigens; one or more mumps antigens selected from the group consisting of fusion glycoprotein F0, nonstructural protein V, RNA-dependent RNA polymerase L, hemagglutinin-neuraminidase HN, nucleoprotein N (NP), small hydrophobic protein (SH), and other antigens; and one or more rubella antigens selected from the group consisting of nonstructural polyprotein p200, structural polyproteins, and other antigens.

[0071] In one embodiment, the antigens targeted in the varicella vaccine are envelope glycoprotein B, envelope glycoprotein E, capsid scaffold protein 33, envelope protein US9, envelope glycoprotein C, envelope glycoprotein I, large tegument protein deneddylase, major viral transcription factor ICP4 homolog, envelope glycoprotein H, triplex capsid protein 1, small capsomere-interacting protein SCP, envelope glycoprotein M, envelope glycoprotein N, major DNA-binding protein DBP, envelope glycoprotein K, cytoplasmic envelope protein 1 (CEP1), and envelope glycoprotein B. The antigens include one or more of: envelope glycoprotein 1, cytoplasmic encapsulation protein 2, cytoplasmic encapsulation protein 3, envelope glycoprotein L, structural protein 1, membrane protein 0, envelope glycoprotein H, nuclear egress protein 2, capsid vertex component 1, capsid vertex component 2, packaging protein UL32, portal protein, and other antigens.

[0072] The present invention also provides a kit for treating a tumor in a subject. In one embodiment, the kit comprises an intratumoral administration of the present invention.

[0073] In one embodiment, the kit further comprises a vaccine for vaccinating the subject with one or more pathogen antigen proteins before or after administering the intratumoral dose.

[0074] In one embodiment, the kit further comprises an immune checkpoint inhibitor. [Example]

[0075] Example 1 Methods and Materials 1) Cancer cell lines and vaccines The murine 4T1 breast cancer cell line, the murine B16F10 melanoma cell line, and the murine MB49 bladder cancer cell line were used.

[0076] 4T1 and B16F10 cell lines were cultured in RPMI Medium 1640 with 10% fetal bovine serum (FBS). MB49 and CT26 cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). All cell lines were cultured at 37°C in 5% CO2.

[0077] We obtained the remaining Pfizer / BioNTech BNT162b2 mRNA vaccine (Comirnaty) in its original vials after patient use from the Department of Health of the Hong Kong Special Administrative Region Government. We developed a lipid nanoparticle-encapsulated HKU1 CoV spike protein-encoding mRNA vaccine and a lipid nanoparticle-encapsulated SEA protein-encoding mRNA vaccine in-house.

[0078] 2) Mice, tumor challenge, and vaccination 3×10 5 pcs and 2 x 10 5 B16F10 tumor cells were subcutaneously inoculated into female C57BL / 6 mice in the "pre-cancer vaccination with BNT162b2" and "post-cancer vaccination with BNT162b2" models. Diluted BNT162b2 (30µg mRNA in 1.8ml saline) was administered intramuscularly at 50µl per mouse before or after tumor inoculation. Each mouse received 50µl of diluted BNT162b2 intratumorally, while control mice received 50µl of PBS intratumorally.

[0079] 1×10 5MB49 tumor cells were subcutaneously inoculated into male C57BL / 6 mice in the "cancer inoculation followed by BNT162b2 vaccination" model. 50 μl of diluted BNT162b2 was administered intramuscularly per mouse after tumor inoculation. Each mouse received 50 μl of diluted BNT162b2 intratumorally, while control mice received 50 μl of PBS intratumorally.

[0080] 2×10 5 4T1 tumor cells were subcutaneously inoculated into female BalB / c mice in the "cancer inoculation followed by BNT162b2 vaccination" model. 50 μl of diluted BNT162b2 was administered intramuscularly per mouse after tumor inoculation. Each mouse received 50 μl of diluted BNT162b2 intratumorally, while control mice received 50 μl of PBS intratumorally.

[0081] 1.75×10 5 CT26 tumor cells were subcutaneously inoculated into female BalB / c mice in the "cancer inoculation followed by BNT162b2 vaccination" model. 50 μl of diluted BNT162b2 was administered intramuscularly per mouse after tumor inoculation. Each mouse received 50 μl of diluted BNT162b2 intratumorally, while control mice received 50 μl of PBS intratumorally.

[0082] 3) Cytokine profiling Blood samples and tumor tissues were collected from BNT162b2-treated and control mice for cytokine profiling at endpoint (BioLegend, LEGENDplex MU Th Cytokine Panel [12-plex], 741043 and LEGENDplex Mouse Inflammation Panel [13-plex], 740446).

[0083] 4) RNA-seq and data analysis Total RNA was extracted from tumor tissue using Trizol and chloroform, followed by precipitation in 2-propanol and ethanol. Gene expression was quantified using "Salmon" directly from bulk transcriptome sequencing data. Differential gene expression analysis was performed using DESeq2 with default parameters. Gene Ontology (GO) analysis was limited to immune-related biological processes (ontologies belonging to GO:0002376), and GO terms were integrated according to the similarity between each ontology using clusterProfiler. Functional enrichment of immune cells was performed using xCell using mouse gene expression profiles with mouse gene symbols transferred to human gene symbols.

[0084] 5) ELISA assay A 96-well ELISA plate (JET BIOFIL, FEP-100-096) was coated overnight with 0.1 μg / ml spike protein or 10 μg / ml tumor membrane protein in coating buffer. The plate was blocked with blocker in 1x TBST and incubated at room temperature for 2 hours. Serum from each group was diluted 1:150 in blocker buffer for spike protein ELISA and 1:5 in blocker buffer for tumor membrane protein ELISA, and the plate was incubated at room temperature for 2 hours. After washing three times with 1x TBST, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000, GE Healthcare) was added for 1 hour at room temperature. The plate was then washed five times with 1x TBST. 100 μl of HRP substrate (TMB Chromogen Solution (for ELISA), 002023) was added to each well. After 15 minutes of incubation, the reaction was terminated by adding 50 μl of 2 M H2SO4 solution, and the ELISA plate was analyzed at 450 nm wavelength using an absorbance microplate reader (Varioskan Flash, Thermo Scientific). Data analysis was performed using GraphPad Prism, and the ELISA AUC was calculated.

[0085] 6) ELISpot assay ELISpot assays were performed using the Mouse IFN-γ ELISpot PLUS (HRP) Kit (Mabtech, 3321-4HST-2). Splenocytes were obtained from sacrificed mice in the BNT intratumoral administration and control groups. Splenocytes were stimulated with spike protein peptide pools, B16F10 cell lysates, and ML-1 cell lysates, respectively. Splenocytes were incubated with stimulators overnight at 37°C. ELISpot plates were imaged using a CTL ImmunoSpot ELISpot Analyzer. Data were statistically analyzed using Student's t-test.

[0086] 7) Flow cytometry Tumor tissues were obtained from sacrificed mice in each group, digested with collagenase I and IV (Sigma-Aldrich, SCR103 and C5138), and single cells were stained with anti-mouse CD45-Brilliant Violet 605, anti-mouse CD3-FITC, and anti-mouse CD19-Brilliant Violet 421 (Biolegend). Flow cytometry and data analysis were performed using an Agilent NovoCyte Quanteon analyzer.

[0087] 8) Immunofluorescence staining Tumors from sacrificed mice were embedded in Tissue-Tek OCT Compound (SAKURA, 4583) and frozen sections were prepared using a Thermo NX50 microscope. After blocking with 5% BSA, tumor sections were stained with SARS Coronavirus Spike Protein Polyclonal Antibody (Thermo Fisher, PA1-41165) overnight at 4°C. The slides were washed three times with 1x TBST for 5 minutes each time. The slides were then stained with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Fisher, A-11008) for 1 hour at room temperature. The slides were then washed three times with 1x TBST for 5 minutes each time. The slides were then stained with DAPI for 20 minutes at room temperature. The slides were then washed three times with 1x TBST for 5 minutes each time. Images were captured using a Carl Zeiss LSM880 microscope.

[0088] 8) Construction of OVA-high-expressing tumor cell lines and cell line transfection Chicken ovalbumin lentivirus was packaged in the LentiX cell line using the plasmid shown in Figure 2. The chicken ovalbumin lentivirus was harvested twice every 24 hours and concentrated by ultracentrifugation at 126,100 x g for 2 hours at 4°C. The B16F10-OVA cell line was constructed by infection with the concentrated chicken ovalbumin lentivirus. Transfected B16F10 cells with high levels of OVA expression were sorted by flow cytometry (BD FACSAria Fusion).

[0089] 1.2ug BNT162b2 mRNA 2-3 x 10 6 The exosomes were directly added to B16F10-OVA, and the medium was changed 8 hours after transfection. The medium was collected 2-3 times every 24 hours, and all collected medium was stored at 4°C. All exosomes in the medium were separated by ultracentrifugation.

[0090] 9) Exosome isolation Cells were cultured in RPMI-1640 containing 1% penicillin-streptomycin and 10% exosome-depleted FBS (Thermo Scientific, A2720803), and the medium was harvested three times every two days. First, the medium was centrifuged at 2,000 g for 20 minutes. The supernatant was then filtered through a 0.45 μm filter, and the flow-through fraction was centrifuged at 10,000 g for 30 minutes to completely remove cell debris. The supernatant was then ultracentrifuged at 100,000 g for 70 minutes. The entire supernatant was then discarded, and the pellet at the bottom of the ultracentrifuge tube was resuspended in PBS and again ultracentrifuged at 100,000 g for 70 minutes. The pellet in PBS was collected and stored at -80°C for exosome isolation.

[0091] 10) Western Blot Exosomes derived from B16F10 or B16F10-OVA were dissolved in Mammalian Protein Extraction Reagent (78501, Thermo Scientific) with a protease inhibitor cocktail (04693132001, Roche). Proteins were separated by SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% Blotting-Grade Blocker (BIO-RAD, 1706404) for 60 minutes, the PVDF membrane was incubated with primary antibodies overnight at 4°C. Secondary antibodies, horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (ThermoFisher, 31460, 31430), were then incubated with the membrane for 1 hour at room temperature. Protein bands were finally detected using an enhanced chemiluminescence (ECL) detection system (ThermoFisher, Pierce ECL Western Blotting Substrate, 32106).

[0092] Example 2 result 1) BNT162b2 has potent anticancer effects. To investigate whether BNT162b2 can also be used as a cancer therapeutic agent, the therapeutic effect of BNT162b2 was first tested against B16F10 melanoma in BNT162b2-vaccinated mice. Two treatment schemes were established. In the first scheme, mice were vaccinated with BNT16b2 before B16F10 cancer cell transplantation. In the other scheme, mice were inoculated with B16F10 cancer cells before BNT162b2 vaccination. In the first scheme, mice were intramuscularly administered with BNT162b2 vaccine twice, 21 days apart, and 3 × 10 5 B16F10 tumors were subcutaneously inoculated. Then, on days 5, 10, and 17 after tumor inoculation, the same volume of BNT162b2 was administered intratumorally to the treatment group, and PBS was administered intratumorally to the control group (Figure 3A). During treatment, tumor volume was measured every 3 days (Figures 3B, 3C, and 3D). On day 20, tumor growth was significantly inhibited in the BNT it group compared with the PBS it group (Figures 3B, 3C, 3D, and 3E). In the second scheme, 2 × 10 5 B16F10 cancer cells were first inoculated subcutaneously. Subsequently, intramuscular vaccination with BNT162b2 was performed on days 2 and 5 after tumor inoculation, followed by intratumoral administration of BNT162b2 on days 10, 15, and 20 after tumor inoculation (Figure 4A). Tumor volume was measured every 2 days (Figures 4B, 4C, and 4D). Mice were sacrificed on day 23. The tumor growth rate in the BNT it group showed that tumor growth was significantly suppressed compared with the PBS it group (Figures 4B, 4C, 4D, and 4E). In addition, the therapeutic effect was also tested in the MB49 subcutaneous model (Figure 4F), the 4T1 breast cancer orthotopic model (Figure 4K), and the CT26 subcutaneous model (Figure 4R). Tumor growth was monitored and measured every 2 days. The results showed that BNT162b2 treatment could effectively inhibit tumor growth of MB49 bladder cancer (Figures 4G, 4H, 4I, 4J), 4T1 breast cancer (Figures 4L, 4M, 4N, 4O), and CT26 colon cancer (Figures 4S, 4T, 4U, 4V). In addition to the therapeutic effect, the results also demonstrated that BNT162b2 treatment could inhibit breast cancer lung metastasis in a 4T1 orthotopic model (Figures 4P, 4Q).

[0093] 2) BNT162b2 cancer therapy induces potent anti-tumor-specific T cell immune responses. To test specific anti-tumor immune responses, tumor, spleen, and serum samples were collected after BNT162b2 cancer vaccine treatment in the B16F10 model (Figure 5A). We first detected spike protein expression in tumor tissue after intratumoral administration of BNT162b2. Immunofluorescent staining of spike protein in tumor sections showed that SARS-CoV-2 spike protein was expressed at very high levels on the surface and intracellularly in tumor cells in BNT162b2-treated tumors (Figures 5D and 5E). This demonstrates that our method successfully induces high levels of spike protein expression in tumor cells as an artificial "tumor-specific antigen" after BNT162b2 administration.

[0094] Next, the results showed that BNT162b2 treatment mobilized a large number of tumor-infiltrating T cells (Figures 5B, 5C, and 5F). We then determined whether the memory immune response induced by previous BNT162b2 vaccination could induce a potent immune response against tumor cells. To detect anti-spike and other anti-tumor T cell responses, splenocytes were collected after all vaccination and treatment steps, followed by ELISpot assays using the spike protein peptide pool or cell lysates of B16F10 cells not treated with BNT162b2 as stimulators, respectively. The results demonstrated potent anti-spike protein antibodies (Figure 5H) and T cell activity (Figures 5I and 5J) against the SARS-CoV-2 spike protein. Furthermore, specific T cell responses against B16F10 cell lysates containing no spike protein were occasionally detected in mice treated with BNT162b2 intratumorally, but not in the PBS-treated group. This indicates that widespread tumor antigen spread was induced by intratumoral administration of the BNT162b2 vaccine. This tumor antigen spreading was also confirmed in the MB49 model by MB49 neoantigen stimulation (Figures 10A and 10B). Interestingly, T cell responses targeting the hepatoma cell line ML-1, which is unrelated to B16F10 cells, were also detected. This indicates that BNT162b2-induced antigen spreading also triggered antitumor T cell responses against several common tumor antigens (Figures 5I and 5J).

[0095] In addition to T cell responses, we also investigated the role of B cell and antibody responses in this BNT162b2 cancer therapy. To study B cell-related responses, we tested BNT162b2 cancer therapy in μMT mice, which lack mature B lymphocytes

[27] . A subcutaneous B16F10 melanoma model was established in these μMT mice, and vaccine therapy was performed as a second treatment modality for B16F10 melanoma (Figure 5K). The results showed that the therapeutic effect of BNT162b2 in the B16F10 melanoma model in μMT mice was comparable to that in wild-type mice (Figures 5L-5O). However, the therapeutic effect was not significant in the Rag1 mice, which do not produce mature B lymphocytes or mature T lymphocytes. - / -These results suggest that BNT162b2 cancer therapy is independent of mature B cells, but dependent on T cells.

[0096] Furthermore, splenocytes from mice vaccinated twice with BNT162b2 or from unvaccinated mice were incubated with BNT162b2-transfected B16F10. After 20 hours of incubation, rapid T cell activation was detected by flow cytometry (Figure 5P). Flow cytometry results showed that after incubation, BNT162b2-transfected B16F10 could rapidly activate T cells by upregulating CD69 expression and increasing IFN-γ secretion (Figures 5Q and 5R). The results suggested that memory T cells induced by BNT162b2 could be rapidly activated by re-contact with spike protein-expressing tumor cells.

[0097] These results indicated that BNT162b2 cancer therapy primarily relies on T cell responses and induces rapid T cell activation that targets tumor cells expressing the spike protein through BNT162b2 transfection, ultimately triggering an initial attack on tumor cells and then inducing the release of additional tumor antigens, potentially inducing a broader tumor-specific immune response through antigen spread.

[0098] 3) Systemic and intratumoral immune activation can be induced by BNT162b2 cancer therapy. To further investigate whether BNT162b2 treatment could activate intratumoral immune cells and systemic immunity to induce a potent antitumor immune response, we first examined intratumoral immune cells by immunofluorescence staining and flow cytometry (Figures 5G-1, 5G-2, 5G-3, and 5G-4). These results showed that BNT162b2 treatment induced the recruitment of more intratumoral immune cells, including T cells, NK cells, antigen-presenting cells (APCs), macrophages, and neutrophils (Figures 5F, 5G-1, 5G-2, 5G-3, and 5G-4). In addition, intratumoral administration of BNT162b2 also induced the reversal of the tumor-suppressive microenvironment. On the one hand, BNT162b2 treatment improved intratumoral expression of MHC-I / MHC-II and inflammatory cytokine levels in tumors and blood (Figures 6C-1, 6C-2, and 6D). On the other hand, BNT162b2 treatment also reversed intratumoral macrophage phenotype from protumor M2 to antitumor M1 (Figures 6A and 6B). Furthermore, we performed transcriptome sequencing to investigate intratumoral immune activation (Figure 5S). We found that the differential gene expression profiles between the intratumoral BNT162b2-treated group (BNT im-BNT it group) and the control groups (PBS it group and BNT im-PBS it group) at different time points were largely non-overlapping (Figure 5T). This suggests that dynamic and significant changes occurred in the TME. Gene ontology (GO) enrichment analysis revealed that myeloid leukocytes, including neutrophils and macrophages, were activated throughout the entire course of intratumoral BNT162b2 treatment (Figure 5U). Results also showed a significant upregulation of CD28 gene expression and B7 family members (CD80, CD86), with a high correlation between their gene expression. This suggests that costimulatory signals from antigen-presenting cells may be involved in T cell activation (Figure 5V).

[0099] 4) BNT162b2 cancer therapy induces potent tumor antigen dissemination, increased tumor-derived heat shock protein secretion, and tumor antigen-containing exosome secretion by dead tumor cells. The present invention demonstrated that BNT162b2 cancer therapy can induce potent tumor antigen spreading and induce strong tumor-specific T cell responses by killing tumor cells (Figures 5I and 5J).

[0100] Furthermore, another study demonstrated that circulating exosomes containing the SARS-CoV-2 spike protein could be detected after SARS-CoV-2 mRNA vaccination

[29] . This finding led to the hypothesis that intratumoral vaccination with BNT162b2 stimulates tumors to release exosomes containing not only the SARS-CoV-2 spike protein but also tumor-specific antigens. The release of these exosomes could induce antigen spread and tumor-specific T cell responses, which in turn could lead to tumor suppression. To test whether BNT162b2 stimulates the release of tumor antigen-containing exosomes, we transfected the B16F10-OVA tumor cell line (B16F10-OVA: a B16F10 cell line stably expressing OVA protein) with BNT162b2. The supernatant of the B16F10-OVA culture medium was collected, and total exosomes secreted by the B16F10-OVA cell line were isolated. These exosomes were identified by transmission electron microscopy (TEM), flow cytometry, immunofluorescence staining, and Western blot (Figures 7A-7F). Flow cytometry and immunofluorescence staining results indicated that BNT162b2 transfection induced increased exosome secretion (Figures 7A, 7B). Western blot and immunogenicity assay results indicated that these exosomes simultaneously contained exosome markers, spike protein, and OVA protein, suggesting that these exosomes could elicit tumor antigen- and spike-specific T cell responses (Figures 7F-7H). These results provide a possible mechanism for tumor antigen dissemination induced by BNT162b2 cancer therapy.

[0101] Furthermore, studies by other groups have shown that intracellular heat shock proteins (HSPs) act as chaperones for tumor antigen-derived peptides and are involved in tumor antigen transport and presentation

[30] . Our data also suggested that the expression levels of heat shock proteins (HSPs), calreticulin, and HSP70 were increased in tumor cells after BNT162b2 treatment (Figures 7I and 7J).

[0102] To further confirm that BNT162b2 treatment can induce antigen spreading and systemic antitumor immune responses, we tested the therapeutic effect of BNT162b2 in a bilateral tumor model (Figure 7K). The therapeutic effect results showed that BNT162b2 treatment not only inhibited the growth of the treated tumor, but also the growth of the untreated tumor (Figure 7L). These results suggest that BNT162b2 cancer treatment can strongly induce antigen spreading and induce tumor antigen-specific immune responses, and also show strong distal inhibition of tumor growth.

[0103] Furthermore, antigen spreading induced by dead tumor cells was also examined (Figure 7M), and the results suggested that liquid nitrogen-treated (LNT)-B16F10 could strongly induce T cell responses against tumor cells, which also demonstrated a way to induce tumor antigen spreading (Figure 7N).

[0104] 5) The therapeutic effect of BNT162b2 cancer therapy can be enhanced by combining it with anti-PD-L1 therapy. BNT162b2 treatment can significantly inhibit tumor growth but cannot completely eliminate tumors. We investigated whether combination therapy could enhance the therapeutic effect of BNT162b2 in tumors. First, we measured PD-L1 expression levels in tumors after BNT162b2 and PBS treatment (Figure 8A). The results suggested that BNT162b2 treatment increased PD-L1 expression in tumors compared to PBS treatment (Figure 8B). Next, we found that PD-L1 expression was significantly increased in CD45+ leukocytes but not in tumor cells (Figure 8C). Transcriptome sequencing data also showed that the expression of IFNγ (Figure 8F) and pattern recognition receptor signaling pathways (Figure 5U) were significantly upregulated or enriched after intratumoral BNT162b2 administration, similar to the mRNA vaccinology results reported by Arunachalam et al.

[31] . Interestingly, significant upregulation of CD274 (PD-L1) was observed across the first three time points after intratumoral BNT162b2 administration (Figure 8D). Tumor-associated neutrophils and macrophages have been reported to negatively regulate adaptive immunity through increased PD-L1 expression in different tumor types [32-34]. Analysis also suggested that PD-L1 expression was strongly correlated with neutrophil and macrophage activation (Figure 8E). Flow cytometry results also demonstrated that PD-L1 expression on macrophages in tumors was high at the endpoint, regardless of BNT162b2 treatment. Importantly, PD-L1 expression on intratumoral neutrophils was significantly increased in BNT162b2-treated tumors (Figure 8G). Furthermore, immunofluorescence staining showed colocalization of PD-L1 and the neutrophil marker Gr-1, and that the percentage of PD-L1 after BNT162b2 treatment in neutrophils increased not only in total intratumoral neutrophils but also in all live cells ( Figure 8H ).

[0105] Although tumor-derived exosomes can stimulate tumor-specific immune responses as described above, numerous studies have shown that tumor-derived exosomes also promote tumor growth [35-37]. Therefore, we tested the preventive and therapeutic effects of exosomes derived from BNT162b2-treated tumors in a B16F10 subcutaneous model (Figures 8I and 8L). The results showed that exosomes derived from BNT162b2-treated tumors did not have a significant therapeutic effect in the preventive model, but rather significantly promoted tumor growth in the therapeutic model (Figures 8J, 8K, 8M, and 8N).

[0106] Increased PD-L1 expression in intratumoral macrophages and neutrophils increases the number of PD-L1-containing exosomes derived from BNT162b2-treated tumors. We investigated whether combination with anti-PD-L1 therapy could enhance the therapeutic efficacy of BNT162b2 cancer treatment (Figure 9A). The results showed that anti-PD-L1 treatment significantly improved the therapeutic efficacy of BNT162b2 (Figures 9B and 9C), and tumors were completely eliminated in three of five mice receiving the combination therapy (Figure 9B). These results strongly suggested the translatable potential of BNT162b2 and its combination therapy with anti-PD-L1 in various types of cancer. We further tested the therapeutic efficacy of BNT162b2 in an advanced tumor model (Figure 9D). The results demonstrated a strong therapeutic effect in the advanced tumor model, which could be further enhanced by combination with anti-PD-L1 therapy (Figures 9E and 9F).

[0107] 6) Therapeutic efficacy of mRNA vaccines encoding other pathogen antigens In addition to BNT162b2, which encodes the SARS-CoV-2 spike protein, we also investigated the therapeutic efficacy of another coronavirus, HKU1-CoV, using the same therapeutic strategy as BNT162b2 (Figure 11A). Lipid nanoparticle-encapsulated mRNA vaccines encoding the HKU1-CoV spike protein demonstrated superior therapeutic efficacy in a subcutaneous melanoma model (Figures 11B, 11C, 11D, and 11E). Furthermore, lipid nanoparticle-encapsulated mRNA vaccines encoding the SEA protein also demonstrated superior therapeutic efficacy (Figures 12A, 12B, 12C, 12D, 12E, and 12G). Interestingly, because superantigens can directly activate T cells by crosslinking MHC II to TCR, it is hypothesized that introducing superantigens into tumors will induce potent T cell responses that directly target superantigen-expressing tumor cells. The results of vaccine therapy suggested that intratumoral administration of SEA mRNA vaccine alone could also produce therapeutic effects equivalent to those of intramuscular vaccination followed by intratumoral administration of SEA mRNA vaccine (Figures 12A, 12B, 12C, 12D, 12E, 12F, and 12G). These results demonstrated that the cancer immunotherapy strategy of the present invention indeed has excellent therapeutic effects and has been validated in different pathogen antigen-based mRNA vaccines.

[0108] Example 3 Consideration The successful clinical application of immunotherapy helps cancer patients extend their lives. However, not all cancer patients benefit from current immunotherapy due to reduced tumor antigenicity or immunogenicity, which can occur due to tumor antigen loss or antigen presentation problems. Furthermore, the formation of a suppressive tumor microenvironment also leads to the suppression of antitumor immune responses, including increased expression of intratumor regulatory immune cells and immune checkpoints. Meanwhile, an effective tumor immune cycle also demonstrates the importance of tumor antigenicity, immunogenicity, and the transformation of the tumor microenvironment. Therefore, to improve antitumor immunotherapy, strategies are needed not only to induce specific antitumor immune responses but also to help increase tumor antigenicity and immunogenicity while simultaneously transforming the suppressive tumor microenvironment into one favorable for immunotherapy. Effective strategies for repurposing the BNT162b2 SARS-CoV-2 vaccine and other pathogen antigen-based mRNA vaccines as antitumor drugs have been demonstrated.

[0109] BNT162b2 or other pathogen antigen-based mRNA vaccine antitumor therapies have been tested in different tumor models using different strategies. In the B16F10 melanoma model, intramuscular vaccination before or after tumor inoculation demonstrated highly effective treatment. The superior efficacy of BNT162b2 was also observed in the 4T1 breast cancer model, the CT26 colon cancer model, and the MB49 bladder cancer model. Although BNT162b2 treatment was not as effective in the 4T1 model as in the melanoma model, it strongly inhibited lung metastasis of 4T1 cancer cells. Furthermore, intratumoral administration of BNT162b2 was found to recruit immune cells to the tumor and induce the secretion of proinflammatory cytokines, which promotes intratumoral T cell activation. Mechanistic studies also suggested that BNT162b2 antimelanoma treatment remained effective in μMT mice, but the therapeutic effect was lost in Rag1- / - mice. This indicates that this strategy relies on T cell responses but not on mature B cells. To understand the mechanisms underlying BNT162b2-induced tumor antigen spread and tumor-specific immune responses, we investigated whether spike protein- and tumor antigen-containing exosomes were released after BNT162b2 treatment. We found that BNT162b2 treatment induced greater exosome secretion than the untreated group. In addition, spike protein and tumor antigens could be detected in exosomes isolated from the BNT162b2-treated group. These results suggest that BNT162b2 treatment promotes tumor antigen spread by increasing the secretion of tumor-derived exosomes containing tumor antigens, and that these tumor antigen-containing exosomes can then induce tumor antigen-specific immune responses, ultimately leading to more effective antitumor responses. In addition to tumor-derived exosomes, increased tumor cell death in BNT162b2-treated tumors may also induce further tumor antigen-specific responses. Antigen spread was also confirmed in a bilateral tumor model, indicating that BNT162b2 cancer treatment can inhibit the growth of distant and untreated tumors. This indicates activation of a systemic tumor-specific immune response.

[0110] BNT162b2 strongly inhibited tumor growth in tumor-bearing mice, but was unable to completely eliminate tumors. Therefore, we investigated changes in the tumor microenvironment after BNT162b2 treatment. The data showed that intratumoral administration of BNT162b2 induced high levels of PD-L1 expression in leukocytes, particularly in intratumoral macrophages and neutrophils. These results suggest the potential for combination therapy with anti-PD-L1. Treatment trials of BNT162b2 and anti-PD-L1 combination therapy indeed demonstrated potent anticancer efficacy, and interestingly, in some cases, tumors could be completely eliminated after combination therapy.

[0111] In addition to BNT162b2B encoding the SARS-CoV-2 spike protein, other pathogen antigen-based mRNA vaccines for cancer treatment, such as the HKU1 CoV spike protein-encoding mRNA vaccine and the SEA protein-encoding mRNA vaccine, have also been developed and have also shown excellent therapeutic efficacy for cancer treatment.

[0112] Due to the COVID-19 pandemic, governments around the world are rushing to vaccinate their populations to prevent the virus from spreading widely. To date, over 70% of the world's population has been vaccinated, and those who have been vaccinated or infected have memory immunity to the spike protein. In addition, widespread vaccination or common coronavirus and bacterial infections have also induced memory immunity to other viral or bacterial antigens. This offers the possibility of cancer treatment using mRNA vaccines based on these pathogen antigens. Furthermore, compared to traditional cancer vaccine designs, this cancer treatment strategy does not require individual analysis of tumor mutations in cancer patients, and spike protein expression in tumors can be considered a common tumor-specific antigen. These advantages make this cancer treatment strategy using pathogen antigen-based mRNA vaccines more universal and more amenable to clinical application. Meanwhile, BNT162b2 and other SARS-CoV-2 mRNA vaccines have been approved for clinical use, and widespread vaccination is sufficient to demonstrate their efficacy and safety. Therefore, BNT162b2 could be rapidly applied clinically for cancer treatment by expressing spike proteins in tumors. Spike protein expression rapidly induces the activation of anti-spike memory immunity and tumor-specific immune responses, attacking tumors. More importantly, other foreign antigens derived from pathogens (especially infectious diseases for which people have been vaccinated against) could also become commercially available vaccines for various cancer treatments, as confirmed by this invention. In conclusion, this invention not only offers exciting potential for repurposing COVID-19 mRNA vaccines into cancer immunotherapy with potent therapeutic effects, but also proposes a new cancer treatment strategy that evokes and redirects memory immunity induced by infection or vaccination for cancer treatment. These treatment strategies may offer more options and hope to patients.

[0113] Although the details of the present invention have been described above, it should be noted that the invention is not limited thereto but can be embodied in various ways as set forth in the appended claims.

[0114] References 1. Chen, DS and I. Mellman, Oncology meets immunology: the cancer-immunity cycle. Immunity, 2013. 39(1): p. 1-10. 2. Aktar, N., et al., Understanding of Immune Escape Mechanisms and Advances in Cancer Immunotherapy. J Oncol, 2022. 2022: p. 8901326. 3. Beatty, GL and WL Gladney, Immune escape mechanisms as a guide for cancer immunotherapy. Clin Cancer Res, 2015. 21(4): p. 687-92. 4. Newman, JH, et al., Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer. Proc Natl Acad Sci USA, 2020. 117(2): p. 1119-1128. 5. Russell, SJ, et al., Advances in oncolytic virotherapy. Commun Med (Lond), 2022. 2: p. 33. 6. Russell, S.J., K.W. Peng, and J.C. Bell, Oncolytic virotherapy. Nat Biotechnol, 2012. 30(7): p. 658-70. 7. Yu, B., et al., Explicit hypoxia targeting with tumor suppression by creating an ”obligate” anaerobic Salmonella Typhimurium strain. Sci Rep, 2012. 2: p. 436. 8. Ning, B.T., et al., Treatment of Neuroblastoma with an Engineered ”Obligate” Anaerobic Salmonella typhimurium Strain YB1. J Cancer, 2017. 8(9): p. 1609-1618. 9. Lin, Q., et al., IFN-gamma-dependent NK cell activation is essential to metastasis suppression by engineered Salmonella. Nat Commun, 2021. 12(1): p. 2537. 10. DeBruyne, L.A., et al., Direct transfer of a foreign MHC gene into human melanoma alters T cell receptor V beta usage by tumor-infiltrating lymphocytes. Cancer Immunol Immunother, 1996. 43(1): p. 49-58. 11. GREGORY E. PLAUTZ*t, Z.-Y.Y., BEI-YUE WU*, XIANG GAOt,LEAF HUANG*, AND GARY J.NABEL*§, Immunotherapy of malignancy by in vivo gene transfer into tumors (genetherapy / majorhistocompatibiitycomplex / cancer / Tcells / adenocarcinoma). Proc. Natl. Acad. Sci. USA, 1993. 90: p. 4645-4649. 12. Schmidt, W., et al., Transloading of tumor cells with foreign major histocompatibility complex class I peptide ligand: a novel general strategy for the generation of potent cancer vaccines. Proc Natl Acad Sci U S A, 1996. 93(18): p. 9759-63. 13. El-Khoueiry, A.B., et al., Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1 / 2 dose escalation and expansion trial. Lancet, 2017. 389(10088): p. 2492-2502. 14. Brahmer, J.R., et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med, 2012. 366(26): p. 2455-65. 15. Lui, D.T.W., et al., Thyroid Immune-Related Adverse Events in Patients with Cancer Treated with anti-PD1 / anti-CTLA4 Immune Checkpoint Inhibitor Combination: Clinical Course and Outcomes. Endocr Pract, 2021. 27(9): p. 886-893. 16. Yau, T., et al., Efficacy and Safety of Nivolumab Plus Ipilimumab in Patients With Advanced Hepatocellular Carcinoma Previously Treated With Sorafenib: The CheckMate 040 Randomized Clinical Trial. JAMA Oncol, 2020. 6(11): p. e204564. 17. Rafiq, S., C.S. Hackett, and R.J. Brentjens, Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol, 2020. 17(3): p. 147-167. 18. Labanieh, L., R.G. Majzner, and C.L. Mackall, Programming CAR-T cells to kill cancer. Nat Biomed Eng, 2018. 2(6): p. 377-391. 19. Sterner, R.C. and R.M. Sterner, CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J, 2021. 11(4): p. 69. 20. June, C.H., et al., CAR T cell immunotherapy for human cancer. Science, 2018. 359(6382): p. 1361-1365. 21. Wagner, J., et al., CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? Mol Ther, 2020. 28(11): p. 2320-2339. 22. Organization, W.H. Background document on the mRNA vaccine BNT162b2 (Pfizer-BioNTech) against COVID-19. 2021 Available from: https: / / www.who.int / publications / i / item / background-document-on-mrna-vaccine-bnt162b2-(pfizer-biontech)-against-covid-19. 23. Polack, F.P., et al., Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med, 2020. 383(27): p. 2603-2615. 24. Sahin, U., et al., BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans. Nature, 2021. 595(7868): p. 572-577. 25. Li, C., et al., Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nat Immunol, 2022. 23(4): p. 543-555. 26. Hannah Ritchie, E.M., Lucas Rodes-Guirao, Cameron Appel, Charlie Giattino, Esteban Ortiz-Ospina, Joe Hasell, Bobbie Macdonald, Diana Beltekian and Max Roser. Coronavirus Pandemic (COVID-19). 2020; Available from: https: / / ourworldindata.org / coronavirus. 27. Kitamura, D., et al., A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature, 1991. 350(6317): p. 423-6. 28. Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S, Papaioannou VE., RAG-1-deficient mice have no mature B and T lymphocytes. Cell, 1992. 68(5):869-77. 29. Bansal, S., et al., Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines. J Immunol, 2021. 207(10): p. 2405-2410. 30. Binder, R.J., Immunosurveillance of cancer and the heat shock protein-CD91 pathway. Cell Immunol, 2019. 343: p. 103814. 31. Arunachalam, P.S., et al., Systems vaccinology of the BNT162b2 mRNA vaccine in humans. Nature, 2021. 596(7872): p. 410-416. 32. He, G., et al., Peritumoural neutrophils negatively regulate adaptive immunity via the PD-L1 / PD-1 signalling pathway in hepatocellular carcinoma. J Exp Clin Cancer Res, 2015. 34: p. 141. 33. Wang, T.T., et al., Tumour-activated neutrophils in gastric cancer foster immune suppression and disease progression through GM-CSF-PD-L1 pathway. Gut, 2017. 66(11): p. 1900-1911. 34. Liu, Y., et al., Immune Cell PD-L1 Colocalizes with Macrophages and Is Associated with Outcome in PD-1 Pathway Blockade Therapy. Clin Cancer Res, 2020. 26(4): p. 970-977. 35. Li, Y., et al., Targeted inhibition of tumor-derived exosomes as a novel therapeutic option for cancer. Exp Mol Med, 2022. 54(9): p. 1379-1389. 36. TL., W., Tumor-Derived Exosomes and Their Role in Cancer Progression. . Adv Clin Chem., 2016. 74:103-41. 37. Tang, Q., et al., Tumor-derived exosomes in the cancer immune microenvironment and cancer immunotherapy. Cancer Lett, 2022. 548: p. 215823.

Claims

1. A method for increasing the antigenicity or immunogenicity of a tumor in a subject, comprising delivering lipid nanoparticles carrying mRNA sequences encoding one or more pathogen antigen proteins to the tumor.

2. 10. The method of claim 1, wherein the one or more pathogen antigen proteins are antigens targeted in a vaccine previously administered to the subject or a vaccine to be administered to the subject.

3. 2. The method of claim 1, wherein the one or more pathogen antigen proteins are selected from the group consisting of coronavirus proteins, human papillomavirus proteins, respiratory syncytial virus proteins, human immunodeficiency virus proteins, hepatitis viruses, and influenza virus proteins.

4. 3. The method of claim 2, wherein the vaccine is selected from the group consisting of BioNTech BNT162b2, BioNTech COVID-19 Omicron strain bivalent vaccine, and Moderna vaccine: mRNA-1273.

5. 10. The method of claim 1, wherein the lipid nanoparticles are delivered by intratumoral administration.

6. The method of claim 5, wherein the intratumoral administration is an mRNA vaccine.

7. 7. The method of claim 6, wherein the mRNA vaccine is an mRNA vaccine against one or more antigens selected from the group consisting of bacterial antigens, bacterial superantigens, viral antigens, and viral superantigens.

8. 10. The method of claim 1, further comprising immunizing the subject against the one or more pathogen antigenic proteins or variants thereof.

9. 10. The method of claim 1, wherein the subject has previously been immunized against the one or more pathogen antigenic proteins or variants thereof.

10. 2. The method of claim 1, wherein the tumor is selected from the group consisting of melanoma, breast cancer, bladder cancer, colon cancer, gastric cancer, pancreatic cancer, blood cancer, lung cancer, and liver cancer.

11. A method of treating a subject having one or more tumors, comprising increasing the antigenicity or immunogenicity of at least one of the one or more tumors using the method of claim 1.

12. 12. The method of claim 11, wherein the at least one tumor is a primary tumor or a secondary tumor.

13. 12. The method of claim 11, further comprising co-administering an immune checkpoint inhibitor to the subject.

14. 14. The method of claim 13, wherein the immune checkpoint inhibitor is one or more selected from the group consisting of anti-PD-1, anti-CTLA4, and anti-PD-L1.

15. 1. An intratumoral administration for treating a tumor in a subject, comprising lipid nanoparticles loaded with mRNA encoding one or more pathogen antigen proteins.

16. The method of claim 15, wherein the one or more pathogen antigen proteins are antigens targeted in a vaccine previously administered to the subject or a vaccine to be administered to the subject.

17. 16. The intratumoral administration of claim 15, wherein the tumor is selected from the group consisting of melanoma, breast cancer, bladder cancer, colon cancer, gastric cancer, pancreatic cancer, blood cancer, lung cancer, and liver cancer.

18. 16. A kit for treating a tumor in a subject, comprising the intratumoral administration of claim 15.

19. 20. The kit of claim 18, further comprising an immune checkpoint inhibitor.

20. 20. The kit of claim 18, further comprising a vaccine for vaccinating the subject against the one or more pathogen antigenic proteins.

Images