Vaccines, their uses, and cancer vaccine combinations

Abstract

A vaccine is provided that comprises a vector and a transgene. [Solution] The transgene encodes a plurality of peptides and is encapsulated in a vector, the peptides including, in order, a secretory signal peptide, at least one tumor antigen, at least one co-inhibitory peptide, and a Toll-like receptor 9 antagonist sequence (TLR9).

Description

[Technical Field]

[0001] The present disclosure relates to vaccines, uses thereof, and cancer vaccine combinations, and in particular to vaccines specific for tumor antigens, uses thereof, and cancer vaccine combinations comprising said vaccines. [Background technology]

[0002] Cancer treatment primarily involves radiation therapy, chemotherapy, targeted therapy, and surgery. Despite recent advances in drugs and surgical techniques, the five-year survival rate for advanced cancer patients remains quite low, highlighting the importance of developing new therapeutic strategies such as cancer immunotherapy. Immunotherapies primarily aim to enhance the immune response to eliminate tumor cells and include immune checkpoint inhibitors, cell therapy, and cancer vaccines. While immune checkpoint inhibitors have demonstrated favorable clinical responses in various cancers, their application is limited by DNA mismatch repair deficiency (10–15% of cancer patients) and the degree of immune cell infiltration, indicating that most cancer patients are resistant to immune checkpoint inhibition. Therefore, developing new immunotherapy strategies, such as neoantigen-based immunotherapy, is crucial.

[0003] Neoantigens, derived from somatic mutations during cancer progression, can induce tumor-specific immune responses. Breakthroughs in next-generation gene sequencing technology have made it possible to develop cancer vaccines by identifying personalized neoantigens and activating tumor-specific immune responses. Radiation therapy (RT) and chemotherapy (CT) not only increase tumor antigen release but also alter the tumor microenvironment to a more permissive one. Combining neoantigen-based cancer vaccines with radiotherapy and immunogenic chemotherapy may potentially achieve sustainable disease control even in patients resistant to conventional treatments. Summary of the Invention [Problem to be solved by the invention]

[0004] Furthermore, single-agent therapy using tumor neoantigen peptide vaccines alone is not effective enough to eliminate tumors, and most mutations vary from patient to patient, making neoantigens more personalized, resulting in high costs and long treatment times. Therefore, targeting frequently shared neoantigens and optimizing the delivery efficacy of neoantigen-based immunotherapy is a new approach to solving the aforementioned problems. [Means for solving the problem]

[0005] One aspect of the present disclosure provides a vaccine comprising a vector and a transgene. The transgene encodes multiple peptides and is packaged in a vector, the peptides comprising, in order, a secretory signal peptide, at least one tumor antigen, at least one co-inhibitory peptide, and a Toll-like receptor 9 antagonist sequence (TLR9). The at least one tumor antigen is a difference between tumor cells and normal cells. The at least one co-inhibitory peptide comprises a programmed cell death ligand 1 (PD-L1) antagonist, a programmed cell death protein 1 (PD-1) antagonist, or a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antagonist.

[0006] Another aspect of the present disclosure is the use of the vaccine described in the preceding paragraph for use in the treatment of cancer, to induce an anti-tumor immune response in a subject in need of cancer treatment.

[0007] A further aspect of the present disclosure provides a cancer vaccine combination comprising the vaccine described in the preceding paragraph, a booster, and an immunopotentiator, wherein the vaccine is used to induce an immune response against at least one tumor antigen in a subject in need of cancer treatment, the booster is used to enhance local tumor control in the subject, and the immunopotentiator is used to prevent local recurrence and metastasis in the subject. [Brief explanation of the drawings]

[0008] To clarify the purpose of the present disclosure, the following detailed description of the embodiments will be given with reference to the drawings. [Figure 1] FIG. 1 is a schematic diagram showing the construction of a vaccine according to one embodiment of the present disclosure. [Figures 2A-2C] 2A, 2B, and 2C are schematic diagrams illustrating the mechanism by which the vaccine of the present disclosure delivers a transgene to a subject and the encoded peptides interact within the subject. [Figure 3A] FIG. 3A is a schematic diagram showing the construction of a cancer vaccine based on the neoAg peptide of Example 1. [Figure 3B] FIG. 3B is a schematic diagram showing a therapeutic strategy of the neoAg peptide-based cancer vaccine of Example 1 in combination with radiotherapy in an animal therapeutic trial. [Figure 3C] FIG. 3C shows the results of analyzing the therapeutic effect of the neoAg peptide-based cancer vaccine of Example 1 on colorectal cancer. [Figures 4A-4E] 4A, 4B, 4C, 4D, and 4E show the results of analyzing the effect of the neoAg peptide-based cancer vaccine of Example 1 on the anti-tumor immune action due to immune cell infiltration. [Figure 5A] FIG. 5A is a schematic diagram showing the experimental process of ex vivo immunoassay. [Figures 5B-5F] 5B, 5C, 5D, 5E, and 5F show the results of an in vitro immunoassay of the neoAg peptide-based cancer vaccine of Example 1. [Figures 6A-6B] 6A and 6B show the results of analyzing the effect of the neoAg peptide-based cancer vaccine of Example 1 on anti-tumor immune activity due to immune cell infiltration. [Figure 6C-6D] 6C and 6D show the results of an analysis of the effect of the neoAg peptide-based cancer vaccine of Example 1 on the tumor microenvironment (TME) after radiotherapy. [Figure 7A] FIG. 7A is a schematic diagram showing the construction and treatment strategies of AAV-based cancer vaccines of Examples 4, 6, and 8. [Figures 7B-7F]Figures 7B, 7C, 7D, 7E, and 7F show the results of analyzing the therapeutic efficacy of the AAV-based cancer vaccines of Examples 4, 6, and 8 against colorectal cancer. [Figure 8A] FIG. 8A is a schematic diagram showing the construction of a vaccine according to Example 10 of the present disclosure. [Figure 8B] FIG. 8B is a schematic diagram showing a treatment strategy of the vaccine of Example 10 of the present disclosure in combination with radiation therapy in an animal treatment trial. [Figure 8C] FIG. 8C shows the results of an analysis of the therapeutic effect of the vaccine of Example 10 of the present disclosure on colorectal cancer. [Figure 9] FIG. 9 shows the survival curves of colorectal cancer mice treated with the vaccine of Example 10 of the present disclosure. [Figure 10A] FIG. 10A is a schematic diagram showing the construction of the vaccine of Example 13 of the present disclosure and its therapeutic strategy in combination with radiation therapy in animal therapeutic trials. [Figures 10B-10G] Figures 10B, 10C, 10D, 10E, 10F, and 10G show the results of an analysis of the effect of the vaccine of Example 13 of the present disclosure in combination with radiation therapy to treat colorectal cancer. [Figure 11A] FIG. 11A is a schematic diagram showing the construction of the vaccine of Example 16 of the present disclosure and its therapeutic strategy in combination with radiation therapy in animal therapeutic trials. [Figures 11B-11G] Figures 11B, 11C, 11D, 11E, 11F, and 11G show the results of analyzing the therapeutic effect of the vaccine of Example 16 of the present disclosure on breast cancer. [Figure 12A] FIG. 12A is a schematic diagram showing a therapeutic strategy for cancer treatment of a cancer vaccine cocktail according to one embodiment of the present disclosure. [Figure 12B] FIG. 12B is a schematic diagram showing a cancer vaccine cocktail according to an example of an embodiment of the present disclosure. [Figure 13A] FIG. 13A is a schematic diagram showing the treatment strategy in an animal therapeutic test of a cancer vaccine combination according to one embodiment of the present disclosure. [Figures 13B-13D]Figures 13B, 13C, and 13D show the results of an analysis of the therapeutic effect of the cancer vaccine cocktail of the present disclosure on colorectal cancer. DETAILED DESCRIPTION OF THE INVENTION

[0009] Please refer to Figure 1, which shows a schematic diagram of the construction of a vaccine 100 according to one embodiment of the present disclosure. The vaccine 100 comprises a vector 110 and a transgene 120 packaged in the vector 110.

[0010] The vector 110 is used to enhance the expression of tumor antigens with different anisotropy and may be a vaccinia virus vector, an adeno-associated virus (AAV) vector, or a nanoparticle. Preferably, the AAV vector is an adeno-associated virus 2 (AAV2) vector or an adeno-associated virus 6 (AAV6) vector. Nanoparticles include, but are not limited to, liposome delivery systems (e.g., cetyl phosphate-tetraethylenepentamine-based polycationic liposomes (TEPA-PCL), lipoplexes (e.g., DOTMA:cholesterol:TPGS lipoplexes or DDAB:cholesterol:TPGS lipoplexes), cationic liposome-hyaluronic acid (LPH) nanoparticles), lipid nanoparticles (LNPs), polyethyleneimine (PEI) or polyethyleneimine conjugates, dendrimer nanoparticles, polyamide (PAMAM) nanoparticles, polylactic acid and polyglycolic acid copolymer (PLGA) nanoparticles, atelocollagen nanoparticles, and silica nanoparticles.

[0011] The transgene 120 encodes a plurality of peptides, which in turn include a secretory signal peptide 121, at least one tumor antigen 122, a co-inhibitory peptide 123, and a Toll-like receptor 9 (TLR9) antagonist sequence 124.

[0012] The secretory signal peptide 121 is used to assist in the secretion of the tumor antigen. Preferably, the secretory signal peptide 121 may be an interleukin 2 signal peptide (IL2 sp) or an interleukin 12 signal peptide (IL12 sp).

[0013] The at least one tumor antigen 122 is used to increase an anti-tumor immune response in a subject in need of cancer treatment, and the at least one tumor antigen 122 is a difference between tumor cells and normal cells. Preferably, the at least one tumor antigen 122 may be selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), oncogenic mutations, aberrantly expressed tumor-specific antigens (aeTSA), and shared neoantigens (neoAg). Alternatively, the at least one tumor antigen 122 may be selected by comparing the DNA sequences of normal cells and corresponding tumor cells from a subject by whole-exome sequencing to identify tumor-specific somatic mutations (neoantigens), and selecting polynucleotides encoding the neoantigens from an existing neoantigen database. TAAs are overexpressed in tumor cells but underexpressed in normal cells. For example, TAA in breast cancer include, but are not limited to, mammaglobin-A, prostate-specific antigen (PSA), melanoma antigen recognized by T cells (MART1), melanocyte protein PMEL, Bcr / Abl tyrosine kinase, HPV E6, E7, MZ2-E, MAGE-1, and MUC-1, which are overexpressed in breast cancer. On the other hand, TSAs are found only in cancer cells and not in healthy cells. For example, TSAs include, but are not limited to, the driver gene KRAS-G12 / 13 codon mutation hotspot, TP53 mutation hotspot, PIK3CA mutation hotspot, BRAF mutation, and frameshift mutation. AeTSAs result from the aberrant expression of unmutated transcripts that are not expressed in any normal somatic cells, including thymic medullary epithelial cells (mTECs), which regulate central immune tolerance.

[0014] The co-inhibitory peptide 123 is for blocking co-inhibitory signals in dendritic cells (DCs) to increase the antigen-presenting ability of DCs. The co-inhibitory peptide 123 includes a programmed cell death ligand 1 (PD-L1) antagonist, a programmed cell death protein 1 (PD-1) antagonist, or a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antagonist. Preferably, the PD-L1 antagonist is a P D The PD-1 antagonist may comprise a PD-1 trap and a PD-L1 / PD-L2 peptide, and the CTLA4 antagonist may comprise a CTLA4 trap and a CTLA4 antagonist antibody.

[0015] The TLR9 antagonist sequence 124 is an antiviral clearance sequence for attenuating viral clearance by the innate immune system and maintaining a high antigen load. Preferably, the TLR9 antagonist sequence 124 may be selected from a CpG oligonucleotide TLR9 binding domain, a TLR decoy peptide, and a CpG binding sequence.

[0016] Additionally, the vaccine 100 of the present disclosure may further comprise a costimulatory peptide for increasing DC recruitment and activation, wherein the costimulatory peptide may be selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 12 (IL12), and interferons (IFNs).

[0017] According to one embodiment, a method for treating cancer comprises administering the vaccine described in the preceding paragraph to a subject in need of cancer treatment, thereby inducing an anti-tumor immune response in the subject. Preferably, the method for treating cancer in this embodiment can further comprise administering radiation therapy to the subject. Furthermore, the vaccine of the present disclosure can synergistically promote the subject's tumor-specific immune response and synergistically extend the subject's survival time under conditions in which the peptide is expressed.

[0018] "Cancer" refers to a physiological condition in mammals characterized by unregulated cell growth. "Tumor" includes one or more types of cancer cells. Examples of cancer include, but are not limited to, epithelial carcinoma, lymphoma, blastoma, sarcoma, and leukemia, or lymphoid malignancies. More specifically, cancers include breast cancer, colon cancer, rectal cancer, colorectal cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenoma, lung cancer including lung squamous cell carcinoma, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, hepatocellular carcinoma, bladder cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, head and neck cancer, and the like.

[0019] An "effective amount" refers to an amount of the vaccine of the present disclosure that is effective to "treat" a disease / disorder in a subject. An effective amount correlates to some extent with the biological or medical response of a tissue, system, animal, or human to which it is administered, e.g., is sufficient when administered to prevent to some extent the progression of one or more diseases / disorders or to alleviate the symptoms of one or more of the diseases / disorders being treated. The therapeutically effective amount will vary depending on the disease and its severity, as well as the age and weight of the mammal being treated, etc.

[0020] See Figures 2A, 2B, and 2C, which are schematic diagrams illustrating the mechanism by which the vaccine of the present disclosure delivers a transgene to a subject and the encoded peptide interacts in the subject's body. The vaccine of the present disclosure is effective in inhibiting immune checkpoints, increasing tumor antigen presentation, and activating tumor immune responses.

[0021] The present disclosure will be further illustrated by way of specific examples in conjunction with the drawings, but these examples are for illustrative purposes only, and the present disclosure is not limited thereto. For example, although the vector used in the following examples is an AAV vector, the vector is used to deliver a transgene to target cells, and similar effects can be expected when other vectors, such as vaccinia virus vectors or nanoparticles, are used.

[0022] Examples and Comparative Examples

[0023] [Examples 1-3]

[0024] First, we discovered that tumors remaining after chemotherapy and radiotherapy have shared neoantigens (hereafter referred to as "neoAg(s)"), and established a neoAg map for refractory and recurrent tumors. These neoAgs can be used for in vitro diagnostic (IVD) tests and the development of antibody-based immunotherapeutics. Furthermore, these neoAgs can be used for DC vaccines and DC- C These findings can be used as tumor-specific key components to improve IK cell therapy and further develop neoAg peptide-based cancer vaccine immunotherapy to enhance the efficacy of radiation therapy, chemotherapy, and cell therapy. See Table 1, which lists neoAgs in the mouse colon cancer CT26 cell line (hereinafter referred to as "CT26 cells"). Table 1. NeoAgs in CT26 cells [Table 1] We also developed a cancer vaccine based on neoAg peptides, including the above-mentioned neoAgs, and confirmed its therapeutic effect against cancer.

[0025] Please refer to Figure 3A, which shows a schematic diagram of the construction of the neoAg peptide-based cancer vaccine of Example 1. As shown in Figure 3A, the peptide encoded by the transgene in the neoAg peptide-based cancer vaccine of Example 1 is an interleukin-12 signal peptide (IL-12) signal peptide (IL-12), which is constructed by constructing a nucleotide fragment corresponding to the peptide into a CMV-initiated pAAV-CMV expression vector. L2 sp), neoAgs, and two ovalbumin sequences (OVA-CD4 and OVA-CD8). L2 sp is used to increase the amount of neoAgs secreted into cells, and I L2The amino acid sequence of sp is shown in SEQ ID NO:11. The neoAgs include neoAgs 1-8 shown in Table 1, and are fused via a RERK linker. OVA-CD4 and OVA-CD8 were used as positive controls, and the amino acid sequences of OVA-CD4 and OVA-CD8 are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. Comparative Example 1 was an empty pAAV-CMV expression vector, and I L2 sp but does not contain the nucleotide fragment encoding neoAgs.

[0026] See Figure 3B and Table 2. Figure 3B shows a schematic diagram of the treatment strategy in which the neoAg peptide-based cancer vaccine of Example 1 was combined with radiation therapy in an animal treatment test, and Table 2 shows the treatment strategies of Examples 1-2 and Comparative Examples 1-2. Table 2, Treatment Strategies of Examples 1-2 and Comparative Examples 1-2 [Table 2]

[0027] To verify the therapeutic effect of the neoAg peptide-based cancer vaccine of Example 1, a mouse model of colorectal cancer was established in the experiment. 5 CT26 cells and 20% Matrigel (Corning, Union City, CA, USA) were subcutaneously inoculated into the right lower leg of 6-week-old female BALB / c mice. After 8 days, the mice with colorectal cancer were randomly divided into different groups and inoculated with either Example 1 or Comparative Example 1 (1 × 10 8 vg) was intramuscularly injected every 6 days for a total of three times, with a fourth booster injection on the 25th day. For the radiotherapy group, mice with colorectal cancer were fully anesthetized on the 11th day, and then their right limbs were placed in the radiation field. The mice were then given a single 5 Gy fractionated radiotherapy to the local tumor. Meanwhile, tumor volume was measured every 3 days and calculated using the formula V = (L × W) until sacrifice on the 28th day. 2 The tumor volume is calculated by the following formula: ) / 2. The collected tumor tissue is used for subsequent immunoassays.

[0028] Please refer to Figure 3C, which shows the analysis results of the therapeutic effect of the neoAg peptide-based cancer vaccine of Example 1 on colorectal cancer. The results of Figure 3C show that, compared with Comparative Example 1, the treatment using the neoAg peptide-based cancer vaccine alone in Example 1 can significantly inhibit tumor growth, achieving the same effect as Comparative Example 2 (radiotherapy alone). In Example 2, the tumor growth inhibitory effect is more pronounced when the neoAg peptide-based cancer vaccine of Example 1 and radiotherapy are administered simultaneously. The above results indicate that the neoAg peptide-based cancer vaccine of Example 1 can improve the therapeutic effect of radiotherapy.

[0029] To verify the effect of the neoAg peptide-based cancer vaccine of Example 1 on the anti-tumor immune response through immune cell infiltration, tumor-infiltrating lymphocytes were further isolated. In the test, fresh tumors were isolated from the colorectal cancer mice of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, and the tumors were incubated at room temperature for 5 min. L Place the tumor in a 6 cm dish containing RPMI 1640 medium and cut the tumor into chunks approximately 1-2 mm in size with a sterile knife. L Prepare a centrifuge tube, place a 70 μm cell strainer on top, and transfer all of the tumor tissue onto the strainer using a sterile dropper. If any tissue fragments remain, inject RPMI 1640 medium with a 5 mL plastic syringe to pass the tissue fragments through the strainer. Carefully transfer all of the cell solution into a 15 mL conical tube containing Ficoll-Paque at the bottom and centrifuge at 20°C for 20 minutes at a centrifugal force of 1025 × g, slowly accelerating and turning off the brake. Transfer the mononuclear cell layer to a new 50 mL tube using a sterile pipette. L Carefully transfer the mixture to a centrifuge tube, add 10 mL of RPMI 1640 medium, and centrifuge at 20°C for 10 minutes at 650 x g. Remove the supernatant and centrifuge for 10 minutes. L Gently resuspend the cells in complete RPMI 1640 medium and centrifuge again for 10 min at 650 x g at 20 °C. Remove the supernatant and 1 mL of RPMI 1640 medium was added to resuspend the cells, and the result was isolated tumor-infiltrating lymphocytes (TILs).

[0030] See Figures 4A, 4B, 4C, 4D, and 4E, which are graphs showing the analysis results of the effect of the neoAg peptide-based cancer vaccine of Example 1 on the anti-tumor immune activity due to immune cell infiltration, where * indicates p<0.05 and ** indicates p<0.01. The data were analyzed by one-way independent analysis of variance. The results of Figures 4A-4E show that, compared with Comparative Example 1, Comparative Example 2, and Example 1, the treatment of Example 2 in which the neoAg peptide-based cancer vaccine of Example 1 and radiotherapy were simultaneously administered significantly increased CD4 + cells, CD8 + cells, CD44 + It can significantly increase the number of CD4 cells, Treg cells, and myeloid-derived suppressor cells (MDSCs), + The number of cells represents the helper T lymphocyte (Th) response, and CD8 + The number of cells represents the cytotoxic T lymphocyte (CTL) response, and CD44 + The number of cells represents the effector / memory T cell response, and the number of Treg cells and MDSCs represents the immunosuppressive cell response. The above results indicate that the neoAg peptide-based cancer vaccine of Example 1 can confer antitumor immunity by promoting the infiltration of immune cells.

[0031] See Figures 5A, 5B, 5C, 5D, 5E, and 5F. Figure 5A is a schematic diagram showing the experimental process of ex vivo immunoassay, and Figures 5B, 5C, 5D, 5E, and 5F show the analytical results of ex vivo immunoassay of the neoAg peptide-based cancer vaccine of Example 1. In the test, single cell suspensions of mouse spleens were subjected to ex vivo immunoassay using an IFNγ ELISpot detection kit (Abcam). Spleen cells were incubated in 2 mM L-glutamine, 0.5 mM ATP, and 100 mM riboflavin. μ 2.5 x 10 cells were cultured in a 96-well plate containing complete RPMI 1640 medium with 2 μg / mL concanavalin A, and 2 ng / mL m-IL2. 5The cells were seeded at a density of 1000 / well and cultured for 2 days. After removing unattached cells, the medium was replaced with medium containing 1 μg / mL of peptide and stimulated for 24 hours. The positive control group consisted of 1 ng / mL PMA and 500 μg / mL of peptide. The spleen cells were cultured in RPMI 1640 medium supplemented with 250 ng / mL ionomycin. Finally, qualitative measurements were performed to detect the sites of IFNγ production and secretion. As shown in Figures 5B-5F, the results demonstrate that the neoAg peptide-based cancer vaccine of Example 1 inhibits neoAg-specific CD8 + It has been shown that it can induce T cell responses.

[0032] To verify the effect of the neoAg peptide-based cancer vaccine of Example 1 in the tumor microenvironment, the experiment involves the isolation of tumor-infiltrating lymphocytes. Mice with established colorectal cancer were randomly assigned to different groups. The mice were injected with Example 1, Comparative Example 1 (1 x 10 8 vg) and PBS were intramuscularly injected four times. For the radiation therapy group, mice with colorectal cancer were fully anesthetized on days 11 and 18, and their right limbs were placed in the radiation field. Two 5 Gy fractionated radiation therapy sessions were administered to the local tumor. On day 28, mice with colorectal cancer were sacrificed, and tumor tissues were collected for immunological analysis. See Table 3 for the treatment strategies of Examples 1 and 3, Comparative Examples 1 and 3, and Control Groups 1 and 3. Table 3, Treatment Strategies of Examples 1 and 3, Comparative Examples 1 and 3, and Control Groups 1 and 3 [Table 3]

[0033] See Figures 6A, 6B, 6C, and 6D. Figures 6A and 6B are graphs showing the results of analyzing the effect of the neoAg peptide-based cancer vaccine of Example 1 on anti-tumor immune activity through immune cell infiltration, and Figures 6C and 6D are graphs showing the results of analyzing the effect of the neoAg peptide-based cancer vaccine of Example 1 on the tumor microenvironment (TME) after radiation therapy administration. In Figures 6A, 6B, and 6D, * indicates p<0.05, *** indicates p<0.001, and data were analyzed by one-way independent analysis of variance. As shown in Figures 6A-6D, compared with Control Group 1, Control Group 3, Comparative Example 1, Comparative Example 3, and Example 1, simultaneous administration of the neoAg peptide-based cancer vaccine of Example 1 and radiation therapy in Example 3 significantly increased CD8 + T EM cell counts, and IFNγ + CD8 + TILs and IFNγ + CD8 + The above results suggest that radiotherapy significantly increases the ratio of TIL / Tregs to tumor-infiltrating effector / memory and cytotoxic CD8 + This shows that the neoAg peptide-based cancer vaccine of Example 1 can increase T cells and reverse the immunosuppressive state in the tumor microenvironment after radiotherapy administration.

[0034] [Examples 4-9]

[0035] Next, we developed AAV-based cancer vaccines containing TLR9 antagonist sequences and various tumor antigens to confirm their therapeutic efficacy against cancer. See Figure 7A, which shows a schematic diagram of the construction and therapeutic strategy of the AAV-based cancer vaccines described in Examples 4, 6, and 8.

[0036] As shown in Figure 7A, three AAV-based cancer vaccines (Examples 4, 6, and 8) contain two short TLR9 inhibitory sequences (shown as "TLR9i" in Figure 7A) to evade viral clearance by the innate immune system and prolong tumor antigen expression. L2The peptides are designed to be inserted into a pAAV-CMV expression vector containing sp. The peptides encoded by the transgenes in the AAV-based cancer vaccine of Example 4 include the TAA carcinoembryonic antigen (CEA) as at least one tumor antigen, and the amino acid sequence of CEA is shown in SEQ ID NO: 12. The peptides encoded by the transgenes in the AAV-based cancer vaccine of Example 6 include neoAgs 1-8 listed in Table 1, fused with a RERK linker to at least one tumor antigen (shown as "neoAg" in Figure 7A). The peptides encoded by the transgenes in the AAV-based cancer vaccine of Example 8 include aberrantly expressed tumor-specific antigens 1-7 listed in Table 4 as at least one tumor antigen (shown as "aeTSA" in Figure 7A), where ERE is an abbreviation for endogenous reverse transcription factor. The amino acid sequences of two short TLR9 inhibitory sequences are shown in SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Comparative Example 1 is an empty pAAV-CMV expression vector, L2 sp, but does not include a nucleotide fragment encoding a tumor antigen. Table 4. aeTSAs in CT26 cells [Table 4]

[0037] To verify the therapeutic effects of the AAV-based cancer vaccines of Examples 4, 6, and 8, mice with colorectal cancer were randomly assigned to different groups. The AAV-based cancer vaccines of Examples 4, 6, and 8, and Comparative Example 1 (1×10 8 vg) were intramuscularly injected four times on days 8, 14, 21, and 25, respectively. For the group receiving radiation therapy, mice with colorectal cancer were fully anesthetized on day 11, and then the right limb was placed in the radiation field and a single 5 Gy fractionated radiation therapy was administered to the local tumor. Tumor volume was measured every three days and calculated using the formula V = (L × W) until sacrifice on day 30. 2The tumor volume is calculated as follows: ) / 2. The collected tumor tissues are used for subsequent immunoassays. See Table 5 for the treatment strategies of Examples 4-9 and Comparative Examples 1-2. Table 5, Treatment Strategies of Examples 4-9 and Comparative Examples 1-2 [Table 5]

[0038] See Figures 7B, 7C, 7D, 7E, and 7F, which show the analysis results of the therapeutic effects of the AAV-based cancer vaccines of Examples 4, 6, and 8 on colorectal cancer. Here, ** indicates p<0.01, *** indicates p<0.001, and data were analyzed by one-way independent analysis of variance. As shown in Figures 7B-7E, administration of the AAV-based cancer vaccine of Example 4 alone fails to protect mice with colorectal cancer from tumor progression. However, tumor growth is slightly delayed in the groups administered with the AAV-based cancer vaccine of Example 6 or Example 8 alone. However, the groups administered with the AAV-based cancer vaccine and radiation therapy simultaneously (Examples 5, 7, and 9) exhibit a significant tumor growth inhibitory effect. The results in Figure 7F also show that the expression level of the proliferating cell marker Ki67 is significantly reduced in Examples 7 and 9. The above results indicate that the AAV-based cancer vaccines of Examples 4, 6, and 8 significantly improve the therapeutic effect of radiation therapy and induce tumor antigen-specific immune responses to delay tumor growth.

[0039] [Examples 10-12]

[0040] We further constructed the vaccine of the present disclosure and confirmed its therapeutic effect on cancer. Please refer to Figure 8A, which shows a schematic diagram of the construction of the vaccine of Example 10 of the present disclosure. As shown in Figure 8A, the peptide encoded by the transgene in the vaccine of Example 10 of the present disclosure contains I as a secretory signal peptide. L2sp, at least one tumor antigen selected from neoAgs 1-8 listed in Table 1, at least one co-inhibitory peptide selected from PD-1 trap and CTLA4 trap, and a TLR9 antagonist sequence selected from TLR9i. The nucleotide fragments corresponding to the peptides are constructed into a CMV-primed pAAV-CMV expression vector. L2 The amino acid sequence of sp is shown in SEQ ID NO: 11. The amino acid sequences of PD-1 trap and CTLA4 trap are shown in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. TLR9i contains the amino acid sequences shown in SEQ ID NO: 20 and SEQ ID NO: 21. Comparative Example 4 also contains I L2 The pAAV-CMV expression vector contains nucleotide fragments encoding sp, PD-1 trap, CTLA4 trap, and TLR9i.

[0041] See Figure 8B and Table 6. Figure 8B shows a schematic diagram of the treatment strategy for the vaccine of Example 10 of the present disclosure in combination with radiation therapy in an animal treatment test, and Table 6 shows the treatment strategies for Examples 10-12 and Comparative Examples 4-6. Table 6, Treatment Strategies of Examples 10-12 and Comparative Examples 4-6 [Table 6]

[0042] To verify the therapeutic effect of the vaccine of Example 10 of the present disclosure, mice with colorectal cancer were randomly assigned to different groups. 8 vg) were intramuscularly injected four times on days 8, 14, 21, and 25, respectively. For the radiotherapy group, mice with colorectal cancer were fully anesthetized on day 11, and then their right limbs were placed in the radiation field to receive a single 5 Gy fractionated radiotherapy to the local tumor. Alternatively, two doses of radiotherapy were administered in the same manner on days 11 and 17. At the same time, tumor volume was measured every three days and calculated using the formula V = (L × W) until sacrifice on day 28. 2 ) / 2 to calculate tumor volume.

[0043] See Figure 8C, Figure 9, and Table 7. Figure 8C is a graph showing the analysis results of the therapeutic effect of the vaccine of Example 10 of the present disclosure on colorectal cancer, Figure 9 is a survival curve of colorectal cancer mice treated with the vaccine of Example 10 of the present disclosure, and Table 7 shows the complete response (CR) rates of Examples 10-12 and Comparative Examples 4-6. Table 7, complete remission rates of Examples 10-12 and Comparative Examples 4-6 [Table 7]

[0044] The results in Figure 8C show that the tumor volume of Example 12 (administered simultaneously with the vaccine of Example 10 of the present disclosure and radiation therapy) was significantly reduced compared with the other groups, indicating that the vaccine of Example 10 of the present disclosure significantly promoted the therapeutic effect of radiation therapy. The results in Figure 9 and Table 7 show that 40% of the colorectal cancer mice achieved complete remission (2 / 5) after administration of the vaccine of Example 10 of the present disclosure, indicating that the vaccine of Example 10 of the present disclosure significantly extended the survival time of the colorectal cancer mice.

[0045] [Examples 13-15]

[0046] We further constructed a vaccine according to another embodiment of the present disclosure and confirmed its efficacy against cancer. See Figure 10A and Table 8. Figure 10A shows a schematic diagram of the construction of the vaccine according to Example 13 of the present disclosure and a schematic diagram of the treatment strategy in combination with radiation therapy in animal treatment tests, and Table 8 shows the treatment strategies of Examples 13-15, Comparative Examples 7-9, and Control Groups 1-3. Table 8, Examples 13-15, Comparative Examples 7-9, and Control Groups 1-3 Treatment Strategies [Table 8]

[0047] As shown in FIG. 10A, the peptide encoded by the transgene in the vaccine of Example 13 of the present disclosure contains I as a secretory signal peptide.L2 sp, neoAg / aeTSA as at least one tumor antigen, PD-1 trap and PD-L1 miRNA (indicated as "miR" in Figure 10A) as at least one co-inhibitory peptide, and TLR9i as a TLR9 antagonist sequence, and the nucleotide fragments corresponding to the above peptides are constructed into a CMV-primed pAAV-CMV expression vector. L2 The amino acid sequence of sp is shown in SEQ ID NO:11. The neoAg / aeTSA includes neoAgs 1-8 listed in Table 1 and aeTSAs 1-7 listed in Table 4. The amino acid sequence of PD-1 trap is shown in SEQ ID NO:22, and the nucleotide sequence of PD-L1 miRNA is shown in SEQ ID NO:24. TLR9i includes the amino acid sequences shown in SEQ ID NO:20 and SEQ ID NO:21. Comparative Example 7 also includes I L2 sp, PD-1 trap, and TLR9i, but does not include the nucleotide fragment encoding the tumor antigen, or the pAAV-CMV expression vector for PD-L1 miRNA.

[0048] To verify the therapeutic effect of the vaccine of Example 13 of the present disclosure, mice with colorectal cancer were randomly assigned to different groups. 8 vg) and PBS were intramuscularly injected four times on days 8, 14, 21, and 25, respectively. For the radiotherapy group, mice with colorectal cancer were fully anesthetized on day 11, and their right limbs were placed in the radiation field. A single 5 Gy fractionated radiotherapy was administered to the local tumor. Alternatively, two fractionated radiotherapy sessions were administered on days 11 and 18. Additionally, mice with colorectal cancer were injected with 3 × 10 5 CT26 cells and 20% Matrigel were subcutaneously inoculated on day 56. At the same time, tumor volume was measured every 3 days and calculated using the formula V = (L × W 2) / 2 to calculate tumor volume. On the 30th day of the study, analysis was performed by flow cytometry. In addition, after intramuscular injection of the vaccine of Example 13 and the vaccine of Comparative Example 7 of the present disclosure, the Glud1 level in the blood of the mice with colorectal cancer was measured. + Expression levels of CD8 cells are measured in a Glud1 / MHC-I specific tetramer assay.

[0049] See Figures 10B, 10C, 10D, 10E, 10F, 10G, Tables 9 and 10. Figures 10B, 10C, 10D, 10E, 10F, and 10G are graphs showing the analysis results of the effect of combining the vaccine of Example 13 of the present disclosure with radiation therapy to treat colorectal cancer, where * represents p<0.05, ** represents p<0.01, and *** represents p<0.001, and the data was analyzed by one-way independent analysis of variance. Table 9 shows the complete remission rates of Examples 13 and 15, Comparative Examples 7 and 9, and Control Groups 1 and 3, and Table 10 shows the median survival times of Examples 13 and 15, Comparative Examples 7 and 9, and Control Groups 1 and 3. Table 9, complete remission rates for Examples 13 and 15, Comparative Examples 7 and 9, and Control Groups 1 and 3 [Table 9] Table 10, Median survival times for Examples 13 and 15, Comparative Examples 7 and 9, and Control Groups 1 and 3 [Table 10]

[0050] The results in Figures 10B-10D show that, compared with the other groups, the tumor volume and tumor weight of Comparative Example 9 (in which the vaccine of Comparative Example 7 and radiation therapy were administered simultaneously) were significantly reduced by about 70%. However, the tumor volume and tumor weight of Example 15 (in which the vaccine of Example 13 of the present disclosure and radiation therapy were administered simultaneously) were significantly reduced by about 90%. The results in Figures 10E, 10F, Tables 9, and 10 show that 40% of the colorectal cancer mice achieved complete remission (3 / 7) after administration of the vaccine of Example 13 of the present disclosure, and their survival time was significantly extended. Furthermore, no secondary tumors developed even 370 days after reinjection of CT26 cells into tumor-free colorectal cancer mice, indicating that administration of the vaccine of Example 13 of the present disclosure not only improves the therapeutic effect of radiation therapy but also inhibits tumor regeneration. The results in Figure 10G show that neoantigen-specific T cell immune responses are significantly increased in monocytes from colorectal cancer mice vaccinated with the vaccine of Example 13 of the present disclosure. Therefore, the above results indicate that the vaccine of Example 13 of the present disclosure can be combined with radiation therapy to achieve a complete response and inhibit tumor recurrence.

[0051] [Examples 16-17]

[0052] To further demonstrate that the vaccine of the present disclosure induces high neoantigen immunogenicity and improves the therapeutic effect of radiation therapy, another vaccine containing eight mutant TSAs was constructed and inoculated into BALB / c mice bearing 4T1 tumors. See Figure 11A and Table 11. Figure 11A shows a schematic diagram of the construction of the vaccine of Example 16 of the present disclosure and a schematic diagram of the therapeutic strategy in combination with radiation therapy in the animal therapeutic test, and Table 11 shows the therapeutic strategies of Examples 16-17, Comparative Examples 10-11, and control groups 4-5. Treatment strategies in Table 11, Examples 16-17, Comparative Examples 10-11, and Control Groups 4-5 [Table 11]

[0053] As shown in FIG. 11A, the peptide encoded by the transgene in the vaccine of Example 16 of the present disclosure contains I as a secretory signal peptide. L2 sp, neoAg as at least one tumor antigen, PD-1 trap and PD-L1 miRNA (indicated as "miR" in Figure 11A) as at least one co-inhibitory peptide, and TLR9i as a TLR9 antagonist sequence, and the nucleotide fragments corresponding to the above peptides are constructed into a CMV-primed pAAV-CMV expression vector. L2 The amino acid sequence of sp is shown in SEQ ID NO:11, and neoAg includes neoAgs 9-16 listed in Table 12. The amino acid sequence of PD-1 trap is shown in SEQ ID NO:22, the nucleotide sequence of PD-L1 miRNA is shown in SEQ ID NO:24, and TLR9i includes the amino acid sequences shown in SEQ ID NO:20 and SEQ ID NO:21. Comparative Example 10 also includes I L2 sp, PD-1 trap, and TLR9i, but does not include the nucleotide fragment encoding the tumor antigen, or the pAAV-CMV expression vector for PD-L1 miRNA. Table 12. NeoAgs in mouse mammary 4T1 cell line (hereinafter abbreviated as "4T1 cells") [Table 12]

[0054] To verify the therapeutic effect of the vaccine of Example 16 of the present disclosure, a breast cancer mouse model was established in the experiment. 5 4T1 cells and 20% Matrigel (Corning, Union City, CA, USA) were subcutaneously inoculated into 6-week-old female BALB / c mice to obtain BALB / c mice bearing 4T1 tumors, which are low-immunogenic breast cancer cells. After 8 days, the breast cancer mice were randomly divided into different groups and administered the vaccine of Example 16 of the present disclosure, Comparative Example 10 (1 × 10 8vg) and PBS were intramuscularly injected four times on days 8, 14, 21, and 25, respectively. For the radiotherapy group, the breast cancer mice were fully anesthetized on days 11 and 18, then placed in the radiation field and administered two 5 Gy fractionated radiotherapy sessions to the local tumor. At the same time, tumor volume was measured every three days and calculated using the formula V = (L × W 2 Tumor volume is calculated as follows: ) / 2. On day 31 of the study, tumors are analyzed by flow cytometry.

[0055] See Figures 11B, 11C, 11D, 11E, 11F, and 11G, which are graphs showing the analysis results of the therapeutic effect of the vaccine of Example 16 of the present disclosure on breast cancer, where * represents p<0.05, ** represents p<0.01, and the data are analyzed by one-way independent analysis of variance. As shown in Figures 11B and 11C, the tumor regression rate and tumor weight of Example 17 (in which the vaccine of Example 16 of the present disclosure and radiotherapy are administered simultaneously) are significantly reduced by about 80%. The results of Figures 11D-11F show that the tumor-infiltrating CD4+ in the residual tumor of Example 17 + cells, CD8 + cells, CD4 + T EM cells, CD8 + T EM cells, and IFNγ + CD8 + The results in Figure 11G show that Example 17 also significantly reduces PD-L1 expression levels in tumor-infiltrating DCs. These results indicate that the vaccine of Example 16 of the present disclosure can inhibit PD-L1 expression in DCs, resulting in better antigen presentation and T cell-mediated immune responses. This indicates that the vaccine of Example 16 of the present disclosure improves the therapeutic efficacy of radiation therapy in a poorly immunogenic breast cancer animal model.

[0056] [Examples 18-22]

[0057] Please refer to Figures 12A and 12B. Figure 12A shows a schematic diagram of a treatment strategy for cancer treatment of a cancer vaccine combination according to one embodiment of the present disclosure, and Figure 12B shows a schematic diagram of an example of a cancer vaccine combination according to one embodiment of the present disclosure.

[0058] According to another embodiment of the present disclosure, the present disclosure provides a cancer vaccine combination comprising the vaccine described in the preceding paragraph, a booster, and an immune booster. The vaccine is used to induce an immune response against at least one tumor antigen in a subject in need of cancer treatment. The booster is used to enhance local tumor control in the subject. The immune booster is used to prevent local recurrence and metastasis in the subject.

[0059] The at least one tumor antigen may be selected from tumor-associated antigens (TAA), tumor-specific antigens (TSA), oncogenic mutations, aberrantly expressed tumor-specific antigens (aeTSA), and shared neoantigens (neoAg). The enhancing agent may be radiation, a chemotherapeutic agent, an immunomodulatory agent, a targeted therapy agent, an antibody drug, or a combination thereof. The immune enhancing agent may be a cancer vaccine comprising at least one tumor antigen or a therapeutic cell comprising at least one tumor antigen. Preferably, the cancer vaccine may be a dendritic cell-based cancer vaccine or a virus-based cancer vaccine, and the therapeutic cell comprising at least one tumor antigen may be a cytokine-induced killer cell (CIK), a dendritic cell-bound cytokine-induced killer cell (DC-CIK), or a neoAg-pulsed DC-CIK. The immune enhancing agent may be a therapeutic cell comprising an immune checkpoint protein, an immunosuppressant, and / or an immunostimulator. Preferably, the therapeutic cells comprising an immune checkpoint protein may be chimeric antigen receptor-T cells (CAR-T), chimeric antigen receptor-natural killer cells (CAR-NK), or adoptive T cells.

[0060] See Figure 13A and Table 13. Figure 13A shows a schematic diagram of the treatment strategies in animal treatment tests of a cancer vaccine mixture according to one embodiment of the present disclosure, and Table 13 shows the treatment strategies of Examples 18-22 and Comparative Example 12. Treatment strategies in Table 13, Examples 18-22 and Comparative Example 12 [Table 13]

[0061] To verify the therapeutic effect of the cancer vaccine mixture of the present disclosure, mice with colorectal cancer were randomly assigned to different groups. The vaccine of Example 13 of the present disclosure and Comparative Example 7 (1×10 8 vg) were intramuscularly injected twice on days 8 and 14, respectively. In this study, radiation therapy was used as an boosting agent. In the boosting agent group, mice with colorectal cancer were fully anesthetized on days 11, 18, and 25, and then their right limbs were placed in the radiation field. Three 5 Gy fractionated radiation therapy sessions were administered to the local tumor. In this study, neoAg-DC-CIK was used as an immune boosting agent. In the immune boosting agent group, neoAg-DC-CIK was intramuscularly injected twice on days 21 and 31. αPD-1 was also administered as an immune checkpoint inhibitor (ICB) twice on days 16 and 23. At the same time, tumor volume was measured every three days and calculated using the formula V = (L × W) until sacrifice on day 40. 2 ) / 2 to calculate tumor volume.

[0062] See Figure 13B, Figure 13C, Figure 13D, and Table 14. Figure 13B, Figure 13C, and Figure 13D are analysis results of the therapeutic effect of the cancer vaccine mixture of the present disclosure on colorectal cancer, where *** represents p<0.001, and the data is analyzed by one-way independent analysis of variance. Table 14 shows the complete remission rates of Examples 18-22 and Comparative Example 12. Table 14, complete remission rates of Examples 18-22 and Comparative Example 12 [Table 14]

[0063] The results in Figure 13B show that the tumor volumes of Examples 19 to 22 were all significantly reduced compared to the other groups. Furthermore, approximately 33% of the colorectal cancer mice in Example 20 achieved complete remission (2 / 6), and approximately 83% of the colorectal cancer mice in Example 22 achieved complete remission (5 / 6). The results in Figures 13C and 13D show that the mononuclear cells of the colorectal cancer mice in Example 22 significantly increased tumor antigen-specific T cell immune responses. These results demonstrate that the cancer vaccine mixture of the present disclosure can achieve a complete response and induce tumor antigen-specific T cell immune responses.

[0064] As described above, the vaccine of the present disclosure co-expresses at least one co-inhibitory peptide and a TLR9 antagonist sequence to increase the expression of at least one tumor antigen and activate tumor antigen-specific T cell immune responses. Therefore, the vaccine of the present disclosure has sufficient transgene expression, reduces viral clearance, and increases tumor antigen expression levels. It is clinically safe and has great potential and advantages. Furthermore, the vaccine of the present disclosure can improve the therapeutic effect of radiation therapy for cancer and synergistically increase the therapeutic effect of radiation therapy and the vaccine of the present disclosure containing the co-inhibitory peptide, providing a novel, safe, and effective tumor antigen-based immunotherapy. Furthermore, therapy with the cancer vaccine combination of the present disclosure, which includes administering the vaccine of the present disclosure, a booster, and an immune booster, is effective in inhibiting tumor growth and tumor recurrence.

[0065] While the present disclosure has been disclosed in detail in the above embodiments, other embodiments are possible, and therefore, the spirit and scope of the appended claims should not be limited to the description of the embodiments herein.

[0066] Obviously, those skilled in the art can make various changes and modifications to the structure of the present disclosure without departing from the scope or spirit of the disclosure. In view of the foregoing, it is intended that this specification cover modifications and variations of the present disclosure that fall within the scope of the appended claims. [Explanation of symbols]

[0067] 100 vaccines 110 Vector 120 transgenes 121 Secretory signal peptide 122 Tumor antigens 123 Co-inhibitory peptides 124 Toll-like receptor 9 antagonist sequences

Claims

1. Vector and a transgene encoding a plurality of peptides and packaged in the vector; Including, The peptide is a secretory signal peptide; At least one tumor antigen is a shared neoantigen (neoAg) or is a neoAg / aeTSA comprising a shared neoantigen (neoAg) and an aberrantly expressed tumor-specific antigen (aeTSA); at least one co-inhibitory peptide selected from the group consisting of programmed cell death-ligand 1 (PD-L1) antagonists, programmed cell death protein 1 (PD-1) antagonists, and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antagonists; Toll-like receptor 9 (TLR9) antagonists, in order, The neoAg comprises the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32; The neoAg / aeTSA comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19; vaccine.

2. 2. The vaccine of claim 1, further comprising a costimulatory peptide between the at least one co-inhibitory peptide and the TLR9 antagonist sequence, wherein the costimulatory peptide is selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 12 (IL12), and interferons (IFNs).

3. 2. The vaccine of claim 1, wherein the vector is a vaccinia virus vector, an adeno-associated virus (AAV) vector, or a nanoparticle.

4. The vaccine of claim 1 , wherein the secretory signal peptide is an interleukin 2 signal peptide (IL2 sp) or an interleukin 12 signal peptide (IL12 sp).

5. 2. The vaccine of claim 1, wherein the at least one tumor antigen is a DNA sequence selected by comparing whole exome sequencing of the normal cells and corresponding tumor cells of the subject to identify tumor-specific somatic mutations.

6. The vaccine of claim 1, wherein the PD-L1 antagonist comprises a PD-L1 trap.

7. The vaccine of claim 1 , wherein the PD-1 antagonist comprises a PD-1 trap.

8. The vaccine of claim 1 , wherein the CTLA4 antagonist comprises a CTLA4 trap and a CTLA4 antagonist antibody.

9. 2. The vaccine of claim 1, wherein the TLR9 antagonist sequence is selected from a CpG oligonucleotide TLR9 binding domain, a TLR decoy peptide, and a CpG binding sequence.

10. 10. The vaccine of claim 1 for use in cancer treatment, wherein the vaccine is used to induce an anti-tumor immune response in a subject in need of cancer treatment.

11. The vaccine for use according to claim 10, wherein the vaccine is used in combination with radiation.

12. The vaccine of claim 1 for use in inducing an immune response against the at least one tumor antigen in a subject in need of cancer treatment; an enhancement agent used to enhance local tumor control in the subject, the enhancement agent being radiation, a chemotherapeutic agent, an antibody drug, or a combination thereof; and an immune enhancing agent used to prevent local recurrence and metastasis in the subject, the immune enhancing agent being a cancer vaccine comprising the at least one tumor antigen or therapeutic cells comprising the at least one tumor antigen.

13. 13. The cancer vaccine mixture of claim 12, wherein the cancer vaccine is a dendritic cell-based cancer vaccine or a virus-based cancer vaccine, and the therapeutic cells are cytokine-induced killer cells (CIKs), dendritic cell-bound cytokine-induced killer cells, or neoAg-loaded DC-CIKs.

14. 13. The cancer vaccine combination of claim 12, further comprising an immune checkpoint inhibitor.

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