Tumor nano vaccine of bionic autophagosome as well as preparation method and application of tumor nano vaccine

Abstract

The invention discloses a tumor nano vaccine (BAPs) of bionic autophagosome as well as a preparation method and application thereof, and the vaccine effectively stimulates anti-tumor immunity by precisely fusing a neoantigen derived from the autophagosome and an immunologic adjuvant targeted lymph node. The BAPs with the redox response characteristic promote the vaccine to be opened in antigen presenting cells, so that an adjuvant and an antigen are exposed, and a strong immune response is stimulated. The BAPs can excite wide T cell reactions, and finally, more than 70% of established tumors are effectively eliminated. BAPs vaccination initiates a lasting T cell reaction and provides powerful protection, all mice are protected in tumor recurrence challenge, and the tumor incidence rate is significantly reduced by nearly 90%. In addition, the BAPs and a checkpoint inhibition therapy have a synergistic effect, and growth of breast cancer with poor immunogenicity is inhibited. The BAPs nano vaccine provides an efficient strategy with clinical transformation potential to enhance the strong immune response of personalized cancer immunotherapy.

Description

Technical Field

[0001] The invention belongs to the technical field of biological nano-medicines, and relates to the research of autophagosome tumor antigens and the development and application research of a class of anti-tumor nano-vaccines co-loaded with autophagosome proteins and immune adjuvants. Background Art

[0002] Tumor vaccines are a breakthrough cancer immunotherapy approach that aims to stimulate and enhance innate and adaptive immune responses against tumor antigens (Ags) and ultimately eliminate tumor cells. In recent years, tumor vaccines have co-delivered tumor-specific antigens with immune adjuvants to antigen-presenting cells (APCs), especially dendritic cells (DCs), to induce specific tumor immune responses. A large number of studies have been devoted to customizing vaccines using autoantigens that are abnormally expressed in tumors, namely tumor-associated antigens (TAAs). However, the use of tumor cell derivatives such as cell lysates, cell-derived nanovesicles, or extracted cell membranes as TAAs has encountered challenges in anti-tumor immune responses, which is mainly attributed to the limited immunogenicity of these antigens. In addition, vaccines derived from autologous tumor cells require tumor resection, which not only limits the access to tumor antigens in cases where tumors cannot be resected, but also increases the risk of metastasis, which becomes a limitation of clinical application.

[0003] Autophagy is a catabolic pathway that is strictly regulated and stress-induced in all eukaryotes and plays an important role in antigen presentation and stimulating host cell immunity. Although tumor cell-derived autophagosome vaccines (TRAPs) can promote immune enhancement, there are still some limitations, such as the large particle size of TRAPs, which is not ideal for endocytosis, and their poor targeting ability to lymph nodes. Therefore, it has become a key challenge to effectively deliver antigens and adjuvants to lymph nodes and enhance antigen cross-presentation to stimulate controllable and potent tumor-specific T cell responses. Summary of the invention

[0004] The purpose of the present invention is to disclose a bionic autophagosome (BAPs) tumor nanovaccine and its preparation method and application, specifically a bionic nano tumor vaccine co-loaded with autophagosome-derived tumor antigens and immune adjuvants and its preparation method, as well as its application in the treatment of tumors, especially solid tumors.

[0005] The present invention adopts the following technical scheme.

[0006] A bionic autophagosome tumor nanovaccine comprises an amphiphilic block polymer and an immune adjuvant and an autophagosome protein loaded by the amphiphilic block polymer, wherein the autophagosome protein is related to the tumor.

[0007] A drug, the active ingredient of which includes the above-mentioned biomimetic autophagosome tumor nanovaccine; further, it may also include other active ingredients. Other active ingredients refer to ingredients that can play an effect on tumor prevention and treatment in addition to the above-mentioned biomimetic autophagosome tumor nanovaccine, such as immune checkpoint inhibitors or cytokines.

[0008] The present invention discloses a method for preparing the above-mentioned bionic autophagosome tumor nanovaccine, which comprises the following steps: using immune adjuvant, autophagosome protein and amphiphilic block polymer as raw materials to prepare the bionic autophagosome tumor nanovaccine.

[0009] The tumor nanovaccine of the bionic autophagosome of the present invention can effectively play a role in preventing and treating tumors, and can also be used in combination with immune checkpoint inhibitors or cytokines to synergistically play a role in preventing and treating tumors, such as in combination with one or more of cytotoxic T lymphocyte-associated protein 4 antibody (anti-CTLA-4), anti-programmed cell death receptor ligand 1 (anti-PD-L1), anti-programmed cell death receptor 1 (anti-PD-1), interleukin 15 (IL-15), IL-2, etc.

[0010] In the present invention, the autophagosome protein is an autophagosome lysate or a genetically engineered protein product; wherein the autophagosome is derived from the supernatant of tumor cell culture, or the autophagosome is derived from the ascites and / or pleural effusion of a metastatic patient. Autophagosomes are obtained from the supernatant of tumor cell culture or from the ascites and pleural effusion of a patient with tumor metastasis. Specifically, when obtaining autophagosomes from the supernatant of tumor cell culture, autophagy is induced in the tumor cells, for example, using an autophagy inducer, hypoxia, etc., and the obtained autophagosomes are enriched by centrifugation or magnetic bead sorting. The operation of obtaining autophagosome proteins is a conventional technique, and a lysate, such as NP-40, RIPA, etc., can be used; a freeze-thaw method, such as a liquid nitrogen freeze-thaw method, can also be used to obtain abundant autophagosome proteins from autophagosomes as tumor antigens.

[0011] In the present invention, the immune adjuvant includes one or more of a Toll-like receptor agonist, a NOD-like receptor agonist, and a STING agonist.

[0012] In the tumor nanovaccine of the biomimetic autophagosome of the present invention, the autophagosome protein is preferably a lysate of the autophagosome of the tumor cell; the preferred immune adjuvant is CpG oligodeoxynucleotide, polyIC, ADU-S100, etc. The feed mass ratio of the autophagosome protein to the immune adjuvant is 1: (0.1-8), the preferred feed mass ratio is 1: (3-8), and the more preferred feed mass ratio is 1: (5-8). In the feed ratio, the amount of adjuvant refers to the total amount of all immune adjuvants. Furthermore, when there are two adjuvants, the mass ratio of the two adjuvants is 1: (1-8), preferably 1: (2-5).

[0013] In the present invention, the amphiphilic block polymer is a hydrophilic segment-P(A-DTC)-cationic segment. The amphiphilic block polymer is composed of a hydrophilic segment, a hydrophobic segment and a cationic segment, wherein the hydrophilic segment is a polyethylene glycol segment (PEG), preferably, the number average molecular weight of PEG is 2000-12000 Da, and the preferred molecular weight is 3000-8000 Da; the hydrophobic segment is a random copolymer segment, expressed as P(A-DTC), and its molecular weight is 2-10 times that of the hydrophilic segment, preferably 2-6 times; wherein A includes an ester monomer and a carbonate monomer, preferably, A is a cyclic ester monomer, a cyclic carbonate monomer, etc., such as a trimethylene carbonate monomer TMC, a lactide monomer LA, a caprolactone monomer CL, etc.; PDTC is polydithiolane trimethylene carbonate, and the molecular weight of the PDTC segment is 5%-40% of the total molecular weight of the hydrophobic segment, preferably 10-30%. The cationic fragment includes poly(ethyleneimine) PEI and spermine SP, etc., and has a molecular weight of 100 to 3000 Da, preferably spermine.

[0014] The present invention discloses the use of the above-mentioned bionic autophagosome tumor nano-vaccine and drug in the preparation of drugs for preventing and treating tumors. The tumor is a solid tumor or a blood tumor, preferably a solid tumor, such as lymphoma, colon cancer, breast cancer, etc., especially in the prevention and / or treatment of colon cancer.

[0015] The biomimetic nano tumor vaccine for anti-tumor co-loading autophagosome protein and immune adjuvant disclosed in the present invention includes an amphiphilic block polymer and the immune adjuvant and autophagosome protein loaded by the amphiphilic block polymer, and the autophagosome protein derived from the autophagosome is used as the tumor antigen; the polymer vesicle formed by the amphiphilic block polymer is loaded with the immune adjuvant and the tumor antigen; the amphiphilic block polymer is a hydrophilic segment-P(A-DTC)-cationic fragment. Wherein, the immune adjuvant includes one or more of Toll-like receptor agonists, NOD-like receptor agonists, and STING agonists; the autophagosome protein is a lysate of tumor autophagosomes.

[0016] The bionic autophagosome tumor nanovaccine of the present invention is assembled and cross-linked by amphiphilic block polymers, and has an asymmetric membrane structure, with an outer shell of polyethylene glycol (PEG), a membrane layer of reversibly cross-linked hydrophobic polycarbonate, and an inner layer of spermine (SP); it can be combined with immune adjuvants and autophagosome proteins through electrostatic interaction, thereby achieving efficient and stable loading of adjuvants and antigens.

[0017] In the present invention, the bionic nano-tumor vaccine can be used in combination with immune checkpoint inhibitors or cytokines, such as cytotoxic T lymphocyte-associated protein 4 antibody (anti-CTLA-4), anti-programmed cell death receptor ligand 1 (anti-PD-L1), anti-programmed cell death receptor 1 (anti-PD-1), interleukin 15 (IL-15), IL-2, etc.

[0018] In the present invention, autophagosomes are obtained from the supernatant of tumor cell culture or from the ascites and pleural effusion of the transferred patient. Among them, obtaining autophagosomes from the supernatant of tumor cell culture requires inducing autophagy in tumor cells, such as using autophagy inducers, hypoxia, etc., and enriching the obtained autophagosomes by centrifugation or magnetic bead sorting, which is specifically the prior art. The operation of obtaining autophagosome lysate is a conventional technique, and a lysis solution, such as NP-40, RIPA, etc., can be used; a freeze-thaw method, such as liquid nitrogen freeze-thaw method, can also be used to obtain rich autophagosome proteins as tumor antigens.

[0019] Autophagosomes contain a wider range of tumor-specific antigens, thereby activating major histocompatibility complex class I (MHC-I) on tumor cells and enhancing immune responses. Studies have shown that autophagosome vaccines (TRAPs) derived from tumor cells are rich in autophagosome-encapsulated cellular proteins, defective ribosomal products (DRiPs), short-lived proteins (SLiPs), and surface markers, and show higher efficacy in mediating tumor immune responses than vaccines derived from tumor cells. More importantly, TRAPs can be extracted from the pleural effusion and ascites of cancer patients.

[0020] The biomimetic nano-tumor vaccine of the present invention has a spontaneous lymph node targeting effect after subcutaneous administration, and effectively promotes the maturation of antigen presenting cells, especially DC. The biologically active immune adjuvant and autophagosome protein in the disulfide cross-linked vesicle lumen can avoid leakage and loss during the transportation process, and under the action of the intracellular reducing agent glutathione (GSH), the disulfide bond is quickly broken to release the active ingredients, effectively activating DC. Furthermore, DC promotes the recognition of tumor antigens by cytotoxic T lymphocytes (CTL), thereby improving the specific killing of tumor cells.

[0021] Compared with the prior art, the present invention has the following advantages: 1. The tumor antigens extracted by the present invention are derived from tumor autophagosomes, which have stronger immunogenicity compared with traditional tumor cell / tissue lysates. The bionic nano-tumor vaccine has a stronger effect on stimulating DC maturation and antigen presentation, and has better therapeutic effect in tumor-bearing mice, thus solving the problem of low immunogenicity of tumor vaccine antigens.

[0022] 2. The present invention designs a biomimetic nano-tumor vaccine that co-loads tumor antigens and immune adjuvants, achieving efficient and stable loading of immune adjuvants and tumor antigens and targeted enrichment of lymph nodes, thereby effectively solving the problem of bioactive molecules being susceptible to enzymatic hydrolysis and inefficient cell uptake. The vesicle membrane uses a reversibly cross-linked biodegradable polymer PTMC, and its side chain is designed as a dithiolane, providing reduction-sensitive reversible cross-linking properties. This design can not only ensure that the drug circulates for a long time in the blood, but also can be rapidly degraded in cells, thereby effectively releasing immune adjuvants and tumor antigens into DCs.

[0023] 3. The bionic nano tumor vaccine disclosed in the present invention exhibits significant DC activation effects both in vivo and in vitro. The vaccine can simultaneously release tumor antigens and immune adjuvants into DC, which not only promotes the maturation of DC, but also improves the efficiency of antigen presentation, thereby enhancing the proliferation of CTL and tumor infiltration. It exhibits efficient anti-tumor activity in vivo, can effectively inhibit the occurrence of tumors, and promote complete regression of tumors. All treated mice acquired lasting immune memory and were able to comprehensively inhibit the recurrence of tumors. In addition, the bionic nano tumor vaccine can also be used as a preventive tumor vaccine, which has a significant preventive effect on tumors and expands the scope of application of the anti-tumor effect.

[0024] 4. The bionic nano-tumor vaccine disclosed in the present invention can be used in combination with commonly used immune checkpoint inhibitors or cytokines in clinical practice, and also has a good therapeutic effect on "cold tumors", providing a new research idea for efficient immunotherapy of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the technical solution of the present invention.

[0026] Figure 2 Schematic diagram of the structure of PEG-P(TMC-DTC)-SP.

[0027] Figure 3 The identification results of TRAP, including (A) size distribution histogram of TRAP derived from MC-38 cells; (B) TEM image of TRAP derived from MC-38 cells, with a scale of 100 nm; (C) Western blot detection of the expression levels of LC3-I and LC3-II in TRAP isolated from MC-38 and 4T1 cell lysates and their culture supernatants.

[0028] Figure 4 Preparation and characterization of biomimetic nano-tumor vaccines (BAPs), including (A) particle size distribution of PS nanovesicles and BAPs and TEM image of BAPs, with a scale of 10 nm; (B) size changes of BAPs in solutions containing FBS or GSH.

[0029] Figure 5 The immunogenicity of Ag from different sources was investigated, including (A) the experimental design for evaluating the therapeutic effect of PS nanovesicles loaded with TRAP or cell lysate Ag; (B) the average tumor growth curve of tumor-bearing mice after different treatments; (C) the individual tumor growth curve of mice after different treatments (n=5). ** P <0.01.

[0030] Figure 6 Figure 2 Flow cytometry results of PS nanovesicles, physical mixing group and BAPs nanovaccine incubated with BMDCs for 24 h, including (A) flow cytometry analysis of the activation percentage of BMDCs; (B) flow cytometry analysis of the expression of MHC-Ⅱ in BMDCs (n=3).

[0031] Figure 7 After 24 h of incubation of PS nanovesicles, physical mixing groups, and BAPs nanovaccines with BMDCs, the culture medium was collected to quantify the expression levels of typical proinflammatory cytokines, including the concentrations of (A) IL-6, (B) IL-12p70, (C) IL-1β, (D) IFN-β, and (E) TNF-α in the culture medium (n = 3).

[0032] Figure 8 The in vivo distribution of BAPs is studied, including (A) in vitro fluorescence imaging of inguinal dLNs. Cy5-labeled Ag or Cy5-labeled TRAP and BAPs were subcutaneously injected at the tail base of mice, and in vitro imaging was performed 24 hours later; (B) representative in vitro fluorescence imaging of each group of mice; (C) average fluorescence intensity of dLNs in Figure A; (D) dLN / liver fluorescence intensity ratio (n=4).

[0033] Fig. 9 Figure 2 shows immune analysis of lymph nodes and spleen within 24 hours after the last injection of mice, including (A) representative flow cytometry images; CD80 in lymph nodes + CD86 + (B) and MHC-Ⅱ + (C) Quantification of DCs; NKG2D in mouse spleen + NK + Quantification of A cells (D) and B cells (E) indicating the systemic immune response induced by different preparations in vivo (n = 3).

[0034] Fig.10In vivo treatment of BAPs nanovaccine in the MC-38 model, including (A) experimental design timetable for evaluating the in vivo therapeutic performance of BAPs; (B) average tumor growth curve of MC-38 mice after treatment with different preparations; (C) single tumor growth curve in Figure B; (D) disease-free survival rate of MC-38 mice in different groups after different treatments (n=7).

[0035] Fig.11 This is the immune analysis of the mouse tumor microenvironment 7 days after the end of treatment, where (A) is the CD8 + Representative flow cytometry images of T cells; (B) is CD8 + Quantification results of T cells; (C) CD4 + T cell quantification; (D) CD4 + CD25 + FoxP3 + Quantification of regulatory T cells (n=4).

[0036] Fig.12 Figure 2 shows the peripheral blood immune analysis of mice after treatment. (A) Effector memory CD8 + T (CD8 + CD44 + CD62L - ) Quantification of cells (n=4); (B) Quantification of effector memory CD4 + T (CD4 + CD44 + CD62L - ) cells (n=3); ELISA method was used to quantify the expression levels of (C) IFN-γ (n=5) and (D) TNF-α in serum (n=4).

[0037] Fig.13 BAPs nanovaccine can prevent tumor re-challenge, where (A) BAPs nanovaccine-immunized mice (tumor completely regressed about 100 days after the first vaccination) were subcutaneously injected with MC-38 cells again, and tumor growth was measured every 4 days; (B) Disease-free survival rate of MC-38-vaccinated mice in different groups; (C) Individual tumor growth curves in Figure A (n=5).

[0038] Fig.14 Effector memory CD8 in spleen (A), PBMCs (B), and LNs (C) + and CD4 + T (CD8 + CD44 + CD62L - and CD4 + CD44+ CD62L - ) Quantification of cells (n=5).

[0039] Fig.15 BAPs nanovaccine prevents tumor occurrence, including: (A) Experimental design timeline for evaluating the preventive effect of BAPs nanovaccine; (B) Body weight changes of mice immunized with BAPs nanovaccine after inoculation with MC-38 cells; (C) Tumor growth measured over time in mice inoculated with MC-38 cells in different groups; (D) Disease-free survival rate of mice in different treatment groups after inoculation with MC-38 cells; (E) Individual tumor growth curves in Figure C (n=8).

[0040] Fig.16 Flow cytometry analysis of peripheral blood CD8 + Cytotoxic T cells (A, B) and effector memory T cells (C, D) (n=6); (E) ELISA analysis of the expression levels of IFN-γ, TNF-α and IL-12p70 in mouse serum (n=5).

[0041] Fig.17 Figure 3. BAPs nanovaccine combined with ICB for the treatment of 4T1 tumors, including (A) schematic diagram of the treatment mechanism and experimental design timeline; (B) average tumor growth curve of 4T1-bearing mice after different treatments; (C) growth curve of a single tumor in B; (D) disease-free survival rate of different treatment groups (n=6).

[0042] Fig.18 This is the immune analysis of the spleen of mice 7 days after the end of treatment, including activated IFN-γ in the spleen + CD8 + Representative flow cytometry plots (A) and statistical data (B) of cytotoxic T cells; flow cytometry analysis of CD8 + Representative flow cytometry plots (C) and statistical data (D) of cytotoxic T cells (n = 5).

[0043] Fig.19 Immune analysis of mice after treatment, including photos of the spleen of mice after different treatments (A) and corresponding weights (B); (C) CD69 in the spleen + CD8 + Quantification of early activated T cells; (D) NKG2D in spleen + NK + Quantification of cells. AD, n=5; (E) ELISA analysis of TNF-α expression level in serum (n=4). DETAILED DESCRIPTION

[0044] Although TRAPs can promote immune enhancement, there are still some limitations, such as the large particle size of TRAPs is not ideal for endocytosis, and its targeting ability to lymph nodes is poor. The present invention discloses a bionic autophagosome tumor nanovaccine (BAPs) and its preparation method and application, specifically a bionic nano tumor vaccine co-loaded with autophagosome-derived tumor antigen (Ag) and immune adjuvant and its preparation method, which effectively delivers antigens and adjuvants to lymph nodes, enhances antigen cross-presentation, and stimulates controllable and powerful tumor-specific T cell response, which has become a key technology.

[0045] The bionic nano-tumor vaccine (BAPs) disclosed in the present invention is prepared from an amphiphilic block polymer, an immune adjuvant and an autophagosome protein. Preferably, the immune adjuvant includes one or more of a TLR9 agonist CpG oligodeoxynucleotide, a TLR3 agonist PolyIC, and a STING agonist ADU-S100; the autophagosome protein is a lysate of autophagosomes of tumor cells; the amphiphilic block polymer is a hydrophilic segment-P(A-DTC)-SP; as an example, the amphiphilic polymer is one or more of PEG-P(TMC-DTC)-SP, PEG-P(LA-DTC)-SP, and PEG-P(CL-DTC)-SP.

[0046] Preferably, the BAPs nano tumor vaccine of the present invention is a polymer nanovesicle co-loaded with immune adjuvants (CpG and ADU-S100) and tumor cell autophagosome lysates, which is obtained by self-assembly and cross-linking of amphiphilic block polymers while loading adjuvants and tumor antigens through electrostatic action; the BAPs nano tumor vaccine of the present invention achieves efficient immunotherapy of subcutaneous solid tumors or recurrent solid tumors by targeting lymph node enrichment, promoting DC maturation and antigen presentation, and enabling T cells to specifically recognize and kill tumor cells, and some mice can be completely cured ( Figure 1 ).

[0047] Furthermore, the biomimetic nano-tumor vaccine of the present invention can be used in combination with immune checkpoint inhibitors or cytokines, such as cytotoxic T lymphocyte-associated protein 4 antibody (anti-CTLA-4), anti-programmed cell death receptor ligand 1 (anti-PD-L1), anti-programmed cell death receptor 1 (anti-PD-1), interleukin 15 (IL-15), IL-2, etc. The dosage of the antibody or cytokine can be 0.5-10 mg / kg, and Kg refers to the weight of the mouse.

[0048] Experimental materials: Rapamycin (>99%), bortezomib (>99%), STING agonist ADU-S100 (>99%) were all purchased from MCE, ammonium chloride (NH4Cl, >99%, Sinopharm Reagent), glutathione (GSH, 99%, Yuanye Biotechnology), CpG (sequence: 5'-TCCATGACGTTCCTGACGTT-3', Shengong Biotechnology). 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bis-1-piperidin H -benzimidazole trihydrochloride staining solution (Hoechst 33342, Biyuntian), Lysotracker red dnd-99 lysosomal red fluorescent probe was purchased from Yisheng Biotechnology. Micro BCA protein detection kit (Pierce, Thermo Scientific, USA) was purchased and used according to the instructions. The antibodies used in the Western Blot experiment included the autophagy marker rabbit LC3B antibody (Novus Biologicals) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L (Abcam), which were used according to the instructions. Cy5-NHS ester (≥95%, water-soluble, Meilun Biotechnology), fluorescein isothiocyanate (isomer I) (>97%, TCI). Mouse interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12p70), interleukin-6 (IL-6), interleukin-1β (IL-1β) ELISA detection kit (Invivogen). Mouse interferon-β (IFN-β) and TNF-α high-sensitivity ELISA detection kit (Lianke Bio). Mouse IFN-γ +ELISpot assay kit (Dakotech) and recombinant mouse GM-CSF (Peprotech) were purchased and used directly. Various mouse fluorescent-labeled antibodies used in flow cytometry analysis (Biolegend): CD16 / 32, CD45-PE, CD45-PerCP / Cy5.5, CD11c-FITC, CD80-APC, CD86-PE / Cy7, MHCⅡ-PerCP / Cy5.5, CD3-FITC, CD3-APC, CD4-PE, CD8α-PE / Cy7, CD8α-PE, IFN-γ-APC, CD107a-Alexa 647, CD69-PE / Cy7, NK1.1-PerCP / Cy5.5, CD314-APC, CD44-FITC, CD62L-BV421, B220-PE, CD25-PE / Cy7, FoxP3-Alexa 647 were purchased and used according to the instructions. Amphiphilic block polymer PEG 5k -P(TMC 15k -DTC 2k )-SP 232 Based on the previous report of our research group, its structure is as follows Figure 2 .

[0049] Cells and animals: Mouse colon cancer cell line MC-38 was purchased from Huzhen Biotechnology, and triple-negative breast cancer cell line 4T1 was purchased from the Cell Bank of the Chinese Academy of Sciences. The mice used in the present invention were 6-8 weeks old female C57BL / 6 or Balb / c, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. All animal experiments and operations were approved by the Experimental Animal Center of Soochow University and the Animal Care and Use Committee of Soochow University.

[0050] The drug loading efficiency test method is as follows: the loading amount of autophagosome Ag is measured using the Micro BCA protein assay kit, and the loading amount of CpG and ADU-S100 is measured by the microvolume UV-visible spectrophotometer Nanodrop (ThermoScientific). The drug loading efficiency (DLE) is calculated as follows:

[0051] The subcutaneous models of colon cancer and breast cancer in mice were established by conventional methods. Each mouse was subcutaneously inoculated with 1.0×10 6 MC-38 cells or 1.0×10 5 4T1 cells, the tumor volume and body weight of mice were measured every two days to evaluate the therapeutic effect.

[0052] The following experiments illustrate the technical progress of the present invention. All raw materials are existing products, specific preparation operations and performance tests are conventional techniques, and data analysis uses conventional statistical methods.

[0053] Example 1 Extraction and identification of TRAP-derived antigens When the tumor cells grew to 80% density, they were treated with 100 nmol / mL rapamycin, 100 nmol / mL bortezomib and 10 mmol / mL ammonium chloride in complete 1640 medium for 24 h, and then the tumor cell culture supernatant was collected. The cells and cell debris were removed by centrifugation at 2000 rpm, 4 °C for 10 min. Next, the supernatant obtained by centrifugation was transferred to a new centrifuge tube, centrifuged at 12000 rpm, 4 °C for 15 min, and the supernatant was discarded to precipitate autophagosomes. Finally, the precipitate was resuspended in PBS and aliquoted. It can be stored at 4 °C for a short period of time or at -80 °C for a long period of time. The autophagosomes obtained from the above were taken, then treated with ultrasound (35kHz, 30 s), subjected to 6 freeze-thaw cycles under liquid nitrogen, centrifuged at 12000 rpm, 4 °C, 15 min to obtain the supernatant, and the autophagosome lysate (also called autophagosome Ag) was obtained. The concentration of autophagosome proteins in the supernatant was determined using a Micro BCA protein assay kit, and 50 mg / mL was used in the following experiments.

[0054] The preparation process of tumor cell lysate is as follows: After digesting tumor cells with trypsin, centrifuge at 1500 rpm for 10 min and discard the supernatant. Add 1 mL of RIPA lysis buffer to resuspend the cells and place them on ice for 30 min. Lyse the cells at low temperature, 12000 rpm, 4 ℃, 15 min, and take the supernatant to obtain tumor cell lysate. The concentration of autophagosome protein in the supernatant was determined using the Micro BCA protein assay kit, and 50 mg / mL was used for the following experiments.

[0055] Western blot was used to detect the expression of LC3 in tumor cell lysates and autophagosomes.

[0056] The results of the size analysis of autophagosomes (TRAP) derived from MC-38 tumor cells showed that the diameter of TRAP was mainly distributed between 200-1000 nm, and the average diameter of the extracellular vesicles was about 580 nm ( Figure 3 A and B). Western blot was used to identify the expression of autophagosome characteristic membrane marker protein LC3-II in MC-38 and 4T1 tumor cells and their secreted autophagosomes. Figure 3As shown in C, TRAP isolated from tumor cell culture supernatant mainly expressed membrane-bound LC3-II, while tumor cell lysate expressed lower levels of LC3-II.

[0057] Example 2 Preparation and Characterization of Bionic Nanoparticle Tumor Vaccines (BAPs) BAPs loaded with CpG / ADU dual immune adjuvant and antigen were prepared by solvent replacement method. The specific experimental method is as follows: Prepare DMF solutions of PEG-P(TMC-DTC)-SP (40 mg / mL), CpG (2 mg / mL), and ADU-S100 (2 mg / mL) in phosphate (PB) buffer (2 mM, pH 6.0). Then, under stirring at room temperature, 100 μL of PEG-P(TMC-DTC)-SP solution was added dropwise to 900 μL of PB buffer (2 mM, pH 6.0) containing 200 μg CpG, 90 μg ADU-S100 and 53 μg autophagosome protein (MC-38 cells or 4T1 cells). After the addition was completed, stirring was continued for 10 min. The sample was then dialyzed using a dialysis bag (MWCO 1000 kDa). First, it was dialyzed with PB (2 mM, pH 6.0) for 2 h, then with PB 6.0 / PB 7.4 (1 / 1, v / v) for 1 h, and finally with PB (10 mM, pH 7.4) for 2 h. The dialysate was changed every hour to remove DMF, unencapsulated proteins and adjuvants, and finally biomimetic autophagosome nanovaccines (BAPs) loaded with Ag, CpG and ADU were prepared. The drug loading efficiencies of Ag, CpG, and ADU were 85%, 90%, and 50%, respectively, and the loading mass ratio was 1:4:1 (Table 1). In the following experiments, unless otherwise specified, autophagosomes were derived from MC-38 cells.

[0058] Referring to the above method, the active ingredient is selected as autophagosome protein, or CpG / autophagosome protein, or ADU-S100 / autophagosome protein, and finally prepared into PS nanovesicles loaded with Ag (PS-Ag) or adjuvant (PS-Ag / CpG and PS-Ag / ADU).

[0059] Referring to the above method, CpG / tumor cell lysate was selected as the active ingredient, and finally PS nanovesicles loaded with adjuvant (PS-Ag / CpG cell lysate) were prepared.

[0060] In the PEG-P(TMC-DTC)-SP (PS) nanovesicles, the autophagosome Ag and two adjuvants were self-assembled into BAPs nanovaccines with an average diameter of 61 nm by electrostatic adsorption, and the vesicle structure was consistent with that in the TEM image of BAPs. The corresponding PS nanovesicles were also prepared ( Figure 4 The stability of BAPs was further studied and it was found that they were stable in a solution containing 10% FBS, with no significant change in particle size within 24 hours, but they rapidly dissociated in a 10 mM GSH solution ( Figure 4 This also indicates that BAPs have good stability but can release drugs rapidly in the reducing cytosol.

[0061] Table 1 Characterization of PS nanovesicles and BAPs nanovaccines

[0062] a Measured by dynamic light scattering (DLS) and electrophoresis (Zetasizer Nano-ZS) in PB (10 mM, pH 7.4) at 25 °C. b Measured by Nanodrop. c Measured by Micro BCA.

[0063] Embodiment 3 To further investigate the effects of Ag derived from MC-38 cell lysates and autophagosomes as well as the Ag content on anti-tumor effects, PS nanovesicles containing different amounts of Ag were subcutaneously injected into the tail base of colon cancer-bearing mice on days 3, 6, 9, 16, and 23. Figure 5 The experimental results showed that CpG-loaded PS nanovesicles inhibited tumor growth to a certain extent, while the addition of Ag significantly enhanced the inhibitory effect ( Figure 5 Compared with the Ag nanovaccine containing cell lysate, the Ag nanovaccine from TRAP showed better tumor inhibition, and its Ag content was only 22.5% of that of Ag from cell lysate, indicating that Ag from TRAP has higher immunogenicity ( Figure 5 C). It can be seen that the advantages of autophagosome vaccines are mainly two points: 1. Autophagosome-derived antigens have stronger immunogenicity, and autophagosome-derived antigens with lower doses have better therapeutic effects; 2. Autophagosomes can be directly extracted from the patient's peritoneal or pleural effusion, avoiding the risks of surgical resection to obtain tumor cells.

[0064] In the prior art, autophagosome vaccines are administered directly using autophagosomes. Due to the large size of the autophagosomes, lymph node targeting and antigen-presenting cell uptake are limited. The present invention uses nanocarriers to deliver autophagosome antigens, significantly improving the efficiency of antigen presentation.

[0065] Example 4 Bionic Nano Tumor Vaccine Stimulates Activation of Bone Marrow-Derived Dendritic Cells (BMDCs) BMDCs were obtained from the bone marrow of C57BL / 6 mice using conventional techniques. Specifically, the femurs and tibiae of mice were dissected in a sterile environment and the bone marrow (BM) cells were obtained by flushing with a syringe. To generate BMDCs, BM cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin, and 20 ng / mL recombinant mouse GM-CSF. Half of the medium was replaced after 72 h. Non-adherent BMDCs were collected on day 6.

[0066] The collected immature BMDCs were placed in a 24-well plate (1.0×10 6 After being cultured in a 37°C incubator overnight, BMDCs were incubated with PBS, PS-Ag, PS-Ag / CpG, PS-Ag / ADU, physical mixture (CpG, ADU, Ag mixed in PBS, mass ratio: 4:1:1), and BAPs nanovaccine, respectively, where the intra-well concentration of CpG was 1 μg / mL, the intra-well concentration of ADU-S100 was 0.25 μg / mL, and the intra-well concentration of Ag was 0.25 μg / mL.

[0067] After 24 h of incubation, the cells were centrifuged, washed with flow buffer (PBS containing 1% FBS), incubated with anti-CD16 / 32 blocking antibody for 10 min on ice (or at 4 °C), and then stained with CD11C-FITC, CD80-APC, CD86-PE / Cy7 and IA / IE-PerCP / Cy5.5 on ice (or at 4 °C) for 30 min. The cells were washed twice with flow buffer and finally detected by flow cytometry. During the experiment, the supernatant of the culture medium after incubation of BMDCs and nanovaccines was collected for ELISA detection of IL-12p70, IL-6, IL-1β, IFN-β and TNF-α.

[0068] PS nanovesicles loaded with Ag and CpG increased the expression of the maturation marker CD86 on BMDCs to some extent. In addition, the introduction of the STING agonist ADU further promoted the activation of BMDCs compared with the other groups ( Figure 6 Middle A). The BAPs nanovaccine can stimulate the activation of BMDCs to the greatest extent, and the activation percentage is higher than that of the physical mixing group. In addition, the BAPs nanovaccine also significantly upregulated the expression of MHC-Ⅱ, and the proportion was significantly increased compared with the PS-Ag / ADU and physical mixing groups ( Figure 6 In summary, BAPs nanovaccine can effectively enhance the activation and antigen presentation ability of BMDCs, thereby promoting the activation of downstream T cells.

[0069] PS nanovesicles, physical mixing groups, and BAPs nanovaccines were incubated with BMDCs for 24 h, and then the culture medium was collected to quantify the expression levels of proinflammatory cytokines secreted by BMDCs. The results showed that PS nanovesicles loaded with Ag and ADU induced the secretion of low levels of interleukin-6 (IL-6), IL-12p70, and IL-1β compared with PS nanovesicles loaded with Ag and CpG ( Figure 7 The secretion of these three cytokines helps to induce a strong and sustained immune response. CpG induces a weak IFN response, and STING ligands can induce type 2 immune responses, such as Figure 7 As shown in D, the synergistic effect of STING agonist ADU and TLR-9 agonist CpG can induce a strong immune response. Therefore, BAPs nanovaccine can promote BMDCs to express high levels of IL-6, IL-12p70, IL-1β, interferon-β (IFN-β), tumor necrosis factor-α (TNF-α) ( Figure 7 These results indicate that co-delivery of antigens and adjuvants stimulates BMDCs more effectively than antigen delivery alone, and biomimetic nano-tumor vaccines co-loaded with tumor antigens, CpG, and ADU can significantly enhance the maturation and antigen presentation ability of BMDCs.

[0070] Example 5 Lymph node targeting and in vivo DC activation of biomimetic nano-tumor vaccines First, the autophagosome Ag and TRAP were labeled with Cy5, and 50 mg / mL of Ag and TRAP were incubated with 1 μg / mL of Cy5 solution at room temperature for 30 min, dialyzed in PBS buffer for 3 days, and freeze-dried to obtain Cy5-labeled Ag and TRAP. Vesicles were prepared according to Example 2.

[0071] Next, C57BL / 6 mice were randomly divided into 4 groups, with 4 mice in each group, and PBS, TRAP-Cy5, free Ag-Cy5, and BAPs nanovaccine loaded with Ag-Cy5 were injected subcutaneously at the tail base, respectively, at a dose of 20 μg / mouse. All mice were killed 24 h after injection, and inguinal lymph nodes and other major organs were obtained for ex vivo imaging ( Figure 8 From the average fluorescence intensity of LNs, BAPs stimulated the highest fluorescence signal in LNs compared with TRAP and Ag ( Figure 8 Middle C). The results of in vitro organ imaging of mice showed that TRAP and Ag groups were more enriched in the liver, while BAPs were less enriched in the liver, with lymph node targeting ability ( Figure 8 Therefore, it is shown that the biomimetic design of the present invention promotes the delivery of nanovaccines to LNs in vivo.

[0072] To investigate the ability of BAPs nanovaccine to activate DCs in the inguinal lymph nodes of mice, mice were inoculated with BAPs nanovaccine or control preparations, and given equal doses of Ag and adjuvants. The draining lymph nodes (dLNs) and spleen were analyzed within 24 h after injection. The results showed that compared with the PS-Ag group and the physical mixing group (same as above), mature DCs (CD80 + CD86 + CD11C + ) and activated DCs (MHCⅡ + CD11C + ) increased ( Fig. 9 The addition of ADU resulted in the activation of NKG2D-positive natural killer (NK) cells ( Fig. 9 (D). Studies have found that B cells can be activated by peptides presented by DCs, leading to the production of specific antibodies against TAAs, which can directly bind to tumor cells and induce memory responses to enhance immune protection. Fig. 9 As shown in Figure E, the percentage of B cells in mice immunized with BAPs was significantly increased compared with other groups. Therefore, BAPs also help to enhance humoral immunity. In summary, BAPs nanovaccines have been shown to be more effective in targeted transport to secondary lymphoid organs, inducing antigen cross-presentation, and eliciting strong immune responses.

[0073] Example 6: Bionic Nano-Tumor Vaccine for Immunotherapy of Colon Cancer The subcutaneous model of mouse colon cancer was established by conventional methods. Each mouse was subcutaneously inoculated with 1.0×10 6 MC-38 cells, that is, day 0. The method of Example 1 was used to extract the corresponding autophagosomes from the MC-38 tumor cells induced by autophagy, and the lysates were obtained to prepare five preparations, namely PS-Ag, PS-Ag / CpG, PS-Ag / ADU, physical mixture group (Physical mixture, CpG, ADU, Ag were mixed in PBS), and BAPs, wherein CpG was 20 μg per mouse, ADU was 5 μg per mouse, and Ag was 5 μg per mouse.

[0074] The vaccine was administered on days 3, 6, 9, 16, and 23. The tumor growth was monitored every three days during the experiment, and the weight and posture of the mice ( Fig.10 (A). Tumor volume calculation formula: tumor volume = length × width × width × 0.5. Mouse death or tumor volume greater than 2000 mm 3 Determine death and draw a survival curve.

[0075] The results showed that the mortality rate of mice in the PBS group was 100% within 30 days. Meanwhile, the PS-Ag, PS-Ag / CpG, and PS-Ag / ADU treatment groups showed suboptimal immunotherapy responses. The physical mixing group showed a moderate tumor inhibition effect ( Fig.10 In addition, about 42.9% of the treated mice in the physical mixture group were tumor-free. In contrast, BAPs nanovaccine treatment led to accelerated tumor regression, with about 71.4% of the treated mice achieving complete regression that lasted for more than 60 days ( Fig.10 Middle D).

[0076] The study of T lymphocyte infiltration in tumor tissues of mice immunized with BAPs nanovaccine and control group found that CD8 + T lymphocytes play a key role in anti-tumor immune response. + The proportion of T cells was significantly increased compared with PS-Ag and PS-Ag / CpG ( Fig.11 A and B). On the other hand, CD4 + Helper T cells can provide help signals to other immune cells and trigger CD8 + CTLs and CD4 + Activation of T cells, which work together to eliminate tumor cells. Fig.11 As shown in C, CD4 + The proportion of T cells was significantly different from that of the single adjuvant groups PS-Ag / CpG and PS-Ag / ADU, and was significantly higher than that of the Physical mixture treatment group CD4 + The proportion of T cells is significantly increased. In addition, the immunosuppressive tumor microenvironment contributes to tumor growth and treatment resistance, thereby promoting cancer development. Regulatory T (Treg) cells are CD4 + The results showed that BAPs can inhibit CD4 + CD25 + FoxP3 + Treg cell infiltration was significantly lower than that of PS-Ag / CpG ( Fig.11 D), which means CD4 + The key role of T cells in tumor regression.

[0077] Next, a simple immune analysis was performed on the peripheral blood of mice treated with different preparations. Peripheral blood mononuclear cells (PBMCs) were collected from each mouse on the 7th day after the last injection, and the phenotype of memory T cells was evaluated by flow cytometry. The study found that effector memory T cells (Tem) expressed higher levels of receptors responsible for migration to inflamed tissues and had stronger immediate effector functions than central memory T cells (Tcm). Tem can come from CD4 + or CD8 + T cells, experimental results show that after vaccination with BAPs nanovaccine, effector memory CD8 + CD44 + CD62L - The expression level of T cells PS-Ag / CpG and PS-Ag / ADU have been significantly improved ( Fig.12 A), and CD4 + CD44 + CD62L - The proportion of T cells also increased significantly ( Fig.12 B). Activated immune cells will seek to kill tumor cells directly by releasing cytokines. Six hours after the last injection, blood was collected from the mouse eye sockets, and serum was collected after centrifugation. The ELISA method was used to measure the concentration of IFN-γ and TNF-α cytokines in the serum. It was found that the secretion of IFN-γ and TNF-α cytokines in the serum of BAPs-immunized mice was significantly increased ( Fig.12 These results reveal the potential of this biomimetic autophagosome vaccine strategy in stimulating a strong anti-tumor immune response and establishing long-lasting immune memory.

[0078] Example 7 Bionic Nano Tumor Vaccine Prevents Tumor Recurrence BAPs nanovaccine has long-term anti-tumor immune memory and prevents tumor recurrence. The specific experiment is as follows: mice cured by BAPs nanovaccine in the treatment experiment of Example 5 and the same number of healthy mice were vaccinated again with 1.0×10 6 Unexpectedly, when the tumor cells of the mice surviving the BAPs nanovaccination group were re-challenged, all re-challenged mice showed a completely tumor-free state, and the tumor inhibition rate reached 100% ( Fig.13 This result indicates that BAPs nanovaccine induces strong immune memory to prevent tumor recurrence.

[0079] The spleen, peripheral blood and lymph nodes of the mice were analyzed by flow cytometry for memory T cell analysis. For example, in spleen cells, the effector memory CD8 + and CD4 + (CD8 +CD44 + CD62L - and CD4 + CD44 + CD62L - ) T cells were significantly increased compared with PBS ( Fig.14 Middle A). Effector memory CD8 + and CD4 + The proportion of T cells also increased significantly ( Fig.14 B and C).

[0080] In addition, BAPs nanovaccine can also be used as a preventive cancer vaccine. BAPs nanovaccine was administered four times on days -28, -21, -14, and -7 (20 μg CpG per mouse, 5 μg ADU per mouse, 4.5 μg Ag per mouse). Mice were randomly divided into two groups and inoculated subcutaneously on the right side on day 0 with 2.0×10 6 MC-38 cells, and the tumor growth and body weight were monitored every other day ( Fig.15 Middle A). The concentrations of IFN-γ, TNF-α and IL-12p70 were quantified in the mouse serum 24 hours after the last injection. The results showed that the weight of the mice did not change significantly during the experiment ( Fig.15 Middle B) illustrates the safety of BAPs nanovaccine. Mice vaccinated with BAPs nanovaccine can significantly prevent tumor formation, leading to complete tumor rejection, and 87.5% of the mice survived for more than 60 days ( Fig.15 CE in the middle). The phenotype of T cells was further explored. Compared with PBS, the flow cytometry results of PBMCs obtained from mice immunized with BAPs showed that the cytotoxic CD8 + The percentage of T cells increased ( Fig.16 A and B). Effector memory T cells can dynamically patrol the host by circulating and entering many tissues, searching for previously encountered antigens, thereby exerting specific anti-tumor effects. Therefore, the expression of effector memory T cells in PBMCs was then analyzed, and it can be seen that the proportion of effector memory T cells expressed in mice vaccinated with BAPs nanovaccine was significantly different from that in PBS ( Fig.16 In addition, BAPs nanovaccines can also promote the secretion of cytokines. Fig.16 As shown in E, 24 h after the last injection, the expression levels of proinflammatory cytokines, including IFN-γ, TNF-α, and IL-12p70, in the serum of mice immunized with BAPs nanovaccine were significantly increased. The above results show that BAPs nanovaccine not only has a specific long-term protective effect, but also has a strong immunity against tumor occurrence.

[0081] Example 8 Biomimetic Nano-Tumor Vaccine Combined with Immune Checkpoint Blockade Immunotherapy To investigate whether BAPs nanovaccine has a good therapeutic effect on "cold tumors" with poor immunogenicity, the 4T1 breast cancer model was used to evaluate the synergistic anticancer potential of BAPs nanovaccine prepared by 4T1 cell autophagosome Ag and FDA-approved immune checkpoint blockade (ICB), namely anti-cytotoxic T lymphocyte-associated protein (aCTLA-4). On day 0, BALB / c mice were subcutaneously inoculated with 1.0×10 5 4T1 cells. The mice were randomly divided into 4 groups, namely PBS, aCTLA4, BAPs, and BAPs+aCTLA4. Specifically, BAPs nanovaccine (CpG 20 μg, ADU 5 μg, Ag 5 μg / mouse) was subcutaneously administered on days 3, 6, 9, and 23 after vaccination, and aCTLA4 antibody (20 μg / mouse) was injected into the tail vein on days 4, 7, 10, and 13. During the experiment, tumor growth and body weight were monitored every 3 days, and the mouse survival curve was plotted ( Fig.17 Middle A).

[0082] The results showed that vaccination with BAPs nanovaccines led to delayed tumor growth, and two of the six mice achieved complete tumor regression. Although aCTLA-4 alone seemed to be less effective and could not significantly inhibit tumor growth, the combined treatment of BAPs nanovaccines and aCTLA-4 showed an unexpected therapeutic effect in inhibiting tumor growth, resulting in a 100-day survival rate of 50% ( Fig.17 Chinese BD).

[0083] The results of immunoassay showed that IFN-γ + CD8 + In terms of the expression of cytotoxic T cells, aCTLA-4 antibody alone had the least effect on the T cell population. IFN-γ + CD8 + The proportion of T cells in the BAPs+aCTLA-4 combined treatment group was significantly higher than that in the aCTLA-4 group ( Fig.18 Similarly, the combination treatment group showed that compared with the BAPs nanovaccine-immunized mice, the CD3 + CD8 + A higher proportion of effector T cells ( Fig.18 C and D).

[0084] In addition, the spleen photos of mice in vitro showed that the mice in the combined treatment group had smaller spleens and lower weights, which also showed that the combined treatment group could effectively reverse the spleen enlargement in 4T1 tumor-bearing mice, indicating that systemic inflammation was reduced to normal levels, which was significantly lower than that in the aCTLA-4 group, which is crucial to ensure the safe use of BAPs nanovaccines ( Fig.19 A and B). Fig.19 As shown in C, CD69 is a marker of early T cell activation. Lymphocytes were obtained from the spleen for analysis and it was found that CD69 in the combination group + CD8 + The proportion of activated T cells increased. NK cells play a key role in innate immunity. The results showed that BAPs nanovaccine treatment of mice led to an increase in the proportion of NKG2D-positive NK cells, and this trend was further enhanced after injection of aCTLA-4 ( Fig.19 At 24 h after the last vaccination, the serum of each mouse was collected to quantify the concentration of TNF-α. The combined treatment group showed the highest level, which was significantly upregulated compared with the aCTLA-4 group, which also suggested further enhancement of adaptive immunity and innate immunity ( Fig.19 In conclusion, the combined immunotherapy of BAPs nanovaccine and aCTLA-4 triggered an effective anti-tumor immune response and promoted the activation of systemic effector cells, thus improving the overall therapeutic effect.

[0085] The present invention discloses a strategy based on biomimetic autophagosomes (BAPs) nano-tumor vaccines to stimulate a strong tumor-specific immune response for personalized cancer immunotherapy. BAPs nano-tumor vaccines are prepared by integrating neoantigens and adjuvants derived from tumor autophagosomes (TRAPs) into self-assembled nanovesicles. Precise regulation of the nano-vaccine components enables accurate dosage control. In addition, these engineered BAPs can protect their payload from degradation by extracellular proteases, thereby promoting their effective transport to draining lymph nodes (dLNs). Through dendritic cell (DCs) endocytosis, neoantigens and adjuvants are responsively released at specific subcellular functional sites, resulting in significantly enhanced maturation of DCs and enhanced immune responses of cross-reactive T cells. In a colorectal cancer mouse model, potent antigen-specific CD8+ cytotoxic T lymphocyte (CTLs) responses, as well as effective immune-mediated tumor shrinkage and the ability to prevent tumor recurrence, were demonstrated. This BAPs-based nano-vaccine can inhibit the growth of poorly immunogenic tumors when used in combination with immune checkpoint blockade (ICB). BAPs nanovaccines provide a highly effective and clinically transformative strategy to enhance strong immune responses for personalized cancer immunotherapy.

Claims

1. A tumor nanovaccine of biomimetic autophagosomes, comprising an amphiphilic block polymer and an immune adjuvant and an autophagosome protein loaded by the amphiphilic block polymer, characterized in that: The autophagosome protein is associated with tumors.

2. The tumor nanovaccine of bionic autophagosome according to claim 1, characterized in that: The autophagosome protein is an autophagosome lysate or a genetically engineered protein product.

3. The tumor nanovaccine of bionic autophagosome according to claim 1, characterized in that: The immune adjuvant includes one or more of a Toll-like receptor agonist, a NOD-like receptor agonist, and a STING agonist.

4. The tumor nanovaccine of biomimetic autophagosome according to claim 1, characterized in that: The amphiphilic block polymer is a hydrophilic segment-P(A-DTC)-cationic segment.

5. The method for preparing the tumor nanovaccine of biomimetic autophagosome according to claim 1, characterized in that: The method comprises the following steps: using immune adjuvant, autophagosome protein and amphiphilic block polymer as raw materials to prepare a tumor nano-vaccine of bionic autophagosome.

6. The method for preparing the tumor nanovaccine of biomimetic autophagosome according to claim 5, characterized in that: The mass ratio of autophagosome protein to immune adjuvant is 1:(0.1~8).

7. A drug, characterized in that The active ingredient comprises the tumor nanovaccine of the bionic autophagosome according to claim 1.

8. The drug according to claim 7, characterized in that The medicament may also include other active ingredients.

9. Use of the tumor nanovaccine of the bionic autophagosome according to claim 1 and the drug according to claim 7 in the preparation of drugs for preventing and treating tumors.

10. The use according to claim 9, characterized in that: The tumor is a solid tumor or a blood tumor.

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