Tumor cell holoantigen nano vaccine as well as preparation method and application thereof

Abstract

The invention discloses a tumor holoantigen nano vaccine as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The nano vaccine comprises a composite liposome, a nucleic acid TLR agonist adjuvant, a tumor cell membrane antigen and a tumor cell soluble antigen. The membrane antigen is embedded into the lipid bilayer, and the soluble antigen and the adjuvant are wrapped in the inner cavity of the lipid to form the nanoparticles with accurate structures. The preparation method comprises the following steps: splitting a tumor cell separation membrane antigen and a soluble antigen; mixing the membrane antigen and lipid to form a lipid phase, and mixing the soluble antigen and an adjuvant to form a water phase; finally, the two phases are self-assembled into the nano vaccine through a micro-fluidic chip. The two antigens are synergistically combined at the nanoscale, the problems that a single antigen is weak in immunogenicity and incomplete in coverage are solved, the synergic anti-tumor effect far better than that of a single antigen vaccine is shown in an animal model, the preparation process is universal and controllable, and an efficient platform is provided for individualized tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of tumor therapeutic vaccines, and relates to a tumor cell whole antigen nanovaccine, its preparation method and application. Background Technology

[0002] The top 10 most common cancers are: lung cancer, digestive system cancers (stomach cancer, colorectal cancer, liver cancer, esophageal cancer, pancreatic cancer), breast cancer, lymphoma, bladder cancer, and thyroid cancer. Biological immunotherapy is another major cancer treatment method after chemotherapy, radiotherapy, and surgery. It has advantages such as safety, low toxicity, significant efficacy, and high patient acceptance. It is considered the fourth major tumor treatment modality after surgery, radiotherapy, and chemotherapy. It is effective for most solid tumors and tumors that are insensitive to radiotherapy and chemotherapy, as well as metastatic tumors. When used in conjunction with radiotherapy and chemotherapy, it can reduce the side effects of these treatments.

[0003] Cancer vaccines are an important means of tumor immunotherapy. As a form of active immunotherapy, cancer vaccines are a crucial component of the field of tumor immunotherapy. They work by stimulating or restoring the body's own immune system to prevent cancer development or eliminate existing tumors. Cancer vaccines mainly include cell vaccines, DNA vaccines, mRNA vaccines, peptide vaccines, dendritic cell vaccines, and nanovaccines. Unlike traditional preventative vaccines, cancer vaccines primarily exert their anti-tumor activity by activating T cells, especially cytotoxic T cells (CTLs). T cells are the main immune mechanism for recognizing and eliminating tumor cells in the body. Cytotoxic T lymphocytes (CTLs), in particular, can specifically recognize tumor antigen epitopes presented by MHC I on the surface of tumor cells, producing various cytotoxic mediators such as perforin and interferon to kill tumor cells.

[0004] Choosing appropriate tumor antigens to activate T cells is crucial for immune clearance of tumors. Tumor antigens include tumor-specific antigens (TSA) and tumor-associated antigens (TAA). T-cell immunotherapy based on TSA has several advantages over T-cell immunotherapy based on TAA, such as high specificity, strong immunogenicity, no off-target effects, and low toxicity. Back in April 2010, the FDA approved Dendreon's Provenge (sipuleucel-T) cancer treatment vaccine for asymptomatic, metastatic prostate cancer. Provenge's main active ingredient is activated dendritic cells (DCs) targeting prostate cancer phosphatase (PAP). The immune response to PAP helps to recognize and kill tumor cells. Clinical studies showed that Provenge extended survival by 4.1 months, with a 3-year survival rate of 32% (vs. 23%). The cost of treating one patient with Provenge is $93,000, and the treatment involves three injections over one month. Provenge is essentially an autologous DC therapeutic vaccine based on tumor-associated antigens (TAA). The lack of use of tumor-specific antigens is one of the main reasons for the vaccine's poor efficacy.

[0005] The main source of non-viral tumor-specific antigens is neoantigens generated by tumor gene mutations. Gene mutations lead to tumor development and progression, while simultaneously producing new antigens that are recognized and attacked by the immune system to eliminate tumor cells. Neoantigen immunotherapy is highly personalized and applicable to all tumor types. Personalized immunotherapy based on tumor neoantigens currently mainly employs next-generation sequencing (NGS) analysis. However, this approach currently faces significant technical limitations. Methods using whole-external sequencing analysis of somatic mutations to predict neoantigens fail to detect up to 90% of neoantigens and also suffer from false positives. Furthermore, the analysis and identification of tumor-associated antigens also present certain challenges.

[0006] Using tumor cells as a source of tumor-specific neoantigens can effectively overcome the problems of antigen omission and false positives in current tumor neoantigen immunotherapy. Furthermore, tumor cells also contain other tumor-specific antigens, such as viral antigens derived from EBV and HPV, as well as some protective tumor-associated antigens (TAAs), such as embryonic antigens, tissue-specific antigens, and certain gene products overexpressed in tumors. Therefore, activating T cells using whole antigens from tumor cells can maximize anti-tumor effects and is suitable for the treatment of any solid tumor. The only requirement is the availability of tumor tissue or cells, and it has broad application prospects in tumor immunotherapy. The autologous therapeutic tumor vaccine (tumor lysate particle-loaded vaccine, TLBO vaccine) developed by Elios Therapeutics in the United States has achieved a 3-year overall survival rate of over 94% for advanced melanoma, with over 60% of patients remaining relapse-free, demonstrating the powerful protective effect of autologous tumor whole antigen T-cell immunotherapy.

[0007] The main obstacle to activating tumor-specific T cells using whole tumor cell antigens is the problem of weak immunogenicity. Because the content of neoantigens or other tumor-specific antigens in tumor cells is very low, it is usually difficult to obtain ideal immune protection by using tumor lysates or direct immunization with whole tumor cells.

[0008] Nanoparticle vaccines use nanomaterials as carriers to deliver specific antigens and adjuvants to achieve therapeutic or preventative purposes. The particle size of nanoparticles is generally between 1 and 1000 nm (usually 10-200 nm). This size advantage makes them easier to concentrate in lymphatic organs such as lymph nodes and spleen. Their size is similar to that of pathogens, making it easy for nanoparticle vaccines to be taken up by antigen-presenting cells (APCs) and activate specific T cells or B cells.

[0009] Our proprietary virus-like nanoparticle (VLNP) technology integrates tumor whole antigens and powerful immune adjuvants, resulting in autologous tumor whole antigen nanovaccines with extremely strong immunogenicity. This technology also solves the problems of low tumor antigen preparation and low tumor antigen immunogenicity. Summary of the Invention

[0010] To address the problems of low immunogenicity in tumor antigen preparation and enhancement in existing technologies, this invention provides a method for preparing a therapeutic nanovaccine based on complete tumor cell antigens and its application. The specific technical solution is as follows: In a first aspect, the present invention provides a therapeutic nanovaccine based on a complete tumor cell antigen. The nanovaccine comprises nanoparticles based on a complex liposome, a tumor cell antigen, and a nucleic acid-based TLR agonist adjuvant molecule; wherein the nanoparticles are loaded with the adjuvant and the tumor antigen.

[0011] Furthermore, the tumor antigen is a combination of tumor cell membrane antigen and soluble tumor cell antigen, or it may be only a tumor cell membrane antigen or only a soluble tumor cell antigen.

[0012] Furthermore, the tumor cell membrane antigen is embedded in the lipid bilayer of the composite liposome, and the soluble tumor cell antigen and the nucleic acid TLR agonist adjuvant are encapsulated in the internal water cavity of the composite liposome.

[0013] Furthermore, the tumor antigen can be derived from tumor cell lines, tumor tissues, or tumor cells cultured in vitro.

[0014] Furthermore, the tumor antigen may be derived from cultured tumor stem cells.

[0015] Furthermore, the tumor antigen can be sourced from induced pluripotent stem cells (iPSCs). Experimental analysis has shown that iPSCs contain a variety of tumor-associated antigens and are an excellent source of broad-spectrum tumor antigens.

[0016] Furthermore, the composite liposome is composed of phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0017] Furthermore, the phospholipid is distearate phosphatidylcholine (DSPC), the polyethylene glycol modified lipid is DMG-PEG2000, and the molar ratio of DSPC, cholesterol and DMG-PEG2000 is (55-65):(35-45):(1-2).

[0018] Secondly, the present invention provides a method for preparing the above-mentioned nano-vaccine, specifically including the following steps: (1) Prepare a complex liposome solution composed of phospholipids, cholesterol and polyethylene glycol modified lipids, wherein the total concentration of the complex liposomes is 5-15 mM; (2) Lyse tumor cells and separate them to obtain a supernatant containing soluble antigens and a precipitate containing membrane antigens; (3) The precipitate containing the membrane antigen is mixed with the composite liposome solution to integrate the membrane antigen into the liposome, thereby obtaining the lipid phase; (4) Mix the nucleic acid TLR agonist adjuvant with the supernatant containing the soluble antigen to obtain an aqueous phase; (5) The lipid phase and the aqueous phase are mixed by a microfluidic chip at a volumetric flow rate ratio to form a nano-vaccine suspension; (6) Purify the suspension to obtain the nano-vaccine.

[0019] Furthermore, the total concentration of the complex liposomes described in step (1) is 10 mM.

[0020] Furthermore, when the tumor antigen is only a tumor cell membrane antigen, the aqueous phase in step (4) does not contain the soluble antigen; when the tumor antigen is only a tumor cell soluble antigen, the lipid phase in step (3) is composed of a complex liposome solution that does not contain the antigen.

[0021] Furthermore, the adjuvant for the nucleic acid TLR agonist mentioned in step (4) is polyinosinic acid.

[0022] Furthermore, the concentration of the nucleic acid-based TLR agonist adjuvant molecule is 0.5-5 mg / ml; Furthermore, the concentration of the nucleic acid-based TLR agonist adjuvant molecule is 2 mg / ml.

[0023] Furthermore, the volumetric flow rate ratio of the lipid phase to the aqueous phase in step (5) is 1:1 to 1:5.

[0024] Thirdly, the present invention provides the application of the above-mentioned antigen vaccine or the vaccine obtained by the above-mentioned preparation method.

[0025] Furthermore, tumor antigen whole-nano vaccines can be used to treat any tumor at any stage of development, as a standalone immunotherapy or in combination with other treatments such as radiotherapy and chemotherapy, or to prevent metastasis and recurrence after surgery.

[0026] Furthermore, nanovaccines prepared from autologous tumors can be used to treat autologous tumors or allogeneic tumors.

[0027] Furthermore, tumor antigen whole-nano vaccines are used for the prevention of any type of tumor.

[0028] This invention has the following beneficial effects: 1. Comprehensive antigen coverage, potent immune response, and low risk of escape: This invention innovatively combines and co-delivers tumor cell membrane antigens and soluble antigens at the nanoparticle level. The soluble antigens contain a richer array of intratumoral antigens, broadening the immune recognition spectrum. Experimental data demonstrate that this "whole antigen" combination produces a significant synergistic enhancement effect, inducing a significantly stronger anti-tumor immune response and better therapeutic efficacy than single membrane antigen or soluble antigen vaccines, effectively preventing immune escape and tumor recurrence due to antigen omission.

[0029] 2. Ingenious structural design mimics innate immune activation: Through microfluidic self-assembly technology, this invention achieves precise localization of antigens and adjuvants: membrane antigens are embedded in the lipid bilayer, while soluble antigens and nucleic acid-based TLR agonist adjuvants are co-encapsulated within the water cavity of the liposome. This structure highly mimics the morphology of natural pathogens, making them easier to recognize and take up, and can simultaneously activate multiple immune signaling pathways, thereby generating strong and durable specific T-cell immunity.

[0030] 3. Highly efficient, controllable, and universally applicable preparation process: This invention provides a standardized and reproducible preparation method. By lysing the same tumor cell sample, membrane antigens and soluble antigens are simultaneously separated, and nano-vaccines are self-assembled in a one-step process using microfluidic technology. The process conditions are mild, maintaining the native conformation of the antigens, and parameters (such as lipid ratio and flow rate) are controllable, ensuring batch-to-batch homogeneity and stability of the vaccine. This method is applicable to various antigen sources, including tumor cell lines, patient tumor tissues, and even induced pluripotent stem cells (iPSCs), providing a universal platform for the preparation of personalized and broad-spectrum tumor vaccines.

[0031] 4. High safety and significant clinical translational potential: This invention utilizes autologous or homologous tumor antigens, exhibiting strong immunogenicity with low off-target toxicity risk. The lipid materials used (DSPC, cholesterol, etc.) demonstrate good biocompatibility and are widely applied clinically. The nanovaccine's size (~100 nm) facilitates targeting lymphoid organs, improving immunogenicity while reducing systemic toxicity. This vaccine can be used as a standalone active immunotherapy or in combination with other therapies such as surgery, radiotherapy, and chemotherapy for tumor treatment and postoperative prevention of recurrence and metastasis, showing broad clinical application prospects.

[0032] Instruction manual illustrations Figure 1 Flowchart of tumor cell whole antigen nanovaccine preparation; Figure 2 Identification of the properties of tumor antigen-based all-nano vaccines; Figure 3 Immunogenicity testing of antigen nanovaccines; Figure 4 Antitumor effects of antigen nano-vaccines (G2-membrane antigen vaccine; G3-soluble antigen vaccine; G4-whole antigen vaccine); Figure 5 Antitumor efficacy of pancreatic cancer cell nanovaccines (A: PBS; B: whole antigen vaccine; C: soluble antigen vaccine; D: membrane antigen vaccine) Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0034] Tumor cell culture preparation The TC-1 mouse lung epithelial cell line is derived from the lung epithelial cells of C57BL / C(H-2b) mice. It was established by transforming HPV-16 E6 and E7 genes and the ras gene. TC-1 cells are cultured for the preparation of whole antigen nanovaccines and for the establishment of mouse HPV-16 tumor models.

[0035] 1. TC-1 cell resuscitation Preheat the water bath to 37°C. Add FBS and penicillin-streptomycin to RPMI 1640 basal medium to prepare a complete medium containing 10% FBS and 1% penicillin-streptomycin. Remove the frozen TC-1 cells from the liquid nitrogen tank and quickly place them in a 37°C water bath to thaw completely. Transfer them to a clean bench and add them to a 15 mL centrifuge tube containing preheated medium. Add preheated medium to a final volume of 10 mL and centrifuge horizontally at 200 rcf for 5 min. After centrifugation, discard the supernatant, add 5 mL of preheated complete medium to resuspend the cells, and transfer them to a T25 culture flask. Incubate horizontally in a 37°C, 5% CO2 saturated humidity incubator. After 24 h of culture, observe the cell status under a microscope, discard the old medium, add fresh 37°C preheated complete medium, and continue culturing.

[0036] 2. Digestion and passage expansion of TC-1 cells When the cells reach a confluence of approximately 80%–90%, passage them, discard the old culture medium, wash once with 2 mL of pre-warmed PBS, then add 1 mL of 0.25% trypsin and digest in a 37°C incubator for 1–2 min. Observe the cell digestion status under a microscope. When the cells shrink and become round, add 2 mL of complete culture medium to stop the digestion. Gently tap the culture flask to detach the cells, and gently pipette them into a single-cell suspension. Collect the cells in a 15 mL sterile centrifuge tube, add complete culture medium to a final volume of 10 mL, centrifuge at 200 rcf for 5 min, discard the supernatant, resuspend the cell pellet in complete culture medium, passage in a new T25 culture flask at a ratio of 1:2–3, and incubate in an incubator.

[0037] 3. Cell preparation Adherent cells in the exponential growth phase were digested with 0.25% trypsin. After digestion was terminated, the cells were washed and resuspended to prepare a cell stock solution. The cell stock solution was mixed with 0.4% trypan blue at a 1:1 (v / v) ratio and incubated at room temperature for 2 min. The total number of cells and cell viability were then calculated using a cell counter.

[0038] Preparation of tumor tissue cell suspension A mouse pancreatic cancer PDX model was used to prepare a whole antigen nanovaccine derived from tumor tissue, and a mouse pancreatic cancer animal model was established to observe the efficacy of the therapeutic vaccine.

[0039] a. When the subcutaneous tumor volume in tumor-bearing mice is approximately 500–600 mm 3At that time, the target mice were euthanized by inhaling an excessive amount of 95% CO2; after the mice were euthanized, they were soaked in 75% alcohol for 5 minutes, then the subcutaneous tumor tissue was peeled off, washed several times in PBS to remove the surface connective tissue; the tumor tissue block was dissected, the calcified tissue in the center of the tumor was removed, and the remaining tissue was weighed.

[0040] b. Weigh 0.4 g of tissue and cut it into pieces of about 2 mm × 2 mm using ophthalmic scissors. Then transfer the pieces to a 5 mL clean EP tube, add 4 mL of trypsin digestion solution and ceramic balls, seal the tube, and place it in a single-cell suspension preparation instrument. Incubate at 37°C for 25 min to prepare a cell suspension.

[0041] c. Transfer the cell suspension to a 15 mL centrifuge tube and add an equal volume of digestion termination solution. Invert the tube to mix and terminate digestion. Use a Pasteur pipette to draw up the mixture and pass it through a 70 μm cell mesh to filter out incompletely digested tissue fragments. Collect the filtrate, centrifuge horizontally at 300 rcf for 10 min, discard the supernatant, add 10 mL of PBS to the pellet to resuspend, centrifuge again at 300 rcf for 10 min to wash the cells, and resuspend the cell pellet in fresh PBS to obtain the tumor tissue cell suspension.

[0042] d. Mix 0.4% trypan blue 1:1 (v / v) with the cell stock solution, incubate at room temperature for 2 min, then use a cell counter to count the cell density and viable cell rate in the tumor tissue cell suspension. Samples with a viable cell rate ≥90% are used for subsequent experiments. Finally, adjust the viable cell concentration to 10⁻⁶ using PBS. 8 / mL, dispensed and stored at -80℃.

[0043] Example 1: Preparation of tumor cell nanovaccines Tumor antigens derived from lysed tumor cells mainly consist of two parts: soluble antigens and membrane-bound antigens. A mixed lipid solution was used to dissolve the membrane-bound antigens as the lipid phase, and adjuvanted soluble antigens were used as the aqueous phase. Microfluidic technology was employed to mix the lipid and aqueous phases to prepare a complete tumor antigen nanovaccine. Figure 1 The preparation of whole-antigen nanovaccines includes the following steps: 1. Lipid formulation Accurately weigh appropriate amounts of DSPC, Cholesterol, and DMG-mPEG-2000, and add them to anhydrous ethanol to prepare a 10 mg / mL stock solution. Then, add the stock solution at a molar ratio of 59.1:39.4:1.5, and adjust the final total lipid concentration to 10 mM with anhydrous ethanol to obtain a composite liposome solution. Finally, filter the solution using a 0.22 μm PVDF membrane filter, dispense it, store it at -20℃, and equilibrate it at room temperature for 1 h before vortexing and mixing.

[0044] 2. Antigen extraction Take 1*10 7 Tumor tissue cell suspensions were repeatedly frozen at -80°C and thawed at room temperature three times. The suspensions were then transferred to ice and subjected to an ultrasonic cell disruptor at 35 W for 2 seconds with 2-second intervals, for a total of 10 minutes to thoroughly break down cell membranes and release cell contents. Finally, the cells were centrifuged at 16200 rcf at 4°C for 10 minutes to separate the supernatant and precipitate. The supernatant was stored at -80°C for later use. The supernatant is the water-soluble antigen extract required for preparing cell-derived nanovaccines, while the precipitate mainly consists of membrane antigens. The separated cell pellet was centrifuged again at 4°C and 16200 rcf for 10 min to remove residual supernatant. 2 mL of the prepared lipid mixture was added to the pellet, and an ultrasonic cell disruptor was used at 35 W for 2 s with a 2 s interval, continuously operating at room temperature for 10 min to ensure complete dissolution of the cell pellet and lipid mixture. Finally, the pellet was centrifuged at 25°C and 16200 rcf for 10 min, and the supernatant (membrane protein-lipid complex) was collected and stored at -80°C. Two volumes of pre-chilled PBS (4°C) were added to the extracted water-soluble antigen extract. Poly(I:C) powder was dissolved using the diluted mixture, or 100 mg / mL Poly(I:C) was added to achieve a final Poly(I:C) concentration of 2 mg / mL. The mixture was stored at 4°C.

[0045] 3. Preparation of tumor nanovaccines The whole-antigen nanovaccine consists of two parts: a soluble cell extract and a precipitated membrane extract, both obtained by lysing the same tumor cell suspension. Membrane antigen nanovaccines or soluble antigen nanovaccines can be prepared alone, or whole-antigen nanovaccines comprising both membrane antigens and soluble antigens can be prepared.

[0046] Connect the microfluidic chip and the nanomedicine preparation system, set the lipid phase:water phase ratio to 1:3, the flow rate to 12 mL / min, and the chip temperature to 30℃. Use a syringe with the system's specifications to take 1 part of lipid and 3 parts of water phase respectively, and then prepare the nanomedicine suspension on the machine.

[0047] The liposome suspension obtained after the extraction contains 25% anhydrous ethanol. This must be immediately diluted with four times the volume of pre-cooled PBS (4°C). The diluted solution is then added to an ultrafiltration tube and centrifuged horizontally at 3000 rcf for 15 min at 4°C. When the ultrafiltrate volume drops to one-quarter of its original volume, replenish with pre-cooled PBS (4°C) to the original volume. Repeat this centrifugation process four times until the anhydrous ethanol content in the system is reduced to below 1%, thus preparing the nano-vaccine. The vaccine can be used immediately after preparation or stored at -80°C.

[0048] Test Example 1: Identification of Nanoparticle Vaccine Characteristics To detect the uniformity and stability of the nanovaccine particles, the particle size and potential of the nanovaccine were measured using a Malvern potentiometer. 20 μL of the nanovaccine was diluted with 980 μL of PBS and then detected using a Malvern Zetasizer particle size analyzer.

[0049] The particle size range of the whole antigen nanovaccine is 68.69–267.2 nm, with particles ranging from 50.79 to 146.1 nm accounting for 71.32% and particles ranging from 79.88 to 125.6 nm accounting for 51.43%. The mean particle size is 128.5 nm, the multivariate distribution coefficient (PI) is 0.0636, and the zeta potential is -3.609 mV. Figure 2 ).

[0050] Test Example 2: Immunogenicity Detection of Tumor Cell Nanoparticle Vaccines To test the immunogenicity of the whole-antigen nanovaccine, particularly its ability to induce T-cell immune responses, mice meeting the immunization criteria were immunized with the vaccine by intramuscular injection of 100 μL on days 0, 7, and 21. On day 7 after the last immunization, the immunized mice were sacrificed, and their spleens were dissected in a clean bench. The dissected spleens were gently ground in a 70 μm cell mesh, with approximately 5 mL of mouse lymphocyte separation medium added to moisten the spleen tissue and the mesh surface. The spleen cell suspension that passed through the mesh was collected; the mesh was for single use, and the cell suspension was stored independently. The separation medium containing the spleen cells was immediately transferred to a 15 mL centrifuge tube. The centrifuge tube was tilted, and 1 mL of RPMI 1640 basal medium was slowly added to cover the cell suspension. After capping, the tube was allowed to stand until the cells separated. The centrifuge was then set to a slow, soft-acceleration and soft-brake setting and centrifuged at room temperature at 800 rcf for 30 min. After centrifugation, the lymphocyte layer between the RPMI 1640 overcoat and the lymphocyte separation medium was aspirated, and then resuspended in 10 mL of RPMI 1640 basal medium. The centrifuge was set to medium-soft acceleration and soft brake, and centrifuged at 250 rcf for 10 min at room temperature. The supernatant was discarded, and the cells were collected. The collected cell pellet was resuspended and diluted using broad-spectrum ELISPOT serum-free medium to adjust the viable cell concentration to 10-1. 7 / mL and 10 6 / mL.

[0051] ELISPOT detection of IFN-γ expression: The procedure was performed according to the instruction manual of Daktronics' Mouse IFN-γ Precoated ELISPOT Kit. Specifically: a. Grouping and Cell Seeding: Each lymphocyte suspension was set up with positive control wells, negative control wells, and experimental wells. Before seeding, 200 μL of RPMI 1640 basal medium was added to each well for 10 min to activate the pre-coated plate. After activation, the medium was discarded, and 100 μL of lymphocyte suspension adjusted to cell density was added to each well, with 10 μL of RPMI 1640 basal medium added to the positive control wells. 5 10 lymphocytes were added to the negative control well, which was the same as the experimental well. 6 One lymphocyte.

[0052] b. Adding stimulants: Add 10 μL of freshly prepared positive stimulant, i.e., a mixture of PMA and Lonomycin, to the positive control well; add 10 μL of PBS to the negative control well; and add 10 μL of the corresponding group of whole antigen nanovaccine to the experimental well.

[0053] c. Cover with a lid and incubate in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 20 h.

[0054] d. Cell lysis: After culture, pour out the liquid in the wells, add 200 μL of pre-cooled deionized water at 4°C to each well, and incubate at 4°C for 10 minutes to lyse the cells.

[0055] e. Washing the plate: Pour out the liquid in the wells, add 260 μL of PBS, let stand for 1 min and then discard. Repeat six times, patting dry on absorbent paper each time.

[0056] f. Antibody incubation test: Add 100 μL of freshly prepared biotin-labeled antibody working solution to each well and incubate at 37°C for 1 h.

[0057] g. Wash the plate: Repeat step e.

[0058] h. Enzyme-linked avidin incubation: Add 100 μL of freshly prepared enzyme-linked avidin working solution to each well and incubate at 37°C for 1 h.

[0059] i. Washing the plate: Repeat step e, and wash the bottom of the membrane and the base with deionized water during the last washing. Use absorbent paper to dry any remaining water and finally wipe the liquid in the holes completely dry.

[0060] j. Color development: Add 100 μL of freshly prepared color development solution to each well, let stand at 37°C in the dark for 30 minutes, check the color development every 5 minutes, and select the time to stop color development according to the spot formation.

[0061] k. Stop color development: Pour out the liquid in the hole, wash the front and back of each solid hole and the base with deionized water 3-5 times to stop color development, and air dry in the ultra-clean table.

[0062] l. ELISPOT plate spot counting: Spot parameters were recorded and statistically analyzed using AID EliSpot Reader.

[0063] Experimental results show that the prepared cell and PDX whole antigen nanovaccines have a strong ability to induce T cell immune responses. Figure 3 ).

[0064] Test Example 3 Construction of subcutaneous tumor-bearing animal model: 1. Preparation of laboratory animals The animal room was set at a temperature of 22℃~26℃ and a relative humidity of 40%~70%, with alternating 12-hour light and 12-hour dark cycles, and free access to food and water. Female C57BL / 6 mice aged 5-6 weeks were selected, and after passing quarantine, they were placed in the animal room. After 3 days of acclimatization, they were weighed, the hair under the right upper limb armpit of the mice was clipped, and the corresponding ear tag was inserted into the right ear of the mice using an ear tagging device.

[0065] 2. Construction of subcutaneous tumor-bearing model Freshly prepared tumor cell suspensions were stored at 4°C and used within 1 hour. Each time, the suspension was gently mixed 5 times by pipetting with a 1 mL pipette. Before tumor implantation, the skin under the right upper limb of the mouse was disinfected with 75% alcohol. Then, 100 μL of tumor cell suspension was drawn into a 1 mL syringe, and air bubbles were gently tapped off the tube wall to prepare for tumor implantation. During tumor implantation, the mouse was held still with one hand, exposing the skin under the right armpit. The syringe was then inserted at an angle into the skin with the other hand, and then gently advanced about 2 / 3 of the way through the skin, injecting the tumor cell suspension at a constant rate. After injection, a small bulge was observed under the skin. After pausing for a few seconds, the needle was slowly withdrawn, and the mouse was held still for a few more seconds to prevent leakage of the cell suspension.

[0066] The subcutaneous PDX tumor-bearing model of pancreatic cancer was prepared by inoculating PDX tumor tissue with an inoculation needle. Before tumor implantation, the tumor-bearing mice were anesthetized intraperitoneally with sodium pentobarbital, and the skin under the right upper limb axilla of the mice was disinfected with povidone-iodine. During tumor implantation, the mouse was restrained with one hand to expose the skin under the right axilla, and the other hand held the sheath needle and inserted it into the skin at an angle. Then, the needle was gently inserted parallel to the skin to the predetermined site. The sheath needle was moved from side to side to dissect the subcutaneous tissue, and then the tumor tissue in the sheath needle was pushed out. During the operation, the mouse's head was kept downward. After the operation, a small bulge was seen to form under the skin. The mouse's head was kept downward for a few seconds to prevent the tissue block from shifting.

[0067] Tumor growth was monitored starting on the 7th day after subcutaneous tumor implantation, with measurements taken 2-3 times per week. The long and short sides of the tumor were measured using calipers, and the tumor volume was calculated. The tumor was considered complete when the average volume reached 100 mm². 3 The tumors were divided into left and right groups for subsequent experiments; when the tumor volume was ≥2000 mm... 3 The time is considered the end point.

[0068] Tumor cell nanovaccine anti-tumor efficacy test: The volume of subcutaneous tumors and body weight of tumor-bearing mice were measured. Twenty-four tumor-bearing mice meeting the immunization criteria were randomly divided into four groups: a control group and an immunization group. The immunization group was further divided into three subgroups: a nanovaccine containing only TC-1 soluble protein, a nanovaccine containing only TC-1 membrane protein, and a whole-antigen nanovaccine containing both soluble and membrane proteins. Each immunization involved an intramuscular injection of 100 μL of vaccine into each hind limb. The day of the first immunization was recorded as D0. Booster immunizations were then administered on D7 and D14. Subcutaneous tumor volume and body weight of the tumor-bearing mice were subsequently observed and recorded.

[0069] Experimental results show that TC-1 membrane protein nanovaccines have significantly better anti-tumor effects than soluble protein nanovaccines. The whole-antigen nanovaccines containing both soluble and membrane proteins exhibit the best anti-tumor effects, with an inhibition rate of over 90% on tumor growth. Figure 4 ).

[0070] The antitumor efficacy of the pancreatic cancer PDX nanovaccine was tested using the same immunization protocol. The nanovaccine prepared from tumor tissue showed a highly similar antitumor efficacy to the TC-1 tumor cell vaccine. The whole antigen vaccine was superior to the single membrane antigen or soluble antigen vaccine. Figure 5 Experiments have shown that whole-antigen nanovaccines prepared using tumor cell lines or tumor tissues all have significant anti-tumor effects.

[0071] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0072] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A tumor cell whole antigen nanovaccine, characterized in that, The invention includes nanoparticles based on composite liposomes, nucleic acid-based TLR agonist adjuvants, and tumor antigens; wherein the nanoparticles are loaded with the adjuvants and the tumor antigens.

2. The nanovaccine according to claim 1, characterized in that, The tumor antigens include tumor cell membrane antigens and / or soluble tumor cell antigens.

3. The nanovaccine according to claim 2, characterized in that, The tumor cell membrane antigen is embedded in the lipid bilayer of the composite liposome, and the soluble tumor cell antigen and the nucleic acid TLR agonist adjuvant are encapsulated in the internal water cavity of the composite liposome.

4. The nano-vaccine according to claims 1-3, characterized in that, The composite liposomes are composed of phospholipids, cholesterol, and polyethylene glycol-modified lipids.

5. The tumor antigen nanovaccine according to claim 4, characterized in that, The phospholipid is distearate phosphatidylcholine (DSPC), the polyethylene glycol modified lipid is DMG-PEG2000, and the molar ratio of DSPC, cholesterol and DMG-PEG2000 is (55-65):(35-45):(1-2).

6. The tumor antigen nanovaccine according to claim 1, characterized in that, The adjuvant for the nucleic acid TLR agonist is polyinosinic acid.

7. A method for preparing a tumor antigen nanovaccine as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Prepare a complex liposome solution composed of phospholipids, cholesterol and polyethylene glycol modified lipids, wherein the total concentration of the complex liposomes is 5-15 mM; (2) Lyse tumor cells and separate them to obtain a supernatant containing soluble antigens and a precipitate containing membrane antigens; (3) The precipitate containing the membrane antigen is mixed with the composite liposome solution to integrate the membrane antigen into the liposome, thereby obtaining the lipid phase; (4) Mix the nucleic acid TLR agonist adjuvant with the supernatant containing the soluble antigen to obtain an aqueous phase; (5) The lipid phase and the aqueous phase are mixed by a microfluidic chip at a volumetric flow rate ratio (1:1 to 1:5) to form a nano-vaccine suspension; (6) Purify the suspension to obtain the nano-vaccine.

8. The method according to claim 7, characterized in that, When the tumor antigen is only a tumor cell membrane antigen, the aqueous phase in step (4) does not contain the soluble antigen; when the tumor antigen is only a tumor cell soluble antigen, the lipid phase in step (3) is composed of a complex liposome solution that does not contain the antigen.

9. The method according to claim 7, characterized in that, The concentration of adjuvants for nucleic acid TLR agonists in aqueous phase is 0.5-5 mg / ml.

10. The use of the tumor antigen nanovaccine according to any one of claims 1-7 in the preparation of a medicament for treating tumors and / or preventing their metastasis or recurrence.

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