Method for preparing tumor vaccine by using magnetic hyperthermia inactivation technology

Abstract

The present disclosure relates to the field of pharmaceuticals, and particularly, to a method for preparing a tumor vaccine by using magnetic hyperthermia inactivation technology. Firstly, a magnetic hyperthermia therapeutic agent is used to generate magnetic heat in tumor cells to induce immunogenic death of the tumor cells. Secondly, the exogenous substance, the magnetic hyperthermia therapeutic agent, is removed by means of magnetic separation technology. Thirdly, two different strategies may be adopted according to needs: one is to add an immunologic adjuvant to prepare a whole-cell tumor vaccine containing various tumor antigens; the other one is to select a specific tumor neoantigen and add the immunologic adjuvant to prepare a tumor neoantigen vaccine with higher specificity. In a mouse tumor vaccine test, the results show that the whole-cell tumor vaccine prepared by the described method can effectively inhibit the growth of homologous tumors, and the tumor-free rate of mice in the vaccination group reached 100%. Thus, the present disclosure provides a new treatment strategy for clinical anti-tumor immunotherapy and possesses good application prospects.

Description

A method for preparing tumor vaccines using magnetic thermal inactivation technology Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a method for preparing a tumor vaccine by utilizing magnetic thermal inactivation technology. Background Art

[0002] Tumor immunotherapy has been used to treat various types of cancer. However, due to tumor heterogeneity and individual patient differences, existing immunotherapies have suboptimal clinical response rates, limiting their therapeutic effectiveness. Therefore, there is an urgent need to develop more effective tumor immunotherapy approaches.

[0003] With a deeper understanding of tumor biology and immunology, therapeutic tumor vaccines have shown great potential in cancer treatment and have become a key research direction in the field of tumor immunotherapy. The goal of tumor vaccines is to activate an active immune response against tumors, leveraging the patient's own immune system to continuously fight the tumor, thereby inhibiting tumor progression and metastasis and preventing recurrence. By carrying specific tumor antigens, tumor vaccines can specifically activate the immune system to attack tumor cells while causing minimal damage to normal cells. Currently, tumor vaccines are primarily used for cancers that are incurable with radical therapy or other immunotherapies, or to prevent tumor recurrence after treatment. Whole-cell tumor vaccines contain multiple tumor antigens and have a wide range of applications in inhibiting tumor initiation, progression, and recurrence. On the other hand, tumor neoantigen vaccines target antigens expressed only on tumor cells, eliciting a specific T cell response against the tumor while minimizing nonspecific damage to normal tissues. These neoantigens, derived from mutated neoepitopes on tumor cells, can bypass central tolerance of T cells to self-epitopes, inducing a more robust immune response. Although some tumor vaccines have demonstrated good immunogenicity and tumor cell-killing abilities in preliminary clinical trials, a major limitation of existing vaccines is the lack of highly immunogenic tumor antigens and the potential for secondary damage to tumor antigens during vaccine preparation, which affects the efficiency of vaccines in activating the immune system. Therefore, the development of new methods for preparing and inactivating tumor vaccines has become crucial. Summary of the Invention

[0004] In order to overcome the shortcoming of low immunogenicity of tumor antigens in existing tumor vaccines, the purpose of the present invention is to provide a method for preparing tumor vaccines using magnetic thermal inactivation technology, which provides a new treatment strategy for clinical tumor immunotherapy. First, a magnetic hyperthermia agent is used to generate magnetic heat in tumor cells, triggering the immunogenic death of tumor cells. This step not only enhances the immunogenicity of tumor cells, but also leads to the release of a large number of tumor antigens. Subsequently, magnetic separation technology is used to remove exogenous substances such as magnetic hyperthermia agents to ensure the purity and safety of the vaccine. Depending on specific treatment needs, the present invention can be used to prepare two types of vaccines: whole-cell tumor vaccines and tumor neoantigen vaccines. Whole-cell vaccines contain multiple tumor antigens and can stimulate a broad immune response, while tumor neoantigen vaccines target specific, personalized neoantigens and provide a more precise immune response. Through this method, the present invention not only improves the efficacy of tumor vaccines, but also provides new possibilities for personalized tumor treatment.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] First, magnetic hyperthermia agents are used to generate magnetic heat within tumor cells, triggering immunogenic death of tumor cells and inducing new antigen mutations in tumor cells. Then, exogenous substances in the magnetic hyperthermia agents are removed through magnetic separation technology. Then, two different strategies can be adopted according to needs: one is to add immune adjuvants to prepare whole-cell tumor vaccines containing multiple tumor antigens; the other is to screen specific tumor neoantigens and combine them with immune adjuvants to make more targeted tumor neoantigen vaccines.

[0007] In one embodiment, tumor cells or ex vivo tumor tissue from a patient are subjected to magnetic thermal inactivation treatment, comprising the following steps:

[0008] Step 1: Tumor cells internalize the magnetic hyperthermia agent, or the ex vivo tumor tissue is dispersed into a single cell suspension, and the tumor cells are sorted and expanded to internalize the magnetic hyperthermia agent;

[0009] Step 2: Apply an alternating magnetic field to the tumor cells that have internalized the magnetic hyperthermia agent, so that the magnetic hyperthermia agent generates magnetic heat in the cells, inducing immunogenic death of the tumor cells.

[0010] Step 3: After the magnetic heat treatment is completed, the magnetic heat treatment agent is removed using magnetic separation technology.

[0011] In one embodiment, after step 3, the tumor cells and their lysates after magnetic heat treatment are collected, separated and purified to obtain highly immunogenic substances, and immune adjuvants are added to prepare whole-cell tumor vaccines containing multiple tumor antigens.

[0012] In one embodiment, after step 3, specific tumor neoantigens are screened, and the highly immunogenic tumor neoantigens produced after magnetic thermal treatment are collected, separated and purified, and immune adjuvants are added to prepare a targeted tumor neoantigen vaccine.

[0013] In one embodiment, a method for screening tumor neoantigens for preparing a tumor neoantigen vaccine comprises the following steps:

[0014] Step 1: Extract DNA and RNA from tumor cells after magnetic thermal treatment, perform exon sequencing and transcriptome sequencing, and preliminarily screen for neotumor antigens.

[0015] Step 2: Conduct an immunogenicity test on the tumor neoantigens that have been preliminarily screened to screen out tumor neoantigens with high immunogenicity.

[0016] In one embodiment, the magnetic thermal inactivation technology uses a magnetic hyperthermia agent concentration of 50-1000 μg / mL, an alternating magnetic field strength of 10-1000 Oe, an alternating magnetic field frequency of 50 kHz-1 MHz, an action time of 10-60 minutes, and a temperature of 39-49°C. Under these magnetic field parameters, the magnetic hyperthermia agent generates a large amount of reactive oxygen species within the cells. This action causes endoplasmic reticulum stress in tumor cells, which in turn affects downstream transcription factors, initiates apoptosis signals, releases damage-associated molecular patterns, and generates new antigenic epitopes.

[0017] In one embodiment, the immune adjuvant includes but is not limited to one or more cytokine adjuvants such as aluminum hydroxide, alum, aluminum phosphate, BCG, cytidylic acid, Freund's adjuvant, polyinosinic-polycytidylic acid, CpG oligodeoxynucleotides, lipopolysaccharide, granulocyte-macrophage colony-stimulating factor and interleukin, and the ratio of the tumor antigen to the immune adjuvant is 1:1-1:20.

[0018] In one embodiment, the magnetic thermal inactivation technology is used to prepare a tumor neoantigen vaccine, and the concentration of the tumor neoantigen is 20-2000 μg / μL.

[0019] In one embodiment, the tumor cell is at least one of a liver cancer cell, a lung cancer cell, a breast cancer cell, a colon cancer cell, a pancreatic cancer cell, a prostate cancer cell, a gastric cancer cell, a kidney cancer cell, and a melanoma cell.

[0020] In one embodiment, the magnetic hyperthermia agent is Fe, FeCo, Fe2C, FePt, MFe2O4 or metal-doped iron-based magnetic nanoparticles, wherein M is Fe, Mn, Co, Ni or Zn, with a particle size of 3 to 500 nanometers and a spherical, cubic or ring-shaped morphology.

[0021] In one embodiment, the surface of the magnetic hyperthermia agent has a hydrophilic or amphiphilic polymer to enable it to be dispersed in an aqueous solution, and the polymer is at least one of 3,4-dihydroxyhydrocinnamic acid, polyethylene glycol, and dopamine.

[0022] Magnetic induction heating (magnetic heat) is an advanced tumor treatment technology that utilizes magnetic hyperthermia agents to generate magnetic heat within tumor cells, inducing apoptosis and simultaneously releasing a large number of antigens, triggering immunogenic cell death. This technology not only enhances the immunogenicity of tumor cells but also exposes a large number of tumor antigens. Magnetic heat integrates tumor cell inactivation with immunogenicity induction, effectively avoiding the secondary damage to tumor antigens that can occur with traditional inactivation methods. After magnetic heat treatment, tumor cells lose their proliferative capacity and exhibit high immunogenicity, making this technology ideal for the preparation of whole-cell tumor vaccines. Furthermore, to meet the needs of personalized treatment, tumor neoantigens after magnetic heat treatment can be further screened for the preparation of tumor neoantigen vaccines. Such vaccines can enhance the duration of specific T cell responses and provide immune memory after treatment, thereby helping to prevent long-term tumor recurrence.

[0023] Compared with the existing technology, the magnetic thermal inactivation technology of the present invention has significant beneficial effects:

[0024] 1. Enhance the immunogenicity of tumor cells: The present invention significantly enhances the immunogenicity of tumor cells by inducing immunogenic death of tumor cells, thereby promoting the release of more tumor antigens in the body.

[0025] 2. Produce new tumor antigens: Magnetic thermal inactivation technology can induce tumor cells to produce new antigen mutations. These new antigens can effectively avoid the tolerance response of the immune system, thereby more effectively activating the immune response against tumors.

[0026] 3. Retain the biological activity of the antigen: The magnetic thermal inactivation method retains the biological activity of the antigen to the maximum extent and enhances the overall efficacy of the vaccine.

[0027] 4. Completely remove foreign substances: The application of magnetic separation technology can completely remove the magnetic hyperthermia agent in the vaccine after magnetic heat treatment, avoiding the potential safety risks brought by foreign substances.

[0028] 5. Wide applicability: The present invention is applicable to the preparation of various types of solid tumor vaccines, showing a high degree of versatility.

[0029] The present invention provides a new tumor vaccine strategy, which is of great significance to clinical tumor immunotherapy and indicates its broad application prospects in the field of cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a schematic diagram of a method for preparing tumor vaccines using magnetic thermal inactivation technology.

[0031] Figure 2 shows the immunofluorescence results of calreticulin externalization in Hepa1-6 cells after magnetic heat treatment.

[0032] FIG3 shows the content of HMGB1 in the supernatant of Hepa1-6 cell culture medium after magnetic heat treatment.

[0033] FIG4 shows the ATP content in the supernatant of Hepa1-6 cell culture medium after magnetic heat treatment.

[0034] FIG5 shows the cell activity of Hepa1-6 cells after magnetic heat treatment.

[0035] Figure 6 shows the preventive effect of whole-cell tumor vaccines on homologous tumor cells. Implementation Method

[0036] The embodiments of the present invention are described in detail below with reference to the accompanying drawings and examples.

[0037] The method of preparing tumor vaccines using magnetic thermal inactivation technology of the present invention can be described as follows with reference to FIG1 :

[0038] (1) Extract tissue and sort tumor cells.

[0039] (2) Magnetic thermal inactivation of tumor cells.

[0040] (3) Magnetic separation to remove magnetic hyperthermia agents.

[0041] (4) Collect whole-cell tumor vaccines; alternatively, extract tumor DNA / RNA and use multi-omics analysis to screen tumor neoantigens to obtain highly immunogenic tumor neoantigens.

[0042] (5) Prepare tumor vaccine preparations with adjuvants.

[0043] Subsequently, the tumor vaccine preparation can be administered to the patient through injection or other methods, and the vaccine can activate T cell anti-tumor immune response in the patient's body.

[0044] The following are several specific embodiments of the present invention from preparation to application. Example

[0045] Induction of immunogenic death of tumor cells by magnetic heat

[0046] 1. Cell Culture and Treatment

[0047] Hepa 1-6 cells were obtained from ATCC. Hepa 1-6 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg / ml streptomycin, and 100 μg / ml penicillin. Hepa 1-6 cell cultures were maintained at 37°C and 5% CO2.

[0048] 2. Magnetic heat treatment

[0049] When tumor cells reach 80% growth, add 50-1000 μg / mL of magnetic iron oxide nanoparticles and incubate with the cells for 6-12 hours. The incubated cells are then exposed to an alternating magnetic field (10-1000 Oe, 50 kHz-1 MHz) for 10-60 minutes. The treated cells are then cultured for another 0-24 hours before use.

[0050] 3. Magnetothermal Induction of Calreticulin Externalization

[0051] When tumor cells die due to immunogenicity, calreticulin is exposed on the cell membrane surface and acts as an "eat-me" signal to promote DC or its precursor cells to phagocytose dying tumor cells, provide rich antigen substances, and promote the maturation of DC cells and their functions. The cells after magnetic heat treatment were blocked in 5% BSA for 15 minutes, the blocking solution was discarded and washed three times with PBS, and then the cells were incubated with recombinant Alexa Fluor ® Incubate with 488 fluorescent anti-calreticulin antibody (abcam) at 4°C for half an hour in the dark. Cell nuclei were stained with DAPI. Cells were imaged using a confocal microscope (NIS-Elements, Ti2). Refer to Figure 2. The experimental results demonstrate that magnetic heating effectively induces calreticulin to flip from the inside out of the cell to the cell membrane surface.

[0052] 4. Magnetic heat-induced HMGB1 and ATP release

[0053] HMGB1 is released extracellularly during immunogenic cell death. Extracellular HMGB1 can activate corresponding signaling pathways and stimulate immunity. The supernatant of the cell culture medium after magnetic heat treatment was collected and centrifuged at 300 × g for 3 minutes at 4°C to remove residual cells and cell debris. The amount of extracellular HMGB1 released was measured using the Mouse HMGB1 / HMG-1 ELISA Kit (Novus Biologicals). Referring to Figure 3, the experimental results show that the extracellular HMGB1 content increased 4.32-fold after magnetic heat treatment.

[0054] Apoptosis in tumor cells triggers the release of intracellular ATP, sending a "find-me" signal to the outside world. This extracellular ATP promotes phagocytosis of dying cells by phagocytes, stimulating a specific anti-tumor immune response. Extracellular ATP levels in the culture supernatant were measured using an ATP assay kit (Solarbio). As shown in Figure 4, the results show that magnetic heat treatment also increased extracellular ATP levels by 1.97-fold. Example

[0055] Inhibition of tumor cell activity by magnetic heat

[0056] Hepa 1-6 cells in good logarithmic growth were taken, and a certain concentration of magnetic hyperthermia agent was added to the cell culture medium and incubated with the Hepa 1-6 cells. Then, magnetic hyperthermia treatment was performed in an alternating magnetic field to test the killing effect of magnetic hyperthermia on the cells. Specifically:

[0057] Hepa 1-6 cells with good growth in the logarithmic phase were obtained and digested into single-cell suspension using trypsin containing 0.25% EDTA. After counting the cells using a hemocytometer, the cells were inoculated into 35 mm cell culture dishes, with 5 x 10 cells per dish. 5 Cells were cultured in a 37°C, 5% CO2 incubator for 24 h. After cell attachment, the culture medium was removed and magnetic hyperthermia agents of different concentrations were added. The cells were cultured in the incubator to allow the cells to internalize the magnetic hyperthermia agents. After treatment in an alternating magnetic field, the cells were digested with trypsin containing 0.25% EDTA to form a single cell suspension. The cells were counted using a hemocytometer and the digested cells were seeded in a 96-well plate at 1 x 10 cells per well. 4 After culturing the cells in a 37°C, 5% CO2 incubator for 24 hours, 100 μL of the prepared CCK-8 reagent was not added, and the OD value was measured using a microplate reader using a dual-wavelength method (detection wavelength 450-490 nm, reference wavelength 600-650 nm).

[0058] Calculate cell viability using the following formula: Cell viability = (OD 实验 –OD 对照 ) / (OD 对照 –OD 空白 )×100%.

[0059] As shown in Figure 5, when the concentration of magnetic hyperthermia agent is 100 μg / mL, the cell activity can be reduced to below 10%. As the particle concentration increases, magnetic heat can effectively inactivate tumor cells. Example

[0060] Screening of tumor neoantigens

[0061] ① Extraction of total DNA from tumor cells: Wash tumor cells with TBS, centrifuge at 4000 × g for 5 minutes, and remove the supernatant. Add 10 volumes of lysis buffer. Incubate in a 50-55°C water bath for 1-2 hours. Add an equal volume of saturated phenol to the sample treatment solution, mix thoroughly, and let stand for 3 minutes. Centrifuge at 5000 × g for 10 minutes. Transfer the upper aqueous phase to another 1.5 mL EP tube. Add an equal volume of phenol / chloroform, gently mix, centrifuge at 5000 × g for 10 minutes, and transfer the upper aqueous phase to another EP tube. Add an equal volume of chloroform, gently mix, centrifuge at 5000 × g for 10 minutes, and transfer the upper aqueous phase to another EP tube. Add 1 / 10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of anhydrous ethanol, and gently invert to mix. Once flocs appear, centrifuge at 5000 × g for 5 minutes and discard the supernatant. The precipitate was washed with 75% ethanol and centrifuged at 5000 × g for 3 minutes. The supernatant was discarded. The ethanol was evaporated at room temperature and dissolved in 50 mL of TE. The resulting DNA was tumor DNA.

[0062] ② Tumor Cell RNA Extraction: Total RNA from samples was extracted using the TRIzol method. After treatment, adherent cells were washed with PBS. 1 mL of TRIzol was added to the culture dish and pipetted repeatedly until cells were completely lysed. The cells were transferred to a 2 mL polypropylene tube and mixed by inversion. The tube was allowed to stand at room temperature for 5 minutes. 200 μL of chloroform was added to the tube, mixed by inversion for 30 seconds, and allowed to stand at room temperature for 5 minutes. The tube was centrifuged at 12,000 × g at 4°C for 15 minutes. The upper aqueous phase was transferred to a new polypropylene tube, an equal volume of isopropanol was added, mixed by inversion, and allowed to stand at room temperature for 10 minutes. The tube was centrifuged at 12,000 × g at 4°C for 10 minutes. The supernatant was discarded and the pellet was washed with 1 mL of cold 75% ethanol (prepared with DEPC water) to resuspend the pellet. The pellet was centrifuged at 7,500 × g at 4°C for 5 minutes. The pellet was retained and the washing step was repeated twice. Open the EP tube in a sterile and enzyme-free environment, dry it at room temperature until the ethanol evaporates, add 20 μL of DEPC water, and pipette to dissolve the precipitate to obtain the total cellular RNA.

[0063] ③ Perform transcriptome sequencing on cell RNA and exome sequencing on cell DNA to compare and confirm tumor-specific mutations, then select dominant mutant peptides based on HLA affinity, and finally perform T cell reactivity analysis in vitro. Example

[0064] Tumor vaccine preparation process

[0065] An appropriate amount of tumor cells or digested and dispersed tumor tissue is cultured in vitro. An appropriate amount of magnetic hyperthermia agent is then added to the cells and cultured to ensure sufficient internalization of the agent. An alternating magnetic field is then applied to induce immunogenic death of the tumor cells and magnetically inactivate them. Following magnetic inactivation, magnetic separation is used to completely remove the exogenous magnetic hyperthermia agent. Highly immunogenic tumor cells and their lysates are then isolated and purified. An immunoadjuvant is then added to the cells and their lysates at a volume ratio of 1:1-1:20 to prepare a whole-cell tumor vaccine. Alternatively, DNA and RNA are extracted from the magnetically inactivated tumor cells. Multi-omics analysis is then used to screen for tumor neoantigens produced by magnetic inactivation. These selected tumor antigens are then tested for immunogenicity to identify one or more highly immunogenic neoantigens. The tumor neoantigen vaccine contains 20-2000 μg / μL of the tumor neoantigen, and an immunoadjuvant is added to the tumor neoantigen at a volume ratio of 1:1-1:20 to prepare the tumor neoantigen vaccine. The tumor vaccine was sterilized, tested for pathogens, packaged, and stored at 4°C. Example

[0066] Preventive effect of whole-cell tumor vaccines on homologous tumors

[0067] Mouse tumor vaccine experiments used 6-8 week old C57BL / 6 male mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) to verify the resistance of whole-cell tumor vaccines to homologous tumor cells. Twelve mice were randomly divided into two groups (control group and vaccination group). On the 0th, 7th, and 14th days of the experiment, each mouse was subcutaneously injected with whole-cell tumor vaccine (on the left side of the back). The control group was injected with normal saline. On the 21st day, each mouse was injected with 1×10 6 Hepa1-6 cells (right back), and the mice were subsequently observed for tumor growth, as shown in Figure 6A. The results show that after injection of normal homologous tumor cells, the control group mice maintained tumor growth, with a tumor-free rate of 0%. In contrast, the mice in the vaccinated group showed no tumor growth over 38 consecutive days of observation, with a tumor-free rate of 100% (Figure 6B). These experimental results demonstrate that the whole-cell tumor vaccine treated with magnetic thermal inactivation has excellent tumor prevention efficacy, significantly inhibiting the growth of homologous tumor cells.

[0068] It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a tumor vaccine using magnetic thermal inactivation technology, characterized in that: The tumor vaccine is a whole cell tumor vaccine or a tumor neoantigen vaccine, and the preparation process is as follows: Step 1, tumor cells internalize magnetic hyperthermia agents, or disperse ex vivo tumor tissue into single cell suspension, sort and expand tumor cells, and then internalize magnetic hyperthermia agents; Step 2, applying an alternating magnetic field to cause the magnetic hyperthermia agent to generate magnetic heat in the cells, triggering immunogenic death of tumor cells and inducing new antigen mutations in tumor cells; Step 3, using magnetic separation technology to remove the magnetic hyperthermia agent; Step 4: Perform one of the following steps: Step 4a, collecting, separating and purifying tumor cells and their lysates to obtain highly immunogenic substances, adding immune adjuvants, and preparing whole-cell tumor vaccines containing multiple tumor antigens; Step 4b: screen specific tumor neoantigens, collect, separate and purify them, add immune adjuvants, and prepare targeted tumor neoantigen vaccines.

2. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 1, characterized in that: The tumor cell is at least one of liver cancer cells, lung cancer cells, breast cancer cells, colon cancer cells, pancreatic cancer cells, prostate cancer cells, gastric cancer cells, kidney cancer cells and melanoma cells.

3. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 1, characterized in that: The magnetic hyperthermia agent is Fe, FeCo, Fe2C, FePt, MFe2O4 or metal-doped iron-based magnetic nanoparticles, wherein M is Fe, Mn, Co, Ni or Zn, with a particle size of 3 to 500 nanometers and a spherical, cubic or ring-shaped morphology.

4. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 3, characterized in that: The surface of the magnetic hyperthermia agent has a hydrophilic or amphiphilic polymer so that it can be dispersed in an aqueous solution.

5. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 4, characterized in that: The polymer is at least one of 3,4-dihydroxyhydrocinnamic acid, polyethylene glycol and dopamine.

6. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 1, characterized in that: The immune adjuvant is one or more of cytokine adjuvants such as aluminum hydroxide, alum, aluminum phosphate, BCG, cytidylic acid, Freund's adjuvant, polyinosinic-polycytidylic acid, CpG oligodeoxynucleotide, lipopolysaccharide, granulocyte-macrophage colony stimulating factor and interleukin, and the ratio of the tumor vaccine to the immune adjuvant is 1:1-1:

20.

7. The method for preparing tumor vaccine by using magnetic thermal inactivation technology according to any one of claims 1 to 6, characterized in that: The concentration range of the magnetic hyperthermia agent is 50-1000 μg / mL, the alternating magnetic field strength is 10-1000 Oe, the alternating magnetic field frequency is 50 kHz-1 MHz, the action time is 10-60 minutes, and the temperature is 39-49°C.

8. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 1, characterized in that: The method for screening specific tumor neoantigens is: extracting DNA and RNA of tumor cells after magnetic thermal treatment, performing exon sequencing and transcriptome sequencing, and thus preliminarily screening tumor neoantigens.

9. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 8, characterized in that: The immunogenicity of the tumor neoantigens initially screened out is tested to screen out tumor neoantigens with high immunogenicity.

10. The method for preparing tumor vaccine using magnetic thermal inactivation technology according to claim 1, characterized in that: The concentration of the tumor neoantigen in the tumor neoantigen vaccine is 20-2000 μg / μL.

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