Whole tumor cell vaccine as well as preparation method and application thereof

Abstract

The invention provides a whole tumor cell vaccine as well as a preparation method and application thereof, and relates to the technical field of cancer immunotherapy. The preparation method of the whole tumor cell vaccine provided by the invention comprises the following steps: obtaining CD47 gene knockout tumor cells, inserting an adjuvant into the surfaces of the tumor cells, and carrying out heating inactivation treatment to prepare the whole tumor cell vaccine, the temperature of the heating inactivation treatment is 40-80 DEG C. According to the preparation method, CD47 genes of tumor cells are knocked out, so that endocytosis of macrophages to vaccines is enhanced; an adjuvant is inserted into the surface of the tumor cell after the CD47 gene is knocked out, so that the immune activation effect of the vaccine is enhanced; the cells are inactivated by heating and inactivating treatment at a proper temperature, so that the safety of the vaccine is ensured, and meanwhile, the cells which die due to immunogenicity can also have a stronger immune activation effect. The preparation method is simple and convenient, and the prepared whole tumor cell vaccine can be used for preventing and treating tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer immunotherapy, and in particular to a whole tumor cell vaccine and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy mainly activates the host's immune system, thereby mobilizing collective specific immune cells to identify and kill tumors, and because the body's immune system has memory, it can effectively inhibit tumor recurrence and metastasis. Among them, tumor vaccines have always been considered an effective means of tumor treatment, which mainly enhances the body's specific T cell response through active immunity to achieve the effect of killing tumors. However, to achieve this process, antigen presenting cells (APCs), especially dendritic cells (DCs), need to take up a large amount of tumor-specific antigens. Therefore, discovering more types of tumor antigens and improving the delivery efficiency of APCs will greatly benefit the therapeutic effect of tumor vaccines.

[0003] Whole tumor cell vaccines use tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) from tumor cells removed from the patient (autologous tumor cells) or cultured tumor cells (allogeneic tumor cells) as antigen sources. A major advantage of using tumor cells as a source of antigens is that due to the presence of a series of mutated tumor antigens on tumor cells, they are more immunogenic than universal tumor antigens and can synergistically enhance the body's specific immune response. Individualized treatment can also be achieved for autologous tumor cells. However, the development of whole tumor cell vaccines is limited by problems such as poor immune activation and weak uptake of vaccines by immune cells. Therefore, there is an urgent need to develop a whole tumor cell vaccine that can be actively taken up by immune cells and has strong immunogenicity.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first purpose of the present invention is to provide a method for preparing a whole tumor cell vaccine to solve the problem of weak immune cell uptake and poor immune activation effect of the whole tumor cell vaccine.

[0006] The second object of the present invention is to provide a whole tumor cell vaccine.

[0007] The third object of the present invention is to provide the use of the above whole tumor cell vaccine in tumor prevention.

[0008] In order to achieve the above objectives, the following technical solutions are adopted:

[0009] In a first aspect, the present invention provides a method for preparing a whole tumor cell vaccine, comprising the following steps:

[0010] Obtain CD47 gene knockout tumor cells, then insert adjuvants on the surface of tumor cells, and then heat-inactivate them to prepare whole tumor cell vaccines;

[0011] The temperature of the heating inactivation treatment is 40-80°C.

[0012] As a further technical solution, gene editing technology was used to knock out the CD47 gene in tumor cells;

[0013] The gene editing technologies include transcription activator-like effector nuclease technology, zinc finger nuclease technology, CRISPR / Cas9 technology and siRNA gene silencing technology.

[0014] As a further technical solution, the tumor cells include melanoma cells, pancreatic cancer cells and breast cancer cells.

[0015] As a further technical solution, the adjuvant includes an adjuvant modified with a DSPE-PEG group.

[0016] As a further technical solution, the adjuvant includes DSPE-PEG-Mannose (phospholipid polyethylene glycol mannose) and DSPE-PEG-IMDQ (phospholipid polyethylene glycol imidazoquinoline).

[0017] As a further technical solution, the step of inserting an adjuvant into the surface of tumor cells comprises: mixing and incubating the tumor cells with the adjuvant to prepare tumor cells with the adjuvant inserted into the surface.

[0018] As a further technical solution, the incubation temperature is 4 to 37°C;

[0019] The incubation time is 0.5 to 8 hours.

[0020] As a further technical solution, the heating inactivation treatment time is 2 to 30 minutes.

[0021] In a second aspect, the present invention provides a whole tumor cell vaccine prepared by the above-mentioned preparation method.

[0022] In a third aspect, the present invention provides the use of the above-mentioned whole tumor cell vaccine in tumor prevention.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The preparation method of the whole tumor cell vaccine provided by the present invention is to enhance the endocytosis of the vaccine by macrophages by knocking out the CD47 gene of the tumor cells; to enhance the immune activation effect of the vaccine by inserting adjuvants into the surface of the tumor cells after the CD47 gene is knocked out; and to inactivate the cells by heating at a suitable temperature to ensure the safety of the vaccine, while the immunogenic dead cells can also have a stronger immune activation effect. The preparation method is simple and convenient, and the prepared whole tumor cell vaccine can be used for the prevention and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a diagram showing the gene knockout effect of CD47KO B16F10 cells detected by flow cytometry in Example 2;

[0027] Figure 2 This is a diagram showing the effect of gene knockout in the agarose gel electrophoresis experiment in Example 2;

[0028] Figure 3 This is a diagram showing the results of gene sequencing in Example 2;

[0029] Figure 4 This is a live-dead staining result of cells after heat treatment in Example 3;

[0030] Figure 5 This is a morphological characterization diagram of cells after heat treatment in Example 3;

[0031] Figure 6 This is a flow cytometry result diagram of CRT release in Example 4;

[0032] Figure 7 This is the confocal imaging result of HMGB1 in Example 4;

[0033] Figure 8 This is a CLSM photo of the effect of inserting a fluorescently labeled adjuvant into the cell membrane in Example 5;

[0034] Fig. 9 This is a graph showing the results of the whole tumor cell vaccine preventing melanoma in mice in Example 7;

[0035] Fig.10 This is a graph showing the results of whole tumor cell vaccine treatment of melanoma in mice in Example 8;

[0036] Fig.11 This is a diagram showing the results of the immune activation experiment on BMDC cells in Example 5. DETAILED DESCRIPTION

[0037] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0038] In a first aspect, the present invention provides a method for preparing a whole tumor cell vaccine, comprising the following steps:

[0039] Obtain CD47 gene knockout tumor cells, then insert adjuvants on the surface of tumor cells, and then heat-inactivate them to prepare whole tumor cell vaccines;

[0040] The temperature of the heat inactivation treatment may be, for example, but not limited to, 40°C, 50°C, 60°C, 70°C or 80°C.

[0041] The preparation method of the whole tumor cell vaccine provided by the present invention is to enhance the endocytosis of the vaccine by macrophages by knocking out the CD47 gene of the tumor cells; to enhance the immune activation effect of the vaccine by inserting adjuvants into the surface of the tumor cells after the CD47 gene is knocked out; and to inactivate the cells by heating at a suitable temperature to ensure the safety of the vaccine, while the immunogenic dead cells can also have a stronger immune activation effect. The preparation method is simple and convenient, and the prepared whole tumor cell vaccine can be used for the prevention and treatment of tumors.

[0042] In addition, DSPE-PEG-Mannose and cell membrane can complete membrane insertion through the similarity and hydrophobicity of DSPE structure and cell membrane phospholipid bilayer, and heat treatment will more or less damage the structure of cell membrane, resulting in changes in the membrane structure after heat treatment, and the efficiency of insertion will be affected to a certain extent, and it is also impossible to ensure that the adjuvant is located in the cell membrane and the environment. The change in membrane structure caused by heat treatment may cause the adjuvant to be inserted into the cell interior, thereby losing some of its effectiveness. For the above reasons, the preparation method of the present invention first inserts the adjuvant, and then inactivates it.

[0043] In some optional embodiments, gene editing technology is used to knock out the CD47 gene of tumor cells;

[0044] The gene editing technology includes but is not limited to transcription activator-like effector nuclease technology, zinc finger nuclease technology, CRISPR / Cas9 technology and siRNA gene silencing technology.

[0045] In some optional embodiments, in the CRISPR / Cas9 technology, the forms of delivery and expression of Cas9 protein in the CRISPR / Cas9 system include but are not limited to: DNA, mRNA and recombinant protein.

[0046] In some optional embodiments, the tumor cells include but are not limited to human or mouse tumor cells such as melanoma (B16F10), pancreatic cancer (Pan02), and breast cancer (4T1).

[0047] In some optional embodiments, the adjuvant includes an adjuvant modified with a DSPE-PEG group.

[0048] The adjuvant can bind to the phospholipid bilayer of the cell membrane through hydrophobic interaction.

[0049] In some optional embodiments, the adjuvant includes but is not limited to DSPE-PEG-Mannose and DSPE-PEG-IMDQ, or other adjuvants well known to those skilled in the art.

[0050] The above-mentioned adjuvants can stimulate the maturation of DC cells and enhance the presentation of vaccine antigens by DC cells.

[0051] In some optional embodiments, the step of inserting an adjuvant into the surface of tumor cells comprises: mixing and incubating the tumor cells with the adjuvant to prepare tumor cells with the adjuvant inserted into the surface.

[0052] In some optional embodiments, the incubation temperature may be, for example, but not limited to, 4°C, 10°C, 20°C, 30°C or 37°C;

[0053] The incubation time may be, for example, but not limited to, 0.5 h, 1 h, 2 h, 4 h or 8 h.

[0054] In an optional embodiment, the specific steps of inserting the adjuvant on the surface of the tumor cell are as follows:

[0055] A) Digest adherent tumor cells (B16F10 for example) into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 4 min, discard the supernatant, resuspend the cells in 1-3 mL PBS, count, and take 1-10 million cells into a 1.5 mL centrifuge tube for later use.

[0056] B) Take the DSPE-PEG-Mannose powder out of the -20°C refrigerator and equilibrate it to room temperature for 10-20 minutes. Weigh 1-50 mg and place it in a 1.5 mL centrifuge tube. Add 50-1000 μL DMSO to dissolve it and ultrasonically vibrate it for 3 minutes.

[0057] C) Pipette 1-20 μL of DSPE-PEG-Mannose solution and add it to the prepared tumor cell suspension. Mix well by pipetting 3-6 times and then start incubation. The incubation temperature is 4-37° C. and the incubation time is 0.5-8 h.

[0058] D) After the incubation, the supernatant was removed and the suspension was resuspended in 1 mL of PBS solution. The suspension was mixed by pipetting 3 to 5 times. The suspension was centrifuged at 1000 rpm for 4 min and the supernatant was removed. The suspension was resuspended in 200 to 1000 μL of PBS solution and mixed by pipetting to obtain membrane-inserted cells.

[0059] In some optional embodiments, the time of the heat inactivation treatment may be, for example, but not limited to, 2 min, 5 min, 10 min, 20 min or 30 min.

[0060] In some optional embodiments, the heat inactivation treatment includes water bath heating, which can inactivate cells and ensure the safety of the vaccine. In addition, water bath heat treatment causes immunogenic cell death to release substances such as ATP, CRT, and HMGB1, which can enhance the vaccine's activation effect on the immune system.

[0061] In a second aspect, the present invention provides a whole tumor cell vaccine prepared by the above-mentioned preparation method.

[0062] The whole tumor cell vaccine can be actively taken up by immune cells and has strong immunogenicity.

[0063] In a third aspect, the present invention provides the use of the above-mentioned whole tumor cell vaccine in tumor prevention.

[0064] The whole tumor cell vaccine provided by the present invention can be used for the prevention and treatment of tumors.

[0065] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0066] Example 1 CD47 gene knockout in B16F10 cells

[0067] B16F10 cells were plated at 8 × 10 4The cells were seeded in a 12-well plate at a density of 10 cells / mL and cultured overnight. The next day, a transfection reagent (Lipofectamine TM 3000 Transfection Reagent, Catalog No.: L3000008). The final concentration of the plasmid was 0.5-3.0 μg / mL. Subsequently, the cells were cultured normally for 5 days and single cells were sorted to obtain CD47 cells with complete knockout of the CD47 gene. KO B16F10 cell line.

[0068] Example 2: CD47 KO Characterization of gene knockout effect in B16F10 cells

[0069] 1. Flow cytometry

[0070] CD47 KO B16F10, normal B16F10 cells were digested with trypsin and resuspended in PBS, counted, and 200,000 cells were taken and resuspended in 50 μL PBS in a 1.5 mL centrifuge tube. One group of normal B16F10 cells was not stained with antibodies as a negative control group, and the other two groups (CD47 KO B16F10 cells and normal B16F10 cells) were added with 0.5 μL of anti-CD47 antibody to the cell suspension and incubated at 4°C for 40 min for staining. After staining, 900 μL of PBS was added for resuspending and washing, and the cells were centrifuged at 1000 rpm for 3 min. The above washing operation was repeated once, and the cells were resuspended with 200 μL of PBS for flow cytometry analysis. Table 1 and Figure 1 Given CD47 KO Knockdown effect in B16F10 cells.

[0071] Table 1CD47 KO Flow cytometry analysis of gene knockout effect in B16F10 cells

[0072] Cell Type CD47 positive rate % Unstained cells 0.97 B16F10 cells 96.6 <![CDATA[CD47 KO B16F10 cells]]> 0.29

[0073] 2. Agarose gel electrophoresis experiment

[0074] CD47 was extracted using Tiangen DNA extraction kit KO B16F10, the genome of normal B16F10 cells, amplify the CD47 gene fragment by PCR and set aside. Use a pipette to transfer 6 mL of 50×TAE solution to 294 mL of ddHO. 2Prepare 300mL 1×TAE buffer solution in 1×TAE buffer solution. Weigh 1g agarose, add 50mL 1×TAE buffer solution to dissolve, boil until slightly boiling, add 5μL 10000×SolarRed nucleic acid dye, shake well, pour into the mold while hot, and solidify naturally at room temperature. After 15 minutes, pull out the comb, put the agarose gel into the electrophoresis device containing 1×TAE buffer solution, and carefully add the sample to be tested to the gel hole. Run the gel at 80V, 50min. After the gel run is completed, place the gel in the imaging device for observation. The results are as follows: Figure 2 shown.

[0075] 3. Gene sequencing

[0076] CD47 was extracted using Tiangen DNA extraction kit KO B16F10, the genome of normal B16F10 cells, the CD47 gene fragment was amplified by PCR and then sequenced by the first generation. Figure 3 shown.

[0077] Example 3: CD47 KO B16F10 cells heated in water bath

[0078] CD47 KO B16F10 cells were digested with trypsin and resuspended in PBS. The cells were counted and 1 million cells were resuspended in 100 μL PBS into a 1.5 mL centrifuge tube. The centrifuge tube was placed in a 60°C water bath and incubated for 5 min, 10 min, and 15 min. The staining results at different incubation times are shown in Figure 2. Figure 4 The morphology characterization results are shown in Figure 5 shown.

[0079] like Figure 4 As shown, the PBS group is the untreated control group, and the other three groups are the results after treatment at 60℃ for 5min, 10min, and 15min, respectively. The Calcein AM dye represented by green fluorescence can mark living cells, and the PI dye represented by red fluorescence can mark dead cells. The experimental results show that treatment at 60℃ for 5 to 10min can successfully cause cell death, verifying the feasibility of the heat treatment method and the safety of the heat treatment method for preparing vaccines in vitro.

[0080] Example 4: CD47 KO ICD Characterization of B16F10 Cells Heated in Water Bath

[0081] 1. CRT release detection

[0082] CD47 after heat treatment KOB16F10 cells and untreated cells were stained with CRT antibody, and the expression of CRT was detected by flow cytometry, as shown in Tables 2 and Figure 6 .

[0083] Table 2CD47 KO Results of CRT flow cytometry detection of B16F10 cells after heat treatment

[0084]

[0085]

[0086] The results showed that CRT was released from heat-treated cells.

[0087] 2. HMGB1 release detection

[0088] Heat-treated CD47 KO B16F10 cells were treated as follows: washed once with PBS, fixed with 4% PFA at room temperature for 15 minutes, washed again, then permeabilized with 0.1% triton X-100 at room temperature for 10 minutes, washed twice again, and nonspecific sites were blocked with blocking solution at room temperature for 60 minutes. Then, anti-HMGB1 antibody was stained overnight at 4°C, and then washed twice. DAPI was then added to stain the cell nucleus. Finally, after washing with PBS, BMDCs were observed using CLSM.

[0089] The results show that ( Figure 7 ), significant HMGB1 release can be observed under CLSM.

[0090] Example 5CD47 KO B16F10 cell membrane insertion adjuvant

[0091] CD47 KODigest B16F10 cells into a 15mL centrifuge tube, centrifuge at 1000rpm for 4min, discard the supernatant, add 1mL PBS to resuspend the cells, count them, and take 1 million cells into a 1.5mL centrifuge tube for later use. Take the DSPE-PEG-Mannose powder out of the -20℃ refrigerator and equilibrate it to room temperature for 10-20min, weigh 20mg and place it in a 1.5mL centrifuge tube, add 300μL DMSO to dissolve, and ultrasonically vibrate for 3min. Use a pipette to transfer 10μL DSPE-PEG-Mannose solution to the prepared tumor cell suspension, blow 3-6 times to mix evenly, and then start incubation. The incubation temperature is 37℃ and the incubation time is 4h. After incubation, centrifuge at 1000 rpm for 5 min, remove the supernatant, add 1 mL of PBS solution to resuspend, pipette 3 to 5 times to mix evenly, centrifuge at 1000 rpm for 4 min, remove the supernatant, resuspend with 200 to 1000 μL PBS solution, pipette and mix to obtain membrane-inserted adjuvant tumor cells.

[0092] DSPE-PEG-Cy5 was selected as a verification method to test the feasibility of the membrane insertion method. First, the structures of DSPE-PEG-Cy5 and adjuvant DSPE-PEG-Mannose inserted into the cell membrane are both DSPE parts. By verifying the insertion effect of DSPE-PEG-Cy5, the insertion effect of DSPE-PEG-Mannose as an adjuvant can be indirectly reflected. Figure 8 As shown, under the optimized insertion conditions (the same as the conditions for inserting DSPE-PEG-Mannose as mentioned above), the Cy5 fluorescence signal can be detected to be evenly distributed on the cell membrane, verifying the feasibility of the adjuvant membrane insertion method.

[0093] Example 6 Whole tumor cell vaccine is used to stimulate BMDCs maturation

[0094] The membrane-inserted adjuvanted tumor cells obtained in Example 5 were heated in a water bath (temperature of 60°C for 5 min) to prepare a whole tumor cell vaccine, which was incubated in BMDCs for 24 h. BMDCs were collected and treated as follows: washed once with PBS, and then BMDCs were stained with antibodies (anti-CD11c antibody, anti-CD80 antibody, anti-CD86 antibody, and anti-MHC II antibody). Then, the cells were washed twice with PBS. Finally, BMDCs were resuspended in 4% PFA for FACS analysis. Table 3 shows the activation levels of BMDCs mediated by the vaccine-treated group and the untreated group.

[0095] Table 3 BMDCs maturation levels mediated by vaccine-treated and untreated groups

[0096] Untreated group Vaccine treatment group <![CDATA[CD80 + BMDCs]]> 19.8％ 49.1％ <![CDATA[CD86 + BMDCs]]> 15.7％ 50.2％ <![CDATA[MHC II + BMDCs]]> 14.8％ 44.1％

[0097] In addition, set the following processing groups:

[0098] The PBS group served as the control group;

[0099] The B16F10 group consisted of wild-type B16F10 cells heat-treated and then co-cultured with BMDC cells;

[0100] CD47 KO The B16F10 group consisted of CD47 knockout B16F10 cells heat-treated and then co-cultured with BMDC cells;

[0101] The Mannose group was incubated with only DSPE-PEG-Mannose adjuvant added to BMDC cells;

[0102] In the vaccine group, CD47KO B16F10 cells inserted with DSPE-PEG-Mannose were heat-treated and then co-cultured with BMDC cells.

[0103] The results are as follows Fig.11 As shown, it can be seen that the activation effect of BMDC was significantly enhanced after the addition of adjuvant to whole tumor cell antigen.

[0104] Example 7 Study on the prevention of melanoma in mice by whole tumor cell vaccine

[0105] The whole tumor cell vaccine was injected into the right dorsal flank of female C57BL / 6J mice. The dose per mouse was 1 million CD47 containing 30 μg DSPE-PEG-Mannose. KO B16F10 whole tumor cell vaccine was administered once a week for a total of 3 times. 6 B16F10 cells were injected into the right dorsal flank of mice, and the tumor growth of each group of mice was recorded. The mice were killed on the 16th day for analysis.

[0106] The experimental results show that ( Fig. 9 ), relative to the untreated group (each mouse was given 100 μL of PBS once a week for a total of 3 times) and pure CD47 KO B16F10 cell group (in the prevention experiment, only CD47 cells killed by heat treatment were injected KO B16F10 cells, which have no membrane-inserted adjuvant, are denoted as CD47 KO B16F10. The specific treatment method is to administer 1 million CD47 KOB16F10 tumor cells (100 μL volume), administered once a week for a total of 3 times), after treatment with a membrane-inserted adjuvant-modified whole tumor cell vaccine (Vaccine), the melanoma in mice was significantly reduced. This indicates that the whole tumor cell vaccine can effectively inhibit the growth of mouse melanoma.

[0107] Example 8 Study on the treatment of melanoma in mice with whole tumor cell vaccine

[0108] 1.0×10 6 B16F10 cells were injected into the right dorsal flank of female C57BL / 6J mice to establish a B16F10 mouse melanoma tumor model. When the tumor volume grew to about 80-120 mm 3 At the same time, B16F10 tumor-bearing mice were randomly divided into PBS, CD47 KO B16F10, Vaccine (debris) and Vaccine. The dose of Vaccine was 1 million CD47 containing 30 μg DSPE-PEG-Mannose per mouse. KO The B16F10 whole tumor cell vaccine, with a total volume of 100 μL, was administered once a week for a total of 3 times.

[0109] Vaccine (debris) is a vaccine prepared by inserting the membrane into the adjuvant and then heat-treating it, and then breaking it. The specific breaking method is to place the vaccine in liquid nitrogen for 5 minutes, then in a water bath at 37°C for 5 minutes, and repeat this cycle 3 times, and then ultrasonicate for 2 minutes (0°C, 60W). The injection method is the same as that of Vaccine.

[0110] CD47 KO B16F10: Injection of CD47 cells that have been killed by heat treatment KO B16F10 cells, which have no adjuvant inserted into the cell membrane. The injection method is the same as that of Vaccine.

[0111] PBS: Each mouse was administered 100 μL of PBS once a week for a total of 3 times.

[0112] The tumor growth of mice in each group was recorded during the administration period, and the mice were killed on the 16th day for analysis.

[0113] The experimental results show that ( Fig.10 ), relative to PBS, CD47 KO In the B16F10 and Vaccine (debris) groups, the melanoma of mice was significantly reduced after treatment with the whole tumor cell vaccine modified with membrane-inserted adjuvant. This indicates that the whole tumor cell vaccine can effectively inhibit the growth of melanoma in mice.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a whole tumor cell vaccine, characterized in that: The following steps are involved: Obtain CD47 gene knockout tumor cells, then insert adjuvants on the surface of tumor cells, and then heat-inactivate them to prepare whole tumor cell vaccines; The temperature of the heating inactivation treatment is 40-80°C.

2. The preparation method according to claim 1, characterized in that: Use gene editing technology to knock out the CD47 gene in tumor cells; The gene editing technologies include transcription activator-like effector nuclease technology, zinc finger nuclease technology, CRISPR / Cas9 technology and siRNA gene silencing technology.

3. The preparation method according to claim 1, characterized in that: The tumor cells include melanoma cells, pancreatic cancer cells and breast cancer cells.

4. The preparation method according to claim 1, characterized in that: The adjuvant includes an adjuvant modified with a DSPE-PEG group.

5. The preparation method according to claim 4, characterized in that: The adjuvants include DSPE-PEG-Mannose and DSPE-PEG-IMDQ.

6. The preparation method according to claim 5, characterized in that: The step of inserting the adjuvant on the surface of the tumor cells comprises: mixing and incubating the tumor cells with the adjuvant to prepare tumor cells with the adjuvant inserted on the surface.

7. The preparation method according to claim 6, characterized in that: The incubation temperature is 4 to 37°C; The incubation time is 0.5 to 8 hours.

8. The preparation method according to claim 1, characterized in that: The time of the heat inactivation treatment is 2 to 30 minutes.

9. A whole tumor cell vaccine, characterized in that: The preparation method is described in any one of claims 1 to 8.

10. Use of the whole tumor cell vaccine according to claim 9 in tumor prevention.

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