Immune-enhanced DC recruitment tumor nano vaccine as well as preparation method and application thereof

Abstract

The invention discloses an immune-enhanced DC recruitment tumor nano vaccine and a preparation method and application thereof.The immune-enhanced DC recruitment tumor nano vaccine is obtained by combining tumor antigen, CCL20 and RADA16-I polypeptide to obtain tumor antigen gel, then adding a Ca < 2 + > solution and conducting extrusion through a filter membrane; the mass ratio of the tumor antigen to the CCL20 to the RADA16-I polypeptide is (175 to 225) to 1 to (110 to 140). The tumor nano vaccine can efficiently recruit DC cells, promote the DC cells to take more tumor antigens, promote the DC cells to be mature, enable the mature DC cells carrying the tumor antigens to migrate to lymph nodes to be presented to T cells and activate the T cells, and promote the number of the activated T cells in tumor tissues to be greatly increased, so that tumor growth can be obviously inhibited, and the tumor growth rate is increased. The composition has no obvious side effect on the body and has good biological safety.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and more specifically, relates to an immune-enhancing DC-recruiting tumor nanovaccine and a preparation method and application thereof. Background Art

[0002] Malignant tumors have high morbidity and mortality rates worldwide, posing a significant threat to human health. Current cancer treatments primarily include surgery, chemotherapy, radiotherapy, and immunotherapy. Compared to other cancer treatments, immunotherapy activates the immune system, restoring immune function and providing a sustained killing effect on tumor cells, making it a hot topic in cancer treatment.

[0003] Tumor vaccines are currently a popular form of tumor immunotherapy. Their principle is to introduce tumor antigens into the patient's body in various forms, such as tumor cells, tumor-associated proteins or peptides, or genes expressing tumor antigens. This approach overcomes the immunosuppression caused by the tumor, enhances immunogenicity, activates the patient's own immune system, and induces both cellular and humoral immune responses, thereby achieving the goal of controlling or eliminating the tumor. However, the effectiveness of a single tumor antigen vaccine in enhancing immune responses is often very limited.

[0004] An attractive alternative to single-antigen vaccines is to use cell lysates obtained by lysing tumor cells from homologous individuals as an antigen source. Tumor lysates can yield all potential tumor-associated antigens. Injecting these antigens into the cancerous environment can reverse the immunosuppressive state of the tumor microenvironment, activate immune responses, and inhibit tumor growth. However, simple tumor cell lysates have not shown significant clinical efficacy due to insufficient activation and antigen presentation by dendritic cells (DCs), key antigen-presenting cells in tumor immunity. Tumor antigens must be taken up, processed, and presented on the cell membrane by DCs for recognition by T cells, activating them to kill tumors. Although various strategies have been developed to enhance DC activation and antigen presentation, such as immune adjuvants or nanodelivery systems, these technologies often focus solely on improving DC activation without addressing the importance of enhancing DC antigen uptake efficiency. Consequently, they struggle to induce a sustained and potent immune response, resulting in limited overall therapeutic efficacy. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a tumor nanovaccine that can efficiently recruit DC cells and improve the antigen presentation effect of DC cells.

[0006] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] The first aspect of the present invention provides an immune-enhancing DC-recruited tumor nanovaccine, which is prepared by mixing tumor antigen, CCL20 and RADA16-I polypeptide to obtain tumor antigen gel, and then adding Ca 2+ The solution is obtained by extruding through a filter membrane; the mass ratio of the tumor antigen, CCL20 and RADA16-Ⅰ polypeptide is 175-225:1:110-140.

[0008] The second aspect of the present invention provides a method for preparing an immune-enhancing DC-recruiting tumor nanovaccine, comprising the following steps:

[0009] (1) Tumor antigen, CCL20, and RADA16-I polypeptide were added to ultrapure water to concentrations of 7 mg / mL to 9 mg / mL, 30 μg / mL to 60 μg / mL, and 4.4 mg / mL to 5.6 mg / mL, respectively, and placed at 3°C to 5°C for 10 to 14 hours to obtain a tumor antigen gel;

[0010] (2) Add tumor antigen gel to Ca 2+ The solution is squeezed out through a filter membrane to obtain the product.

[0011] The third aspect of the present invention provides the use of the above-mentioned immune-enhancing DC-recruiting tumor nanovaccine in the preparation of tumor immunotherapy drugs.

[0012] The DC recruitment tumor nanovaccine of the present invention is to combine tumor antigens and DC recruitment factor CCL20 by using RADA16-I polypeptide, and then add Ca 2+ It is then prepared by extrusion through a filter membrane. It can efficiently recruit DC cells, promote DC cells to take up more tumor antigens, and at the same time promote DC cell maturation. Mature DC cells carrying tumor antigens migrate to the lymph nodes to present them to T cells and activate T cells, thereby causing a large increase in the number of activated T cells in tumor tissues, thereby better inhibiting tumor growth and showing a good anti-tumor effect.

[0013] Furthermore, the present invention uses doxorubicin, cisplatin, and citronellol to induce immunogenic death of tumor cells, so that tumor cells can express more immunogenic molecules and have stronger pro-immunity potential, thereby preparing three immune-enhancing DC-recruiting tumor nanovaccines (apoptosis-type Apo-TNV, ferroptosis-type Fer-TNV, and necrosis-type Nec-TNV). Among them, the apoptosis-type DC-recruiting tumor nanovaccine Apo-TNV has better ability to promote DC cell maturation and activate T cells, and the killing CD8 + The number of T cells increased significantly, showing a better tumor suppression effect.

[0014] The four tumor nanovaccines of the present invention have obvious anti-tumor effects. After administration, they can significantly inhibit tumor growth without obvious side effects on the body. They have good biosafety, and the preparation method is simple, time-saving and low-cost, which is conducive to promoting the clinical development of tumor vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The average particle size and Zeta potential results of the DC recruitment tumor nanovaccine TNV prepared in Example 1 of the present invention.

[0016] Figure 2 This is the result of the nanovaccine TNV recruiting DC cells in Example 2 of the present invention.

[0017] Figure 3 This is the result of the nanovaccine TNV's ability to affect DC cell uptake of tumor antigens in Example 3 of the present invention.

[0018] Figure 4 These are the expression results of MHCⅡ antibodies and CD80 antibodies in DC cells after stimulation with the nanovaccine TNV in Example 4 of the present invention.

[0019] Figure 5 This is a graph showing the anti-tumor growth effect of the nanovaccine TNV in Example 5 of the present invention.

[0020] Figure 6 The results show the mRNA expression of some genes in LLC cells cultured in complete medium containing doxorubicin, cisplatin and citronellol for 24 hours and normal LLC cells in Example 6 of the present invention.

[0021] Figure 7 These are the expression results of CD86, MHCⅠ double-positive cells and CD80, MHCⅡ double-positive cells in DC cells after stimulation with DC-recruited tumor nanovaccine and three immune-enhancing DC-recruited tumor nanovaccines in Example 7 of the present invention.

[0022] Figure 8 These are the results of the effects of DC-recruited tumor nanovaccine and three immune-enhancing DC-recruited tumor nanovaccines on T cell activation in Example 8 of the present invention.

[0023] Figure 9 These are the results of the effects of the DC-recruited tumor nanovaccine and three immune-enhancing DC-recruited tumor nanovaccines on tumor growth in C57 mice in Example 9 of the present invention.

[0024] Figure 10 These are the results of the effects of the DC-recruited tumor nanovaccine and three immune-enhancing DC-recruited tumor nanovaccines on the body weight of C57 mice in Example 9 of the present invention.

[0025] Figure 11The DC-recruited tumor nanovaccine and three immune-enhancing DC-recruited tumor nanovaccines in Example 10 of the present invention have an effect on CD8 + The effect of T cell content on the results. DETAILED DESCRIPTION

[0026] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or the conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0029] In some embodiments of the present invention, an immune-enhancing DC-recruiting tumor nanovaccine is disclosed, which is prepared by mixing tumor antigen, CCL20 and RADA16-I polypeptide to obtain tumor antigen gel, and then adding Ca 2+ The solution is obtained by extruding through a filter membrane; the mass ratio of the tumor antigen, CCL20 and RADA16-Ⅰ polypeptide is 175-225:1:110-140.

[0030] In some embodiments, the mass ratio of the tumor antigen, CCL20 and RADA16-Ⅰ polypeptide is 190-210:1:120-130.

[0031] In some embodiments, the mass ratio of the tumor antigen, CCL20 and RADA16-Ⅰ polypeptide is 195-205:1:124-126.

[0032] In some embodiments, the tumor antigen is prepared by the following method: culturing tumor cells until the monolayer cell sheet reaches 75% to 85% confluence, collecting adherent tumor cells, and lysing them to obtain the tumor antigen.

[0033] In some embodiments, the lysis is performed by a freeze-thaw cycle method, wherein the parameters of the freeze-thaw cycle method are: freeze-thaw cycles at -82°C to -78°C and 36°C to 38°C for 4 to 5 times.

[0034] In some embodiments, the tumor antigen is an apoptotic tumor antigen, a ferroptosis tumor antigen, or a necrotic tumor antigen.

[0035] In some embodiments, the tumor antigen is an apoptotic tumor antigen.

[0036] In some embodiments, the tumor antigen is prepared by the following method: when the monolayer cell sheet reaches 75% to 85% confluence, the culture is continued for 22 hours to 26 hours using a complete culture medium containing a cell death inducer, and then the adherent tumor cells are collected and lysed to obtain the tumor antigen.

[0037] In some embodiments, the lysis is performed by a freeze-thaw cycle method, wherein the parameters of the freeze-thaw cycle method are: freeze-thaw cycles at -82°C to -78°C and 36°C to 38°C for 4 to 5 times.

[0038] In some embodiments, the cell death inducing agent is doxorubicin, cis-diamminedichloroplatinum (II) or citronellol.

[0039] In some embodiments, the cell death-inducing agent is doxorubicin.

[0040] In some embodiments, the final concentration of doxorubicin in the complete culture medium is 1.4 μM to 1.6 μM.

[0041] In some embodiments, the final concentration of doxorubicin in the complete culture medium is 1.45 μM to 1.55 μM.

[0042] In some embodiments, the final concentration of cis-diamminedichloroplatinum (II) in the complete culture medium is 230 μM to 250 μM.

[0043] In some embodiments, the final concentration of cis-diamminedichloroplatinum (II) in the complete culture medium is 235 μM to 245 μM.

[0044] In some embodiments, the final concentration of citronellol in the complete culture medium is 1150 μM to 1250 μM.

[0045] In some embodiments, the final concentration of citronellol in the complete culture medium is 1180 μM to 1220 μM.

[0046] In some embodiments, the Ca 2+ The solution is a 14.5mM to 15.5mM CaCl2 solution.

[0047] In some embodiments, the tumor antigen gel is 2+ The volume ratio of the solution is 0.8-1.2:1.

[0048] In some embodiments, the tumor antigen gel is 2+ The volume ratio of the solution is 0.9 to 1.1:1.

[0049] In some embodiments, the tumor antigen gel is 2+ The volume ratio of the solution is 0.95-1.05:1.

[0050] In other embodiments of the present invention, a method for preparing the above-mentioned immune-enhancing DC-recruiting tumor nanovaccine is disclosed, comprising the following steps:

[0051] (1) Tumor antigen, CCL20, and RADA16-I polypeptide were added to ultrapure water to concentrations of 7 mg / mL to 9 mg / mL, 30 μg / mL to 60 μg / mL, and 4.4 mg / mL to 5.6 mg / mL, respectively, and placed at 3°C to 5°C for 10 to 14 hours to obtain a tumor antigen gel;

[0052] (2) Add tumor antigen gel to Ca 2+ The solution is squeezed out through a filter membrane to obtain the product.

[0053] In some embodiments, the pore size of the filter membrane in step (2) is 0.22 μm.

[0054] In other embodiments of the present invention, the use of the above-mentioned immune-enhancing DC-recruiting tumor nanovaccine in the preparation of tumor immunotherapy drugs is disclosed.

[0055] In some embodiments, the tumor is lung cancer, breast cancer, colorectal cancer or lymphoma.

[0056] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0057] Example 1 Synthesis and Characterization of DC-recruiting Tumor Nanovaccine (TNV)

[0058] The following steps are involved:

[0059] 1. LLC mouse lung cancer cells (purchased from ATCC) were subcultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0060] 2. When the LLC monolayer cell sheet reaches 80% confluence, collect the adherent tumor cells and centrifuge them at 500g for 10 min. Resuspend the cells in 0.25× PBS and freeze-thaw them five times at -80°C and 37°C to obtain tumor antigens.

[0061] 3. Add 4 mg of tumor antigen, 20 μg of CCL20, and 2.5 mg of RADA16-Ⅰ peptide to ultrapure water to a final volume of 0.5 mL and incubate at 4°C for 12 h to obtain tumor antigen gel.

[0062] 4. Add the tumor antigen gel to a 15 mM CaCl2 aqueous solution at a volume ratio of 1:1 and extrude through a 0.22 μm filter membrane to obtain the DC-recruiting tumor nanovaccine TNV.

[0063] The DC recruitment tumor nanovaccine TNV prepared in this example has an average particle size of 110 nm and an average Zeta potential of +5.25 mV ( Figure 1 ).

[0064] Example 2 Test of the effect of nano vaccine TNV in recruiting DC cells

[0065] This example uses a Transwell with an 8 μm pore size to test the recruitment of DCs by the nanovaccine TNV prepared in Example 1. The following steps are included:

[0066] 1. Calcein-AM-labeled dendritic cells (DCs) were planted in the upper chamber, PBS was added to the lower chamber of the control group, tumor antigens (Example 1, step 2) were added to the lower chamber of the Antigen group, and the nanovaccine TNV of Example 1 was added to the lower chamber of the TNV group. The cells were cultured in a cell culture incubator.

[0067] 2. After 24 hours, the number of DC cells that migrated to the lower chamber was observed under a fluorescence microscope. Green fluorescence represents DC cells that migrated to the lower chamber.

[0068] The experimental results are as follows Figure 2 As shown, Figure 2 It was shown that after being recruited by TNV, DC cells in the upper chamber were able to migrate more to the lower chamber, and the number of migrated DC cells was significantly greater than that in the control group and the Antigen group. This result indicated that the nanovaccine TNV prepared in Example 1 could better recruit DC cells.

[0069] Example 3 Effect of Nanovaccine TVN on the Ability of DC Cells to Uptake Tumor Antigens

[0070] Dendritic cells (DCs) are the most important antigen-presenting cells in tumor immunity. DCs are required to present ingested antigens to T cells to trigger their anti-tumor immune function. Therefore, the ability of DCs to take up tumor antigens is crucial. This example tested the ability of the nanovaccine TNV prepared in Example 1 to inhibit DCs from taking up tumor antigens. The following steps were involved:

[0071] 1. Hoechst-labeled dendritic cells (DCs) were seeded in 12-well plates and divided into three groups: control group (Control), antigen group (Antigen) and nanovaccine group (TNV).

[0072] 2. Cy3 fluorescently labeled tumor antigens were added to the Antigen group, Cy3 fluorescently labeled nanovaccine TNV was added to the TNV group, and the same volume of PBS was added to the control group. After culturing in the incubator for 6 hours, the fluorescence intensity was observed under a confocal fiber microscope.

[0073] Hoechst-labeled DC cells appear blue, and Cy3-labeled antigens show red fluorescence. The intensity of red fluorescence reflects the ability of DC cells to take up antigens. Figure 3 As shown, from Figure 3 It can be seen that compared with the Antigen group, the DC cells in the TNV group were able to take up more tumor antigens. We analyzed that the main reason is that DC cells tend to take up nanoparticles, and DC cells have an affinity for arginine in the RADA16-Ⅰ polypeptide, so the efficiency of taking up TNV is higher.

[0074] Example 4 Effect of Nanovaccine TVN on Promoting DC Cell Maturation

[0075] Mature DCs are able to present antigens to T cells. This example tested the effect of the nanovaccine TNV prepared in Example 1 on promoting DC maturation. The following steps were included:

[0076] 1. Dendritic cells (DCs) were seeded in 12-well plates and divided into three groups: a control group (culture medium added), an antigen group (tumor antigen added), and a nanovaccine group (TNV, TNV prepared in Example 1 added).

[0077] 2. After 24 hours, remove the plate, collect DC cells by centrifugation, resuspend them in 100 μL of PBS containing 2% FBS, add a certain amount of CD11c antibody to label DC cells, add MHCⅡ antibody and CD80 antibody to label MHCⅡ and CD80 molecules on the cell surface, respectively, and incubate at 4°C in the dark for 30 minutes.

[0078] 3. Subsequently, wash twice with PBS containing 2% FBS. Collect the stained cells, add 0.5 mL of cell staining buffer to resuspend, and analyze by flow cytometry.

[0079] The results are as follows Figure 4The results showed that compared with the control group and the antigen group, after stimulation with the nanovaccine TNV, DC cells were able to express more MHCⅡ and CD80 molecules, both of which are markers of DC cell maturation. Therefore, the nanovaccine TNV prepared by the present invention can promote DC maturation and improve its ability to present antigens.

[0080] Example 5 Comparison of the anti-tumor effects of nanovaccine TNV and tumor antigens

[0081] This example compares the anti-tumor effects of nano-vaccine TNV and tumor antigens, and includes the following steps:

[0082] 1. Establishment of LLC subcutaneous tumor mouse model: LLC cells were cultured at a rate of 1*10 6 The dose of 100mg / mouse was implanted subcutaneously into C57BL / 6 mice.

[0083] On the day of modeling, mice were randomly divided into three groups: control, antigen, and TNV. Tumor antigens or the nanovaccine TNV were injected subcutaneously near the lymph nodes. The control group received the same volume of saline. Tumor size was measured and recorded every two days using a vernier caliper throughout the experiment.

[0084] The results of tumor efficacy evaluation are as follows Figure 5 As shown, in vivo experiments in mice found that after subcutaneous vaccination with the nanovaccine TNV, the TNV group had a more obvious anti-tumor growth effect compared with the control group injected with normal saline, and this effect was also stronger than the Antigen antigen group.

[0085] The results of this example show that the nanovaccine TNV of the present invention promotes the maturation of DC cells by efficiently recruiting DC cells and promoting the uptake of antigens by DC cells. Mature DC cells carrying antigens migrate to the lymph nodes to present to T cells and activate T cells, thereby significantly increasing the number of activated T cells in tumor tissues, thereby exhibiting a stronger anti-tumor effect.

[0086] Example 6 Preparation of Immunoenhanced DC-Recruited Tumor Nanovaccine

[0087] In this example, three immune-enhancing DC-recruiting tumor nanovaccines were prepared using three chemical drugs (doxorubicin that induces apoptosis of tumor cells, cisplatin that induces ferroptosis of tumor cells, and citronellol that induces necrosis of tumor cells).

[0088] 1. Apo-TNV (apoptosis-induced DC-recruiting tumor nanovaccine) (doxorubicin-induced)

[0089] (1) Same as step 1 of Example 1.

[0090] (2) When the LLC monolayer reached 80% confluence, the cell culture medium was replaced with complete medium containing 1.5 μM doxorubicin and cultured for another 24 h. The supernatant and adherent tumor cells were collected by centrifugation at 500 g for 10 min, resuspended in 0.25× PBS, and repeatedly frozen and thawed five times at -80°C and 37°C to obtain tumor antigens.

[0091] Subsequent steps 3 and 4 are the same as in Example 1.

[0092] 2. Ferroptosis-induced DC-recruiting tumor nanovaccine Fer-TNV (ferroptosis-induced DC-recruiting tumor nanovaccine) (cisplatin, cisplatin)

[0093] (1) Same as step 1 of Example 1.

[0094] (2) When the LLC monolayer reached 80% confluence, the cell culture medium was replaced with complete medium containing 240 μM cisplatin and cultured for another 24 h. The supernatant and adherent tumor cells were collected by centrifugation at 500 g for 10 min, resuspended in 0.25× PBS, and repeatedly frozen and thawed five times at -80°C and 37°C to obtain tumor antigens.

[0095] Subsequent steps 3 and 4 are the same as in Example 1.

[0096] 3. Nec-TNV (necroptosis-induced DC-recruiting tumor nanovaccine) (citronellol)

[0097] (1) Same as step 1 of Example 1.

[0098] (2) When the LLC cell monolayer reached 80% confluence, the cell culture medium was replaced with complete medium containing 1200 μM citronellol and cultured for another 24 h. The supernatant and adherent tumor cells were collected and centrifuged at 500 g for 10 min. The cells were resuspended in 0.25× PBS and repeatedly frozen and thawed five times at -80°C and 37°C to obtain tumor antigens.

[0099] Subsequent steps 3 and 4 are the same as in Example 1.

[0100] Untreated LLC cells and LLC cells treated with doxorubicin, cisplatin, and citronellol for 24 h were collected by centrifugation, cellular RNA was extracted, and the mRNA expression of some genes in the cells was tested using a reverse transcription kit and a qPCR kit.

[0101] The results are as follows Figure 6 Results showed that LLC tumor cells that underwent immunogenic death induced by chemical drugs expressed more immunogenic damage-associated molecular patterns (DAMPs), including high-mobility group protein B1 (HMGB1), calreticulin, and heat shock proteins (HSPs). These molecules can better activate DCs. Among the three chemical drugs, the immunogenic molecules formed by doxorubicin-induced apoptosis were significantly higher than those in the other two groups, indicating its potential to effectively activate immune cells.

[0102] Example 7 Effect of immune-enhancing DC recruitment tumor nanovaccine on DC cell maturation

[0103] This example tested the effects of the three immune-enhancing DC-recruiting tumor nanovaccines prepared in Example 6 and the DC-recruiting tumor nanovaccine prepared in Example 1 on promoting DC cell maturation. The following steps were included:

[0104] 1. Dendritic cells (DCs) were seeded in 12-well plates and divided into five groups: a control group (culture medium added), a nanovaccine group (TNV, TNV prepared in Example 1 added), and an immune-enhancing nanovaccine group (Apo-TNV, Fer-TNV, and Nec-TNV prepared in Example 6 added).

[0105] After 24 hours, remove the plate and collect DCs by centrifugation. Resuspend in 100 μL of PBS containing 2% FBS, add a certain amount of CD11c antibody to label DCs, add MHC I and CD86 antibodies, or MHC II and CD80 antibodies, and incubate at 4°C in the dark for 30 minutes.

[0106] 3. Wash the cells twice with cell staining buffer. Collect the stained cells, resuspend them in 0.5 mL of cell staining buffer, and analyze them by flow cytometry.

[0107] The results are as follows Figure 7As shown, the results showed that the DC-recruiting tumor nanovaccine prepared in Example 1 and the three immune-enhancing DC-recruiting tumor nanovaccines prepared in Example 6 all promoted the maturation of DC cells, with a significant increase in the number of CD86, MHC I double-positive cells and CD80, MHC II double-positive cells. Among them, Apo-TNV was significantly better than the other groups in activating DC cells, indicating that doxorubicin-induced tumor cell apoptosis can enhance the immunogenicity of tumor antigens and has a better ability to promote DC maturation.

[0108] Example 8 Effect of DC recruitment of tumor nanovaccines on T cell activation

[0109] This example tested the effects of the three immune-enhancing DC-recruiting tumor nanovaccines (Apo-TNV, Fer-TNV, and Nec-TNV) prepared in Example 6 and the DC-recruiting tumor nanovaccine (TNV) prepared in Example 1 on activated T cells. The following steps were included:

[0110] 1. Inoculate spleen cells into 6-well plates and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0111] 2. After the cells stabilize, add TNV, Apo-TNV, Fer-TNV, and Nec-TNV respectively. Add the same volume of PBS to the control group and continue culturing in the incubator.

[0112] 3. Collect the spleen cells of each group by centrifugation, extract the cellular RNA, and use the reverse transcription kit and qPCR kit to test the mRNA expression of related genes in the cells.

[0113] The results are as follows Figure 8 The results showed that all four nanovaccines could promote the secretion of cytotoxic cytokines (IFNγ, TNFα, TNFβ) by T cells in spleen cells to a certain extent, and all of them could activate T cells. Among them, Apo-TNV had the most significant activation ability, which could promote T cells to secrete a large amount of cytotoxic cytokines.

[0114] Example 9 Effect of DC recruitment of tumor nanovaccine on inhibition of tumor growth

[0115] This example compares the effects of three immune-enhancing DC-recruiting tumor nanovaccines (Apo-TNV, Fer-TNV, and Nec-TNV) prepared in Example 6 and the DC-recruiting tumor nanovaccine (TNV) prepared in Example 1 on inhibiting tumor growth. The following steps are included:

[0116] 1. Establishment of LLC subcutaneous tumor mouse model: LLC cells were cultured at a rate of 1*10 6 The dose of 100mg / mouse was implanted subcutaneously into C57BL / 6 mice.

[0117] On the day of modeling, mice were randomly divided into five groups: control, TNV, Apo-TNV, Fer-TNV, and Nec-TNV. The corresponding nanovaccines were injected subcutaneously near the lymph nodes, while the control group received the same volume of saline. Tumor size and body weight were measured every two days throughout the experiment using a vernier caliper.

[0118] The results of tumor efficacy evaluation are as follows Figure 9 and Figure 10 As shown, in vivo experiments in mice revealed that subcutaneous administration of the four nanovaccines, TNV, significantly inhibited tumor growth compared to the saline-injected control group. Apo-TNV was particularly effective in suppressing tumor growth, surpassing the other three groups. This suggests that after recruiting DCs, the abundant tumor antigens carried by Apo-TNV more effectively activated DCs, leading to enhanced T cell activation and ultimately demonstrating excellent tumor suppression. All four nanovaccines had no significant effect on mouse body weight, exhibited minimal toxicity and side effects, and demonstrated excellent biosafety.

[0119] Example 10 DC recruits tumor nanovaccine to CD8 + Effect of T cell content

[0120] CD8 + T cells are the main effector T cells that kill tumors. This example compares the effects of three immune-enhancing DC-recruited tumor nanovaccines (Apo-TNV, Fer-TNV, and Nec-TNV) prepared in Example 6 and the DC-recruited tumor nanovaccine (TNV) prepared in Example 1 on CD8 + Effect of T cell content.

[0121] The following steps are involved:

[0122] 1. 14 days after administration in Example 9, the subcutaneous tumors of the mice in each group were collected, the obtained tumors were ground, and the cells were filtered through a 300-mesh sieve. The obtained cell suspension was centrifuged to obtain a cell pellet;

[0123] 2. Add 2 mL of red blood cell lysis buffer to resuspend the cells. After 3 minutes, add 3 mL of culture medium to dilute the cells. Centrifuge at 500g for 5 minutes to obtain a cell pellet.

[0124] 3. Resuspend with 100uL cell staining buffer and add CD3 and CD8 antibodies to mark T cells and CD8 respectively. + T cells were incubated at 4°C in the dark for 30 min.

[0125] 4. Wash the cells twice with cell staining buffer. Collect the stained cells, resuspend them in 0.5 mL of cell staining buffer, and analyze them by flow cytometry.

[0126] The results are as follows Figure 11 As shown, the results showed that CD8 + The number of T cells in the Apo-TNV group was significantly increased. + The highest number of T cells indicates that it has the strongest ability to activate immunity.

[0127] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. An immune-enhancing DC-recruiting tumor nanovaccine, characterized in that: The method is to mix tumor antigen, CCL20 and RADA16-Ⅰ polypeptide to obtain tumor antigen gel, and then add Ca 2+ The solution is obtained by extruding through a filter membrane; the mass ratio of the tumor antigen, CCL20 and RADA16-Ⅰ polypeptide is 175-225:1:110-140.

2. The immune-enhancing DC-recruiting tumor nanovaccine according to claim 1, characterized in that The mass ratio of the tumor antigen, CCL20 and RADA16-I polypeptide is 190-210:1:120-130, preferably, the mass ratio of the tumor antigen, CCL20 and RADA16-I polypeptide is 195-205:1:124-126; And / or, the tumor antigen is prepared by the following method: culturing tumor cells until the monolayer cell sheet reaches 75% to 85% confluence, collecting the adherent tumor cells, and lysing them to obtain the tumor antigen; preferably, lysis is performed using a repeated freeze-thaw method, and the parameters of the repeated freeze-thaw method are: repeated freeze-thaw cycles at -82°C to -78°C and 36°C to 38°C for 4 to 5 times.

3. The immune-enhancing DC-recruiting tumor nanovaccine according to claim 1, characterized in that The tumor antigen is an apoptotic tumor antigen, a ferroptosis tumor antigen, or a necrosis tumor antigen, preferably an apoptotic tumor antigen.

4. The immune-enhancing DC-recruiting tumor nanovaccine according to claim 3, characterized in that The mass ratio of the tumor antigen, CCL20 and RADA16-I polypeptide is 190-210:1:120-130, preferably, the mass ratio of the tumor antigen, CCL20 and RADA16-I polypeptide is 195-205:1:124-126; And / or, the tumor antigen is prepared by the following method: when the monolayer cell sheet reaches 75% to 85% confluence, continue culturing for 22 hours to 26 hours using complete culture medium containing a cell death inducer, then collect the adherent tumor cells and lyse them to obtain the tumor antigen; preferably, lysis is performed using a repeated freeze-thaw method, and the parameters of the repeated freeze-thaw method are: repeated freeze-thaw cycles at -82°C to -78°C and 36°C to 38°C for 4 to 5 times.

5. The immune-enhancing DC-recruiting tumor nanovaccine according to claim 4, characterized in that: The cell death inducer is doxorubicin, cis-diaminedichloroplatinum (II) or citronellol, preferably doxorubicin; The final concentration of doxorubicin in the complete culture medium is 1.4 μM to 1.6 μM, preferably 1.45 μM to 1.55 μM; The final concentration of cis-diamminedichloroplatinum (II) in the complete culture medium is 230 μM to 250 μM, preferably 235 μM to 245 μM; The final concentration of citronellol in the complete culture medium is 1150 μM to 1250 μM, preferably 1180 μM to 1220 μM.

6. The immune-enhancing DC-recruiting tumor nanovaccine according to any one of claims 1 to 5, characterized in that: The Ca 2+ The solution is a 14.5mM to 15.5mM CaCl2 solution, wherein the tumor antigen gel and Ca 2+ The volume ratio of the solution is 0.8 to 1.2:1; preferably, the tumor antigen gel and Ca 2+ The volume ratio of the solution is 0.9 to 1.1:

1. More preferably, the tumor antigen gel and Ca 2+ The volume ratio of the solution is 0.95~1.05:1 。 7. A method for preparing an immune-enhancing DC-recruiting tumor nanovaccine, characterized in that: The following steps are involved: (1) Tumor antigen, CCL20, and RADA16-I polypeptide were added to ultrapure water to concentrations of 7 mg / mL to 9 mg / mL, 30 μg / mL to 60 μg / mL, and 4.4 mg / mL to 5.6 mg / mL, respectively, and placed at 3°C to 5°C for 10 to 14 hours to obtain a tumor antigen gel; (2) Add tumor antigen gel to Ca 2+ The solution is squeezed out through a filter membrane to obtain the product.

8. The method for preparing the immune-enhancing DC-recruiting tumor nanovaccine according to claim 7, characterized in that: The pore size of the filter membrane in step (2) is 0.22 μm.

9. Use of the immune-enhancing DC-recruiting tumor nanovaccine according to any one of claims 1 to 6 in the preparation of tumor immunotherapy drugs.

10. The use according to claim 9, characterized in that The tumor is lung cancer, breast cancer, colorectal cancer or lymphoma.

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