CN115300617A - Multivalent aptamer modified tumor vaccine loaded with STING agonist and preparation method and application thereof - Google Patents

Abstract

The invention discloses a multivalent aptamer-modified tumor vaccine loaded with a STING agonist. The tumor vaccine is loaded with a STING agonist and modified with a multivalent aptamer. The preparation method of the tumor vaccine: incubate the model cells with the STING agonist for 48 hours to obtain the model cells loaded with the STING agonist; connect the linear DNA template with the model cells loaded with the STING agonist obtained in the first step for 1 hour ; Amplification in RCA reaction buffer; after ultrasonication, after centrifugation, a multivalent aptamer-modified tumor vaccine loaded with STING agonist was obtained. Tumor vaccines are used in anti-tumor immunotherapy. In the present invention, the tumor vaccine is prepared by performing the RCA rolling circle amplification process on the surface of the tumor cell membrane for the first time; the STING agonist is used in advance to activate the STING pathway in the tumor cell for the first time, and then the extracted tumor vaccine can be loaded with the STING agonist to effectively activate the antigen presenting cells; First use of GMP‑DCapt‑TV as a tumor vaccine to treat tumors.

Description

A multivalent aptamer-modified tumor vaccine loaded with STING agonist and its Preparation method and application

technical field

The invention relates to the technical field of myocardial infarction, in particular to a multivalent aptamer-modified tumor vaccine loaded with a STING agonist and its preparation method, use and application.

Background technique

Cancer immunotherapy against cancer by modulating the patient's own immune system holds great promise for clinical cancer treatment. In immunotherapy, cancer vaccines that activate a patient's adaptive immune system against specific tumor antigens to control tumor growth have received increasing attention. However, even cancers of the same type can exhibit significant genetic heterogeneity among patients, so conventional cancer vaccines that only possess single antigens (such as tumor-specific proteins, peptides, and deoxyribonucleic acid DNA fragments) cannot induce Strong immune response.

Whole tumor cell lysates (WTCLs) can provide a comprehensive collection of patient-specific tumor-associated antigens (TAAs), which can elicit stronger immunity against the immune system against a broad spectrum of tumor-associated antigens. WTCL has been used as a tumor antigen for cancer vaccine development. However, the ability of tumor cells to activate antigen-presenting cells (APCs) is tightly controlled to evade immune surveillance systems through various mechanisms, thereby limiting the efficacy of potential WTCL-based tumor vaccines. Therefore, modification of naive tumor cells to improve their immunogenicity and APCs sensitivity is expected to enhance the antitumor immune effect of WTCL-based tumor vaccines.

The most similar solution to the present invention is to directly modify tumor cell membranes with cholesterol-modified monovalent aptamers, thereby preparing tumor vaccines. However, the aptamers prepared by this method are monovalent, and the binding efficiency to the target is very low. Our method enables the preparation of functional nucleic acids on the cell surface in a multivalent state that enhances binding to the target. In addition, we have also loaded STING agonists in tumor vaccines, which can activate antigen-presenting cells better, while the previous schemes did not have relevant designs. The monovalent form of the aptamer used in the existing technology has low targeting to DC cells, which affects the effect. In addition, DC cells cannot be effectively activated, which limits the antigen presentation process. The present invention performs RCA reaction on tumor cells for the first time, thereby endowing tumor vaccines with multivalent functional nucleic acids, and at the same time uses STING agonists to activate tumor cells, so that the subsequently derived tumor vaccines can better activate antigen-presenting cells and better Activate the immune system in the body. Therefore, it is necessary to design a multivalent aptamer-modified tumor vaccine loaded with a STING agonist and its preparation method and application.

Contents of the invention

In order to overcome the defects in the prior art, a multivalent aptamer-modified tumor vaccine loaded with a STING agonist and its preparation method and use are provided.

The present invention realizes by following scheme:

A multivalent aptamer-modified tumor vaccine loaded with a STING agonist. The tumor vaccine is loaded with a STING agonist and modified with a multivalent aptamer.

A method for preparing a multivalent aptamer-modified tumor vaccine loaded with a STING agonist, the method for preparing the tumor vaccine comprises the following steps:

The first step is to incubate the model cells with the STING agonist for 48 hours to obtain the model cells loaded with the STING agonist;

In the second step, at room temperature, the linear DNA template is connected to the model cell loaded with the STING agonist obtained in the first step for 1 hour, and the linear DNA template is complementary to the DNA sequence of the multivalent aptamer;

In the third step, the product obtained in the second step is put into the RCA reaction buffer for amplification;

In the fourth step, the product obtained in the third step is sonicated and centrifuged to obtain a multivalent aptamer-modified tumor vaccine loaded with a STING agonist.

In the first step, the model cells are B16-OVA cells.

In the first step, the STING agonist is C-di-GMP.

In the second step, the circular closure of the linear DNA template is performed by ligation of the annealed template and primers by T4 DNA ligase.

In the second step, the 5' end of the primer was labeled with cholesterol and the 3' end of the primer was labeled with Cy5.

In the third step, the RCA reaction buffer contains DNTP, phi29DNApolymerase and reaction buffer.

The amplification was carried out at 30°C for 4 hours.

In the fourth step, the centrifugation is a multi-step density gradient ultracentrifugation.

A multivalent aptamer-modified tumor vaccine loaded with STING agonist for anti-tumor immunotherapy.

The beneficial effects of the present invention are:

1. Whole tumor cell lysate (WTCL)-based tumor vaccines contain comprehensive patient-specific tumor-associated antigens (TAAs), which are expected to be used in anti-tumor immunotherapy. However, low immunogenicity and low targeting to DC cells severely limit its therapeutic efficacy. Herein, a multivalent aptamer-modified STING agonist-loaded tumor vaccine was developed from WTCL of highly immunogenic tumor cells to develop a multivalent functional DNA engineered tumor vaccine for anti-tumor immunotherapy . Efficient cytoplasmic delivery of the STING agonist c-di-GMP triggers tumor cell-intrinsic STING signaling and increases tumor cell immunogenicity, thereby enhancing dendritic cell (DC) antigen presentation responses to derived tumor vaccines .

2. Furthermore, we first designed multivalent DCs-targeting aptamers through an enzymatic amplification process on the surface of tumor cells, endowing the derived tumor vaccine with the ability to target DCs. In vivo, a multivalent aptamer-modified tumor vaccine GMP-DCapt-TV loaded with a STING agonist of the present invention can effectively accumulate in lymph nodes to promote DC uptake and activate the STING pathway to mediate DC maturation and promote T cells primed. A multivalent aptamer-modified tumor vaccine GMP-DCapt-TV loaded with a STING agonist of the present invention has significant preventive and inhibitory effects on tumor growth and the occurrence of B16-OVA melanoma.

3. In addition, in the B16-OVA melanoma lung metastasis model, the combination of tumor GMP-DCapt-TV with anti-PD-L1 antibody further showed improved anti-tumor efficacy.

Description of drawings

Fig. 1 is the schematic diagram of the preparation process of the tumor vaccine of the present application;

Figure 2 is a schematic diagram of GMP-DCapt-TV targeting DC cells and lymph nodes in vivo and in vitro;

Figure 3 is a representative flow cytometry data and statistical data graph;

Figure 4 is a schematic diagram of the preventive effect of GMP-DCapt-TV on B16-OVA melanoma;

Figure 5 is a diagram of the preparation and characterization of GMP-Dapt-TV.

Detailed ways

Preferred embodiments of the present invention are further described below:

In this application: C-di-GMP is the abbreviation of cyclic diguanylic acid;

DCapt or DC-Aptamer is the abbreviation of multivalent aptamer;

chol-CT/P is the abbreviation of linear DNA template;

phi29 DNA polymerase is the abbreviation of phi29 DNA polymerase;

DNTP is the abbreviation of deoxyribonucleoside triphosphate;

GMP-DCapt-TV is the abbreviation of a multivalent aptamer-modified tumor vaccine loaded with a STING agonist of the present invention; there are also a large number of other English abbreviations appearing in the specification, rights and drawings of this application. The meanings are uniquely determined by those skilled in the art, and will not be repeated here.

Activation of the cGAS-STING sensing pathway can enhance and direct adaptive immune responses to vaccine antigens (e.g. dendritic cell maturation, antitumor macrophage polarization, T cell priming and activation, natural killer cell activation). In addition to immune cells, recent reports suggest that defective STING signaling helps tumor cells escape immune surveillance. Therefore, dying tumor cells exhibit higher immunogenicity if pretreated with STING agonists, which can induce robust APC antigen presentation and antitumor immune responses. Inspired by this, the applicant pretreated tumor cells with STING agonists, and then prepared WTCL-based tumor vaccines. Such a vaccine could exhibit higher immunogenicity and better activate the cGAS-STING sensing pathway in APCs to enhance antigen presentation; to the best of our knowledge, this strategy has not been applied in tumor vaccine development.

Cell engineering modification based on functional nucleic acid (DNA) has the advantages of simple synthesis, convenient modification, high programmability, and good biocompatibility, and has been widely used to endow cells with functions, such as aptamers, deoxyribonucleases, etc. , molecular beacons, etc. However, the monovalent form of conventional functional nucleic acids results in weaker affinity and is easily degraded by nucleases, resulting in poor bioavailability. Rolling circle amplification (RCA) is a method for the isothermal amplification of circular DNA molecules catalyzed by phi29 polymerase, and has been widely used in the synthesis of multivalent functional nucleic acids. We expect to perform RCA reactions on the cell membrane surface to engineer tumor cells, thereby endowing tumor vesicles with multivalent DC-SIGN targeting aptamers to improve DC targeting. However, performing RCA on the cell membrane surface has not been reported and is very challenging.

In the technical proposal of the application, we prepared a novel tumor vaccine (GMP-DCapt-TV) from whole tumor cell lysates, which can target DCs (dendritic cells) and activate the STING pathway in DCs, Personalized immunotherapy for cancer and demonstrated ability to elicit powerful T-cell responses with potent therapeutic effects. By pre-incubation with the STING agonist C-di-GMP, the immunogenicity of tumor cells was increased, and subsequently derived tumor vaccines loaded with STING activators could further activate the STING signaling pathway in DCs. In addition, we used the RCA reaction on the surface of tumor cells to target multivalent DC-SIGN to aptamer-labeled tumor vaccines to enhance the targeting of DCs and improve the efficiency of antigen presentation. In vivo, this tumor vaccine triggered robust antigen cross-presentation and elicited high levels of antitumor T cell responses, thereby effectively suppressing established B16-OVA tumors. Furthermore, the combination of tumor GMP-DCapt-TV with anti-PD-L1 antibody further showed improved antitumor efficacy in the B16-OVA melanoma lung metastasis model. Through enhanced immunogenicity and DNA engineering of primary tumor cells, the anti-tumor immunity of WTCL-derived tumor vaccines was significantly enhanced, providing a new strategy for the development of tumor vaccines.

A multivalent aptamer-modified tumor vaccine loaded with a STING agonist. The tumor vaccine is loaded with a STING agonist and modified with a multivalent aptamer.

As shown in Figure 1, a method for preparing a multivalent aptamer-modified tumor vaccine loaded with a STING agonist, the method for preparing the tumor vaccine comprises the following steps:

The first step is to incubate the model cells with the STING agonist for 48 hours to obtain the model cells loaded with the STING agonist;

In the second step, at room temperature, the linear DNA template is connected to the model cell loaded with the STING agonist obtained in the first step for 1 hour, and the linear DNA template is complementary to the DNA sequence of the multivalent aptamer; in order to produce STING For the agonist-loaded DC-targeted tumor vaccine (GMP-DCapt-TV), we first designed a linear DNA template with a DNA sequence complementary to DCapt. The specific preparation method of the linear DNA template is a well-known technology and will not be repeated here.

In the third step, the product obtained in the second step is put into the RCA reaction buffer for amplification; in practical application, in order to verify the successful RCA reaction on the surface of the model cell B16-OVA cell, FITC-labeled DCapt complementary DNA sequence was added In order to track the available multivalent DCapt, the confocal results showed that the multivalent DCapt was successfully attached to the surface of the cell membrane, and details will not be repeated here.

In the fourth step, the product obtained in the third step is sonicated and centrifuged to obtain a multivalent aptamer-modified tumor vaccine loaded with a STING agonist.

As determined by BCA protein, 108 B16-OVA tumor cells can obtain 21.62±5.21 mg of membrane protein tumor vaccine. Transmission electron microscopy (TEM) images showed that GMP-DCapt-TV had nanoscale vesicle morphology with an average diameter of about 200 nm. Meanwhile, as measured by dynamic light scattering (DLS), the hydrodynamic particle size of the GMP-DCapt-TV nanovaccine was 210 ± 7.5 nm, and the polydispersity index (PDI) was 0.22 ± 0.03, slightly larger than that of GM-TV due to its multivalent adaptability. the existence of the body. It maintains relatively good colloidal stability for 7 days in PBS at 4°C, and maintains relatively good colloidal stability for 24 hours in fetal bovine serum (FBS) at 37°C, showing very good stability.

In the first step, the model cells are B16-OVA cells.

In the first step, the STING agonist is C-di-GMP.

In the second step, the circular closure of the linear DNA template is performed by ligation of the annealed template and primers by T4 DNA ligase.

In the second step, the 5' end of the primer was labeled with cholesterol and the 3' end of the primer was labeled with Cy5. It is worth noting that the 5' ends of the primers in this application are labeled with cholesterol to facilitate embedding into the cell membrane of the model cells. The confocal results showed that the Cy5-labeled chol-CT/P could attach to the cell membrane of the model cell B16-OVA, and details will not be repeated here.

In the third step, the RCA reaction buffer contains DNTP, phi29DNA polymerase and reaction buffer.

The amplification was carried out at 30°C for 4 hours.

In the fourth step, the centrifugation is a multi-step density gradient ultracentrifugation. Total proteins of GMP-DCapt-TV and other tumor vaccine preparations were separated and protein profiles identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that all the synthesized tumor vaccines well retained tumor membrane proteins similar to those of the TV group, indicating the stability of membrane proteins during preparation. Furthermore, using WB assay, a representative OVA antigen of B16-OVA was identified to be present on the surface of all tumor vaccines detected by WB assay. Unlike endogenous tumor-derived exosomes, no significant expression of PDL1, which can inhibit T cell proliferation and cytokine secretion by binding to PD-1, was observed in all tumor vaccine formulations due to the lack of immunosuppressive tumor microbiota. Environmental stimulus B16-OVA upregulates PDL1.

Figure 5 is a diagram of the preparation and characterization of GMP-Dapt-TV, wherein Figure 5(a) is a schematic diagram and a scanning confocal microscope image showing that cy5-labeled Chol-CT/P is coupled to the cell membrane of B16-OVA cells. 500nM of each conjugate was incubated with 5×105B16 cells for 1h at room temperature, and the unbound conjugate was removed by centrifugation. Figure 5(b) Schematic and scanning confocal microscope images showing the RCA reaction on the surface of the B16 cell membrane tracked by FITC-labeled DCapt complementary strands. Figure 5(c) is the TEM imaging morphology of GMP-DCapt-TV under the ratio scale of 200nm. Figure 5(d) shows the DLS results showing the particle size variation of different vaccine formulations. Fig. 5(e) SDS-PAGE detection of protein content in different tumor vaccine preparations. In the figure (1) Marker; (2) TV; (3) DC-TV; (4) GMP-TV; (5) GMP-DC-TV.

A multivalent aptamer-modified tumor vaccine loaded with STING agonist for anti-tumor immunotherapy.

Below in conjunction with specific experimental data this application is further elaborated:

(1) GMP-Dapt-TV showed good targeting to DC cells

Dendritic cells (DCs) are specialized antigen-presenting cells that play key roles in the activation and regulation of innate and adaptive immune responses. Therefore, it is very important to modulate DC function to improve cancer immunotherapy, and one of the strategies is to improve the targeting of vaccines to DCs. Based on the described features, we next evaluated the DCs-targeting ability of tumor vaccines mediated by multivalent aptamers that interact with DC-specific intercellular adhesion molecule (ICAM)-3 non-integrin ( DC-SIGN) with high binding efficiency. Immature bone marrow-derived dendritic cells (BMDCs) were co-cultured with DiO-labeled GMP-DCapt-TV and GMP-TV for 4 hours, and endo/lysosomes were stained with LysoTracker Red. Confocal laser scanning microscopy imaging showed that the GMP-DCapt-TV group showed stronger DiO fluorescence compared with the GMP-TV group, indicating that GMP-DCapt-TV could selectively target BMDCs. Consistent results were observed in flow cytometry results. Complete contact with DCs is a prerequisite for successful antigen presentation in vivo, which requires efficient targeting of GMP-DCapt-TV to LNs. We labeled GMP-DCapt-TV and GMP-TV with a near-infrared optical film dye (DiR) and quantified the biodistribution after subcutaneous (s.c.) injection at the tail base of C57BL/6 mice. In vivo near-infrared fluorescence imaging results showed that GMP-Dapt-TV efficiently accumulated in peripheral lymph nodes within 4 hours and could still be observed even at 48 hours, while the GMP-TV group did not show significant fluorescence signals in peripheral lymph nodes. Subsequently, mice were sacrificed 48 hours after injection, and inguinal and axillary lymph nodes were harvested for ex vivo imaging. Consistent with the in vivo imaging results, fluorescence imaging of ex vivo LNs revealed ~3-fold accumulation of LNs in GMP-DCapt-TV compared with GMP-TV. Then, the lymph nodes were further sectioned and stained for confocal imaging. Higher lymph node retention was observed for DiR-labeled GMP-DCapt-TV compared to GMP-TV). Thanks to its nanoscale size, good stability, and especially the multivalent aptamer, GMP-DCapt-TV can more effectively target LNs and achieve better contacts with DCs, thereby promoting immune stimulation in vivo. See Figure 2 for details, Figure 2 shows the targeting of DC cells and lymph nodes in vivo and in vitro by GMP-DCapt-TV, in which Figure 2(a) treats BMDC cells with dio-labeled GMP-TV and GMP-DCapt-TV (green) for 2 hours Post confocal fluorescence images. Nuclei were stained with Hoechst (blue) and lysosomes with Lysotracker (red). Scale bar = 25 μm. Figure 2(b) panel a DiO fluorescence quantification assay. Figure 2(c-d) Fluorescence intensity and statistical data of CD11c+BMDCs measured by flow cytometry after DiO-labeled GMP-TV and GMP-DCapt-TV were incubated for 2 h.

(2) GMP-Dapt-TV activates DCs

To assess whether c-di-GMP loading in GMP-DCapt-TV could activate the STING pathway and type I IFN production in APCs, we incubated BMDCs with different tumor vaccine formulations for 24 h. Western blot analysis showed that the c-di-GMP-loaded GMP-DCapt-TV and GMP-TV groups had higher levels of STING and phosphorylated TBK1 than the GMP-TV group, indicating activation of the STING pathway. The STING activation profile of GMP-DCapt-TV was measured by detecting the messenger RNA (mRNA) transcript levels of STING-related genes in BMDCs by real-time polymerase chain reaction (RT-PCR). Compared with GMP-TV, GMP-DCapt-TV induced a sharp increase in the expression of IFN-β and CXCL10, while no increase was observed in other groups. Consistent with the PCR results, enzyme-linked immunosorbent assay (ELISA) measurements showed a significant increase in IFN-β in the culture medium of BDMCs treated with GMP-DCapt-TV.

Maturation of DCs is essential for antigen presentation and subsequent initiation of immune responses. The maturation level of BMDCs was assessed by detecting the upregulation of CD80 and CD86 using flow cytometry. Compared with other tumor vaccines, BMDCs treated with GMP-DCapt-TV showed the most up-regulated expression of co-stimulatory factor markers CD80 and CD86, indicating that tumor vaccines can activate DCs, while DCapt and c-di-GMP contributed to higher DC activates. The antigen presentation efficacy of BMDCs was then assessed by detecting the frequency of OVA(SIINFEKL)-H2Kb+ complexes on the DC membrane surface. GMP-Dapt-TV treatment induced a higher proportion of OVA-H2Kb-positive DCs compared to the other groups due to the vaccine-promoted OVA cytosolic release for antigen processing and OVA/major histocompatibility complex presentation I (MHC I).

See Figure 3 for details, Figure 3 is a representative flow cytometry data and statistical data graph, wherein Figure 3(a) and Figure 3(b) are representative flow cytometry data and statistical data respectively , to show the maturation of BMDCs induced by different tumor vaccines. Figure 3(c) Priming of unactivated OVA-CD8+ T cells in vitro. BMDCs treated with different tumor vaccines were co-incubated with CFSE-labeled OVA-TCR transgenic CD8+ T cells.

(3) GMP-Dapt-TV inhibits tumor growth in vivo

The therapeutic effect of the GMP-DCapt-TV tumor vaccine was evaluated using a B16-OVA melanoma model established by subcutaneously injecting 2×105 B16-OVA tumor cells on the right side of each mouse. After the tumor volume reached 50 mm, the tumor ^- bearing mice were injected with various tumor vaccine preparations (TV, DCapt-TV, GMP-TV, and GMP-DCapt-TV) once a week for a total of 3 times, and the tumor volume was monitored every 2 sky. The amount of cell membrane depends on the amount of protein measured by BCA. Tumor progression was slightly inhibited in mice vaccinated with DC-TV, GMP-TV, and GMP-DCapt-TV compared to PBS-injected control and TV-only groups. It is worth noting that the GMP-DCapt-TV treatment group showed the best therapeutic effect, and the survival rate of mice was significantly prolonged. In addition, all tumor vaccine treatments had no significant effect on mouse body weight and major organs detected by hematoxylin-eosin (H&E) staining, indicating that the tumor vaccines had good biological safety. At the end of the antitumor study, terminal deoxynucleotidyl transferase dUTP end-labeling (TUNEL) staining of tumor sections further revealed significant apoptosis and necrosis of tumor cells in the GMP-DCapt-TV group, a mechanism that may contribute to As the tumor regresses. vaccine. Treatment-induced intratumoral infiltration of T lymphocytes was then measured. The frequencies of CD8+T cells (CD3+CD8+) and CD4+T cells (CD3+CD4+) in TV group were 19.6% and 44.9%, respectively. In contrast, vaccination with GMP-DCapt-TV resulted in a 1.8-fold increase in CD8+ T cells and a 2.4-fold decrease in CD4+ T cells, respectively. Likewise, the frequency of CD8+IFN-γ+TNF-α+ effector T cells was increased in the GMP-DCapt-TV group, which was 1.9-fold higher than that in the TV group, indicating the activation of the antitumor immune response. Infiltration of regulatory T cells (Treg) within tumors suppresses the antitumor activity of CTLs and induces an immunosuppressive tumor microenvironment (ITM). The frequency of intratumoral Treg (CD3+CD4+Foxp3+) decreased after GMP-DCapt-TV treatment. Fig. 4 is a schematic diagram of the preventive effect of GMP-DCapt-TV on B16-OVA melanoma, wherein Fig. 4(a) is the preventive immunization scheme for B16-OVA tumors. Figure 4(b) shows the preventive effect of GMP-DCapt-TV on B16-OVA tumors. Figure 4(c) shows the survival time of mice in b16-ova mouse model after immunization. Figure 4(d) is the growth curve of a single tumor in group b.

The present invention is the first to prepare a tumor vaccine by performing RCA rolling circle amplification process on the surface of the tumor cell membrane; for the first time, the STING agonist is used to activate the STING pathway in the tumor cell in advance, and then the extracted tumor vaccine can be loaded with the STING agonist to effectively activate the antigen-presenting cells; For the first time, GMP-DCapt-TV was used as a tumor vaccine to treat tumors. Compared with the previous strategy, this application is the first to prepare multivalent functional nucleic acid by performing RCA rolling circle amplification reaction on the surface of tumor cells, and prepare GMP-DCapt-TV by endowing STING agonist in advance for the development of tumor vaccines. Compared with the prior art, the tumor vaccine prepared by this method can enhance the targeting of DC cells, and can better activate the antigen presentation ability of DC cells.

Although the technical solutions of the present invention have been elaborated and listed in detail, it should be understood that for those skilled in the art, it is necessary for those skilled in the art to make modifications to the above-mentioned embodiments or adopt equivalent alternatives. Obviously, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. A multivalent aptamer-modified tumor vaccine loaded with a STING agonist, characterized in that: the tumor vaccine is loaded with a STING agonist and modified with a multivalent aptamer. 2. a kind of preparation method of the tumor vaccine that is loaded with STING agonist of multivalent aptamer modification as claimed in claim 1, it is characterized in that, the preparation method of this tumor vaccine comprises the following steps: The first step is to incubate the model cells with the STING agonist for 48 hours to obtain the model cells loaded with the STING agonist; In the second step, at room temperature, the linear DNA template is connected to the model cell loaded with the STING agonist obtained in the first step for 1 hour, and the linear DNA template is complementary to the DNA sequence of the multivalent aptamer; In the third step, the product obtained in the second step is put into the RCA reaction buffer for amplification; In the fourth step, the product obtained in the third step is sonicated and centrifuged to obtain a multivalent aptamer-modified tumor vaccine loaded with a STING agonist. 3. The method for preparing a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 2, characterized in that: in the first step, the model cells are B16-OVA cells. 4. The preparation method of a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 2, characterized in that: in the first step, the STING agonist is C-di- GMP. 5. The preparation method of a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 2, characterized in that: in the second step, the closed loop of the linear DNA template is passed through T4 DNA ligase is used to ligate the annealed template and primers. 6. The method for preparing a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 5, characterized in that: in the second step, the 5' end of the primer is labeled as Cholesterol, the 3' end of the primer is labeled Cy5. 7. The preparation method of a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 2, characterized in that: in the third step, the RCA reaction buffer contains DNTP, phi29 DNA polymerase and reaction buffer. 8 . The method for preparing a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 2 , characterized in that: the amplification is reacted at 30° C. for 4 hours. 9. The preparation method of a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to claim 2, characterized in that: in the fourth step, the centrifugation adopts multi-step density gradient overspeed centrifugal. 10. An application of a multivalent aptamer-modified tumor vaccine loaded with a STING agonist according to any one of claims 1-9, characterized in that the tumor vaccine is used in anti-tumor immunotherapy.

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