Engineering potent dendritic cell vaccine as well as preparation method and application thereof

Abstract

The invention discloses an engineered potent dendritic cell vaccine as well as a preparation method and application thereof, tumor antigen and cationic liposome are fused by utilizing a nanoscale antigen delivery mode, stability of a lysosomal membrane is destroyed by virtue of interaction of cationic lipid charges, so that part of the antigen escapes into cytoplasm and is crossly presented through an MHC I (major histocompatibility complex I) way, and the dendritic cell vaccine is prepared. Administering DC to activate the ability of cellular immune response to resist tumors; on the other hand, the DC is endowed with T cell guidance through a synthetic immunology method, firstly, the surface of the DC is modified with an azide group N3, a T cell specific antibody is modified with a dibenzocyclooctyne group DBCO, the T cell specific antibody is coupled to the surface of the mature DC through a cycloaddition reaction of the N3 and the DBCO, the chance of contact between the DC and the T cell is increased, the duration time of contact between the DC and the T cell is prolonged, and the T cell specific antibody is coupled to the surface of the mature DC through the cycloaddition reaction of the N3 and the DBCO. The interaction and signal transmission of DC-T cells are promoted; the combination of the two mechanisms can overcome the problem of low reaction rate of the existing DC vaccine, and maximization of anti-tumor immune response is realized.

Description

Technical Field

[0001] The present invention belongs to the field of dendritic cell vaccines, and particularly relates to an engineered potent dendritic cell vaccine, a preparation method thereof and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to increase some understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Malignant tumors are major public health problems threatening human health. Surgical resection is the preferred method for treating solid tumors, but recurrence and metastasis caused by residual tumor cells often lead to treatment failure. In recent years, personalized dendritic cell (DC) vaccines designed based on patient tumor information have shown great advantages in the adjuvant treatment of tumors after surgery. Autologous or allogeneic monocytes are induced to differentiate into DCs in vitro, and tumor antigens are obtained from surgically resected tumor tissues. After the DCs are loaded with antigens, they are adoptively transferred back into the body to activate T cells to specifically kill residual tumor cells. Currently, in the field of treating hematological tumors and solid tumors, hundreds of DC vaccines are in clinical trials. In particular, DCVax®-L for glioblastoma and DCVAC / OvCa for ovarian cancer have entered the phase III clinical stage. Although DC vaccines have been proven effective in the adjuvant treatment of tumors after surgery, the overall objective response rate rarely exceeds 15%, and clinical application still has quite limitations.

[0004] Existing DC vaccines only simply co-incubate immature DCs with tumor antigens to obtain mature DCs, lacking refined regulation of the antigen presentation process and DC function, resulting in poor efficacy. The treatment bottleneck mainly stems from two interrelated mechanism defects: the insufficient ability of DCs to cross-present antigens through the major histocompatibility complex (MHC) class I pathway, which causes antigens to be degraded in lysosomes and weakens the ability to activate cellular immune responses. At the same time, DCs lack the opportunity and duration of contact with T cells during the antigen presentation process, which weakens the interaction and signal transmission between DCs and T cells and limits subsequent T cell activation. Therefore, deeply exploring the action mechanism and limiting factors of DC vaccines is of great significance for improving the treatment effect of DC vaccines. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention utilizes the method of nano-scale antigen delivery to fuse tumor antigens with cationic liposomes. By means of the charge interaction of cationic lipids, the stability of the lysosomal membrane is disrupted, enabling some antigens to escape into the cytoplasm and be cross-presented through the MHC I pathway, endowing DCs with the ability to activate cellular immune responses against tumors. On the other hand, by means of synthetic immunology, DCs are endowed with T cell tropism. First, azide groups N3 are modified on the surface of DCs, and dibenzocyclooctyne groups DBCO are modified on T cell-specific antibodies. Through the cycloaddition reaction of N3 and DBCO, the T cell-specific antibodies are conjugated to the surface of mature DCs, increasing the opportunity and duration of contact between DCs and T cells, and promoting the interaction and signal transmission between DCs and T cells. The combination of the two mechanisms can overcome the problem of low response rate of existing DC vaccines and maximize the anti-tumor immune response.

[0006] The technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a method for preparing an engineered potent dendritic cell vaccine is provided, and the method comprises the following steps: (1) Preparation of tumor antigen nanoparticles; (2) Preparation of azide nanoparticles: Cationic liposomes are used to encapsulate a metabolic glycoprotein labeling reagent containing azide groups to obtain azide nanoparticles; (3) Preparation of fusion nanoparticles: The antigen nanoparticles in step (1) and the azide nanoparticles in step (2) are co-extruded to obtain fusion nanoparticles; (4) Preparation of mature DCs: The fusion nanoparticles in step (3) are co-incubated with DCs to obtain mature DCs activated by the fusion nanoparticles, and the DCs have N3 groups on their surfaces; (5) Preparation of a potent DC vaccine: The mature DCs in step (4) are co-incubated with T cell-specific antibodies modified with DBCO, and through the azide-alkyne cycloaddition reaction, the T cell-specific antibodies are conjugated to the surface of the mature DCs to obtain a potent DC vaccine.

[0007] In step (1) of the present invention, the preparation of tumor antigen nanoparticles can adopt the prior art. The types and treatment methods of tumor antigens are not limited, and can be tumor whole cell antigens, tumor-specific antigens, tumor-associated antigens, differentiation antigens, embryonic antigens, etc. If it is a tumor whole cell antigen, it can be obtained by various treatment methods such as freeze-thaw cycles, ultraviolet radiation or photothermal ablation of tumor cells.

[0008] In one or some embodiments of the present invention, in step (1), the method for preparing the tumor antigen nanoparticles comprises the following steps: Monodisperse tumor cells are obtained from tumor tissues, centrifuged to collect the supernatant after freeze-thaw cycles, and tumor antigen nanoparticles are obtained after extrusion by a liposome extruder.

[0009] In step (2) of the present invention, in the cationic liposome, the cationic lipid is one of dioleoyltrimethylammonium propane (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), didodecyldimethylammonium bromide (DODAB), cholesterol derivative cationic lipid (DC-Chol), etc. In addition, it also includes neutral co-lipid dioleoylphosphatidylethanolamine (DOPE) and cholesterol. The metabolic glycoprotein labeling reagent containing an azide group is one of tetraacetylated N-azidoacetylmannosamine (Ac4ManNAz), tetraacetylated N-azidoacetylgalactosamine (Ac4GalNAz), N-azidoacetylglucosamine (GlcNAz), azide-modified fucose analog (FucAz), 6-azidosialic acid, etc.

[0010] In one or some embodiments of the present invention, in step (2), the cationic liposome is a mixture of DOTAP, DOPE, and cholesterol, and the metabolic glycoprotein labeling reagent containing an azide group is tetraacetylated N-azidoacetylmannosamine (Ac4ManNAz). Preferably, the mass ratio of DOTAP, DOPE, cholesterol, and Ac4ManNAz is (5-8):(15-20):(1-5):(0.5-2).

[0011] Specifically, the preparation method of azide nanoparticles includes the following steps: dissolving a mixture containing a cationic lipid, DOPE, cholesterol, and a metabolic glycoprotein labeling reagent containing an azide group in an organic solvent, and evaporating under reduced pressure to form a lipid film; hydrating the dried film with PBS, and then incubating to obtain azide nanoparticles.

[0012] In step (3) of the present invention, the mass ratio of the tumor antigen nanoparticles to the azide nanoparticles is (1-5):(1-5).

[0013] In one or some embodiments of the present invention, in step (3), specifically, the preparation method of the fusion nanoparticles includes the following steps: sequentially passing a mixture of antigen nanoparticles and azide nanoparticles through polycarbonate membranes with pore sizes of 400 nm and 200 nm using a liposome extruder to co-extrude and prepare the fusion nanoparticles.

[0014] In step (4) of the present invention, the ratio of the fusion nanoparticles to DC is (1-50) μg: 2×10 5 cells.

[0015] In one or some embodiments of the present invention, in step (4), the fusion nanoparticles and DC are co-incubated for 18-36 h.

[0016] In one or some embodiments of the present invention, in step (5), the T cell-specific antibody is one of anti-CD3 monoclonal antibody, anti-CD4 monoclonal antibody, anti-CD8 monoclonal antibody, and anti-PD-1 monoclonal antibody.

[0017] In step (5) of the present invention, the ratio of dibenzocyclooctyne-T cell-specific antibody to mature DC is (0.1~1) μg: 1×10 6 cells.

[0018] In one or some embodiments of the present invention, in step (5), the mature DC and the DBCO-T cell-specific antibody are co-incubated for (1~4) h.

[0019] In the second aspect of the present invention, there is provided an engineered potent dendritic cell vaccine prepared by the above method.

[0020] In the third aspect of the present invention, there is provided the application of the engineered potent dendritic cell vaccine in the preparation of anti-tumor drugs.

[0021] Compared with the related technologies known to the inventors of the present invention, one of the technical solutions of the present invention has the following beneficial effects: (1) The present invention combines the cross-presentation of tumor antigens in DCs with the DC-T cell contact mediated by T cell-specific antibodies to obtain a potent DC vaccine, which can improve the anti-tumor immune response induced by DCs, enhance the tumor-killing effect mediated by CD8+ T cells, and reduce the immune tolerance caused by Tregs cells.

[0022] (2) The present invention significantly reduces the risk of tumor recurrence after surgery and prolongs the survival period. Compared with the traditional DC vaccine, the tumor inhibition rate is increased by 41.4%.

[0023] (3) The potent DC vaccine obtained by the present invention has good activity. On the basis of fine regulation, its effectiveness is significantly increased, and the cell viability is comparable to that of the traditional DC vaccine obtained by the existing classical preparation process.

[0024] (4) The drugs and materials involved in the present invention have good safety. The ratio and concentration are data optimized for DC cells, which are safe, non-toxic, biodegradable, and have good biocompatibility.

[0025] (5) The fusion nanoparticles prepared by the present invention have an appropriate particle size (about 200 nm), stable process, are simple and feasible, and are easy to industrialize. Description of the Drawings

[0026] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0027] Figure 1 : Flow chart for the preparation of the present invention.

[0028] Figure 2 : Electron micrographs and particle size distributions of antigen nanoparticles, labeled nanoparticles, and fusion nanoparticles.

[0029] Figure 3 : Triple signals of mature DCs: antigen presentation (MHC I), costimulatory molecules (CD80 / CD86), cytokines (TNF-α, IL-6).

[0030] Figure 4 : Characterization of the conjugation of anti-CD3 antibody on the surface of mature DCs by flow cytometry.

[0031] Figure 5 : Characterization of the ability of the potent DC vaccine to accumulate in lymph nodes.

[0032] Figure 6 : Characterization of the ability of the potent DC vaccine to activate T cells.

[0033] Figure 7 : The potent DC vaccine inhibits postoperative tumor recurrence and prolongs the survival of mice. Detailed Description of the Invention

[0034] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, and / or combinations thereof.

[0036] The present invention designed a DC engineering platform to synergistically combine enhanced antigen cross-presentation and DC-T cell contact mediated by T cell-specific antibodies, with the aim of establishing a next-generation potent DC vaccine with enhanced immunotherapeutic efficacy.

[0037] First, tumor antigens are co-extruded with cationic liposomes to form fusion nanoparticles. These fusion nanoparticles are internalized by DCs into lysosomes, where the cationic lipids disrupt the stability of the lysosomal membrane through charge interactions, allowing some antigens to escape into the cytoplasm and be cross-presented via the MHC I pathway, endowing DCs with the ability to activate cellular immune responses against tumors. A metabolic glycoprotein labeling reagent containing an azide group is co-encapsulated in the above-mentioned cationic liposomes, and the azide group is metabolically conjugated to the DC surface glycan during antigen processing. Subsequently, a T cell-specific antibody is modified with dibenzocyclooctyl, and site-specific antibody conjugation is achieved through the cycloaddition reaction of azide and alkyne, that is, the T cell-specific antibody is linked to the DC surface.

[0038] After subcutaneous administration at the inguinal region of mice, the engineered DC vaccine migrates to the lymph nodes, where it enhances MHC I pathway-mediated antigen cross-presentation and antibody-mediated DC-T cell interactions, synergistically activating tumor-specific cellular immune responses and ultimately triggering an effective anti-tumor immune response. Generally speaking, the present invention establishes a new strategy for preparing DC vaccines, combining nanoscale antigen delivery with synthetic immunology methods, providing a translational platform for overcoming the limitations of current cancer immunotherapy.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific examples.

[0040] Example 1 In this example, a drug delivery system and drug combination for obtaining a potent DC vaccine were designed and prepared. First, the tumor antigen was processed. The tumor antigen was obtained from the excised tumor tissue, and the supernatant was collected by centrifugation after freeze-thaw cycles and then extruded through a liposome extruder to obtain nano-antigens. Cationic lipids were used to encapsulate the metabolic glycoprotein labeling reagent Ac4ManNAz to obtain azide nanoparticles. The antigen nanoparticles and azide nanoparticles were co-extruded through a liposome extruder to obtain fusion nanoparticles. Subsequently, the DCs were functionalized. After co-incubation of the above fusion nanoparticles with DCs, the DCs could be stimulated to mature, and the stability of the lysosomal membrane was disrupted by the cationic lipids, allowing some antigens to escape into the cytoplasm and be cross-presented via the MHC I pathway. At the same time, the azide group was metabolically conjugated to the DC surface during antigen processing. Subsequently, the obtained DC-N3 was co-incubated with the DBCO-CD3 antibody, and the CD3 antibody was conjugated to the DC surface through a cycloaddition reaction to obtain a potent DC vaccine.

[0041] The specific preparation method is as follows, and the preparation process is as Figure 1 : (1) Preparation of antigen nanoparticles: B16F10 cells were subcutaneously injected into the right axilla of C57BL / 6 mice to establish a mouse tumor-bearing model. When the tumor volume reached approximately 300 mm3 At this time, the tumor tissue was surgically removed and digested with trypsin at 37 °C for 30 minutes. After washing, monodisperse B16F10 cells were obtained and resuspended in pre-cooled phosphate buffer (PBS, pH 7.4) at a concentration of 2.5×10 7 cells / mL. Subsequently, the cell suspension was subjected to repeated freeze-thaw cycles (rapid freezing in liquid nitrogen followed by thawing at 37 °C), and the tumor antigen in the supernatant was collected by centrifugation at 3000g for 30 min. The supernatant containing the tumor antigen was extruded through a liposome extruder and passed through polycarbonate membranes with different pore sizes (400 nm and 200 nm) successively to obtain antigen nanoparticles.

[0042] (2) Preparation of azide nanoparticles: A mixture containing DOTAP (6.0 mg), DOPE (18.0 mg), cholesterol (3.0 mg) and Ac4ManNAz (1.0 mg) was dissolved in 2 ml of ethanol and evaporated under reduced pressure at 40 °C for 30 minutes to form a thin lipid film. The dried lipid film was hydrated with 2 ml of PBS and incubated at 37 °C for 1 hour to obtain azide nanoparticles.

[0043] (3) Preparation of fusion nanoparticles: The above-obtained tumor antigen nanoparticles and azide nanoparticles were mixed at a mass ratio of 2:1. The mixture was extruded through a liposome extruder and passed through polycarbonate membranes with different pore sizes (400 nm and 200 nm) successively to obtain fusion nanoparticles. Transmission electron microscopy (TEM) was used to observe the morphological characteristics of the nanoparticles, and a Zatasizer Nano ZS analyzer was used to measure the hydrodynamic diameter of the nanoparticles.

[0044] (4) Obtaining DC cells: C57BL / 6 mice at 6-8 weeks of age were euthanized under sterile conditions. The femurs and tibias were taken, and the attached muscle tissues were removed. The ends of the bones were cross-cut with a scalpel, and the bone marrow was rinsed into a culture dish with pre-cooled PBS. The obtained bone marrow suspension was filtered through a 200-mesh nylon mesh to remove bone fragments. Erythrocyte lysis buffer was added and stored at 4 °C for 5 min, and the cells were collected by centrifugation at 1200 rpm. Subsequently, the cells were resuspended in RPMI 1640 medium containing 10% FBS, 20 ng / mL GM-CSF and 10 ng / mL IL-4 and inoculated into a 6-well plate at a concentration of 1×10 6 cells / mL. Half of the medium was replaced with fresh medium containing cytokines every 48 hours. On the 8th day of culture, immature bone marrow-derived DC cells, namely BMDC, were harvested.

[0045] (5) Obtaining mature DC (FmDC): BMDC were seeded at 5×10 5The cells were inoculated at a density of [[[number of cells per well]]] per well in a 12-well plate and pre-cultured for 12 h. Subsequently, normal saline (NS), antigen nanoparticles, or fusion nanoparticles (10 μg / well in terms of antigen content) were added respectively, and they were co-incubated for 24 h under the conditions of 37 °C and 5% CO2 to obtain immature DCs (DC group), antigen-activated mature DCs (mDC group), and fusion nanoparticle-activated mature DCs (FmDC group). The cells were collected, stained with fluorescent dye-labeled anti-CD11c, anti-CD80, anti-CD86, and anti-MHC I antibodies, and analyzed by flow cytometry. Meanwhile, the culture supernatant was collected, and the levels of TNF-α and IL-6 were quantitatively detected using an ELISA kit.

[0046] (6)Obtaining a potent DC vaccine (FmDC-CD3): To obtain a potent DC vaccine, the fusion nanoparticle-activated mature DCs (FmDC group, with azide group -N3 on the surface) were further co-incubated with DBCO-CD3 antibody (1 μg antibody / 10 6 cells) for 2 h. Through the azide-alkyne cycloaddition reaction, the CD3 antibody was conjugated to the surface of FmDC to obtain FmDC-CD3, which is the potent DC vaccine. The conjugation of the CD3 antibody on the surface of FmDC was observed using a confocal microscope.

[0047] The relevant characterization results are as follows: (1)Characterization of nanoparticle morphology and particle size: The physicochemical properties of antigen nanoparticles, azide nanoparticles, and fusion nanoparticles were characterized. As Figure 2 shown, the TEM images showed that the three types of nanoparticles were uniformly distributed at the nanoscale. The particle size measurement results showed that the hydrodynamic sizes of antigen nanoparticles, azide nanoparticles, and fusion nanoparticles were 227.8 ± 10.99 nm, 154.8 ± 8.00 nm, and 211.4 ± 9.38 nm, respectively.

[0048] (2)Characterization of mature DCs: As Figure 3 shown, the molecular expression of BMDC was analyzed by flow cytometry. The MHC I of the FmDC group (91.1 ± 3.42%) was significantly higher than that of the mDC group (49.5 ± 4.60%) ( p <0.001), indicating that the presence of cationic lipids can promote the cross-presentation of antigens via the MHC I pathway. The proportion of mature DCs in the mDC group (45.3 ± 6.61%) was significantly higher than that in the DC group (15.9 ± 3.87%) ( p <0.001), while there was no significant difference among the mDC, FmDC, and FmDC-CD3 groups ( p >0.05, p >0.05, p(>0.05). Meanwhile, compared with the mDC group, the levels of TNF-α and IL-6 in the culture supernatants of the mDC, FmDC, and FmDC-CD3 groups also increased. These results indicate that MHC I-mediated antigen presentation does not affect the maturation of DCs.

[0049] (3) Characterization of CD3 antibody conjugation on DC surface: Click chemistry enables the covalent conjugation of DC-N3 with DBCO-CD3 antibody. To visualize this effect, the DBCO-CD3 antibody is conjugated with Cy5 fluorescent dye. As Figure 4 shown, the confocal fluorescence imaging results show that after co-incubation of DC-N3 with DBCO-CD3-Cy5, there is obvious red fluorescence on the DC surface, indicating the successful modification of the CD3 antibody on the DC surface.

[0050] (4) Characterization of the accumulation of potent DC vaccines in lymph nodes: Different DC vaccine preparations labeled with the near-infrared lipophilic dye DiR were subcutaneously injected at the inguinal region of C57BL / 6 mice. As Figure 5 shown, the in vitro lymph node quantitative fluorescence imaging results show that the signal intensity reaches a peak within 24 - 48 hours. Among them, the lymph node accumulation of FmDC-CD3 is the highest, followed by the FmDC and mDC groups, all significantly exceeding the DC control group, indicating that the potent DC vaccine conjugated with CD3 antibody has excellent lymph node accumulation ability.

[0051] (5) Characterization of the activation of T cells by potent DC vaccines: Further, flow cytometry analysis of lymph node T cells was performed. As Figure 6 shown, compared with the DC group, the proportions of CD4+ T cells and CD8+ T cells in the mDC group and FmDC group were significantly increased. Among them, the increase in CD4+ T cells in the mDC group was more significant ( p <0.05), while the increase in the proportion of CD8+ T cells in the FmDC group was more significant ( p <0.05). The level of CD8+ T cells in the FmDC-CD3 group was the highest (53.5 ± 4.30 %), significantly higher than that of other groups, indicating that the potent DC vaccine can effectively activate cell-mediated immune responses mainly dominated by CD8+ T cells.

[0052] (6) Pharmacodynamic characterization: The potent DC vaccine can significantly inhibit the postoperative recurrence of tumors. Combining with PD-1 monoclonal antibody can further enhance its effect. As Figure 7 shown, the present invention significantly reduces the risk of postoperative tumor recurrence and prolongs the survival period. Compared with the traditional DC vaccine, the tumor inhibition rate is increased by 41.4%.

[0053] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of an engineered potent dendritic cell vaccine, characterized in that, The method comprises the following steps: (1) Preparation of tumor antigen nanoparticles; (2) Preparation of azide nanoparticles: Cationic liposomes are used to encapsulate a metabolic glycoprotein labeling reagent containing an azide group to obtain azide nanoparticles; (3) Preparation of fusion nanoparticles: The antigen nanoparticles in step (1) and the azide nanoparticles in step (2) are co-extruded to obtain fusion nanoparticles; (4) Preparation of mature DCs: The fusion nanoparticles in step (3) are co-incubated with DCs to obtain mature DCs activated by the fusion nanoparticles, and the DCs have N3 groups on their surfaces; (5) Preparation of a potent DC vaccine: The mature DCs in step (4) are co-incubated with dibenzocyclooctyne-T cell specific antibody, and through azide-alkyne cycloaddition reaction, the T cell specific antibody is conjugated to the surface of the mature DCs to obtain a potent DC vaccine.

2. The preparation method of the engineered potent dendritic cell vaccine according to claim 1, characterized in that, In step (1), the method for preparing the tumor antigen nanoparticles comprises the following steps: Monodisperse tumor cells are obtained from a tumor tissue, the supernatant is collected by centrifugation after freeze-thaw cycles, and the tumor antigen nanoparticles are obtained after extrusion by a liposome extruder.

3. The preparation method of the engineered potent dendritic cell vaccine as described in claim 1, characterized in that, In step (2), in the cationic liposomes, there are a cationic lipid, a neutral co-lipid DOPE and cholesterol; the cationic lipid is one of dioleoyl trimethylammonium propane DOTAP, dioleoylpropyl trimethylammonium chloride DOTMA, didodecyldimethylammonium bromide DODAB, cholesterol derivative cationic lipid DC-Chol, and the metabolic glycoprotein labeling reagent containing an azide group is one of tetraacetylated N-azidoacetylmannosamine Ac4ManNAz, tetraacetylated N-azidoacetylgalactosamine Ac4GalNAz, N-azidoacetylglucosamine GlcNAz, azide-modified fucose analogue FucAz, 6-azidosialic acid.

4. The preparation method of the engineered potent dendritic cell vaccine according to claim 1, characterized in that, In step (2), the cationic lipid is a mixture of DOTAP, DOPE and cholesterol; the metabolic glycoprotein labeling reagent containing an azide group is tetraacetylated N-azidoacetylmannosamine Ac4ManNAz; wherein, the mass ratio of DOTAP, DOPE, cholesterol and Ac4ManNAz is (5-8):(15-20):(1-5):(0.5-2).

5. The preparation method of the engineered potent dendritic cell vaccine according to claim 3, characterized in that, In step (2), specifically, the method for preparing the azide nanoparticles comprises the following steps: A mixture containing a cationic lipid, DOPE, cholesterol and a metabolic glycoprotein labeling reagent containing an azide group is dissolved in an organic solvent, evaporated under reduced pressure to form a lipid film; the dried film is hydrated with PBS and then incubated to obtain azide nanoparticles.

6. The preparation method of the engineered potent dendritic cell vaccine according to claim 1, characterized in that, In step (3), the mass ratio of the tumor antigen nanoparticles to the azide nanoparticles is (1-5):(1-5).

7. The preparation method of the engineered potent dendritic cell vaccine according to claim 1, characterized in that, In step (4), the ratio of the fusion nanoparticles to DC is (1 - 50) μg: 2×10 5 cells; the fusion nanoparticles and DC are co-incubated for 18 - 36 h.

8. The preparation method of the engineered potent dendritic cell vaccine according to claim 1, characterized in that, In step (5), the T cell-specific antibody is one of anti-CD3 monoclonal antibody, anti-CD4 monoclonal antibody, anti-CD8 monoclonal antibody, and anti-PD-1 monoclonal antibody; the ratio of dibenzocyclooctyne-T cell-specific antibody to mature DC is (0.1~1) μg: 1×10 6 cells; mature DC and dibenzocyclooctyne-T cell-specific antibody are co-incubated for (1~4) h.

9. An engineered potent dendritic cell vaccine prepared by the method according to any one of claims 1 to 8.

10. Use of the engineered potent dendritic cell vaccine according to claim 9 in the preparation of an anti-tumor drug.

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