An engineered potent dendritic cell vaccine and preparation method and application thereof

Abstract

The application discloses an engineered potent dendritic cell vaccine and a preparation method and application thereof, and utilizes a nano-scale antigen delivery mode to fuse tumor antigens with cationic liposomes, destroys the stability of a lysosome membrane by means of cationic liposome charge interaction, makes part of the antigens escape into a cytoplasm to be cross-presented through an MHC I pathway, gives the DCs the ability to activate a cellular immune response to resist tumors, on the other hand, gives the DCs T cell directivity through a synthetic immunology method, promotes the interaction and signal transmission of DC-T cells, and the combination of the two mechanisms can overcome the low response rate problem of the existing DC vaccine, and maximizes the anti-tumor immune response.

Description

Technical Field

[0001] This invention belongs to the field of dendritic cell vaccines, specifically relating to an engineered, potent dendritic cell vaccine, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Malignant tumors are a major public health problem threatening human health. Surgical resection is the preferred treatment for 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 adjuvant therapy after tumor surgery. These vaccines involve inducing differentiation of autologous or allogeneic mononuclear cells into DCs in vitro, obtaining tumor antigens from surgically removed tumor tissue, and then adoptively reinfusing the DCs loaded with antigens into the body to activate T cells to specifically kill residual tumor cells. Currently, hundreds of DC vaccines are in clinical trials for the treatment of hematologic malignancies and solid tumors. In particular, DCVax®-L for gliomas and DCVAC / OvCa for ovarian cancer have entered Phase III clinical trials. Although DC vaccines have been shown to be effective in adjuvant therapy after tumor surgery, the overall objective response rate rarely exceeds 15%, and their clinical application remains quite limited.

[0004] Current DC vaccines rely on a simple process of co-incubating immature DCs with tumor antigens to obtain mature DCs. This lack of refined regulation of antigen presentation and DC function leads to suboptimal efficacy. The therapeutic bottleneck stems primarily from two interrelated mechanistic defects: insufficient cross-presentation of antigens via the Major Histocompatibility Complex (MHC) Class I pathway, resulting in antigen degradation in lysosomes and weakening the ability to activate cellular immune responses; and a lack of opportunity and duration for DCs to interact with T cells during antigen presentation, which weakens DC-T cell interaction and signal transduction, limiting subsequent T cell initiation. Therefore, in-depth exploration of the mechanism of action and limiting factors of DC vaccines is crucial for improving their therapeutic efficacy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention utilizes nanoscale antigen delivery to fuse tumor antigens with cationic liposomes. The cationic-liposome charge interaction disrupts the stability of the lysosomal membrane, allowing some antigens to escape into the cytoplasm and be cross-presented via the MHC I pathway, thereby activating the cellular immune response in dendritic cells (DCs) to combat tumors. On the other hand, synthetic immunology methods endow DCs with T-cell targeting. First, an azide group (N3) is modified onto the DC surface, and a dibenzocyclooctylene group (DBCO) is modified onto the T-cell-specific antibody. The T-cell-specific antibody is coupled to the mature DC surface through a cycloaddition reaction between N3 and DBCO, increasing the opportunity and duration of DC-T cell contact and promoting DC-T cell interaction and signal transduction. The combination of these two mechanisms overcomes the low response rate of existing DC vaccines, maximizing the anti-tumor immune response.

[0006] The technical solution adopted in this invention is as follows:

[0007] In a first aspect of the present invention, a method for preparing an engineered, potent dendritic cell vaccine is provided, the method comprising the following steps:

[0008] (1) Preparation of tumor antigen nanoparticles;

[0009] (2) Preparation of azide nanoparticles: Azide nanoparticles were obtained by encapsulating azide-containing metabolic glycoprotein labeling reagent with cationic liposomes;

[0010] (3) Preparation of fused nanoparticles: The antigen nanoparticles in step (1) and the azide nanoparticles in step (2) are co-extruded to obtain fused nanoparticles;

[0011] (4) Preparation of mature DC: The fused nanoparticles in step (3) are co-incubated with DC to obtain mature DC activated by fused nanoparticles, and the surface of DC has N3 groups;

[0012] (5) Preparation of potent DC vaccine: The mature DC in step (4) is co-incubated with DBCO-modified T cell-specific antibody. The T cell-specific antibody is coupled to the surface of the mature DC through an azide-acetylsene cycloaddition reaction to obtain a potent DC vaccine.

[0013] In step (1) of this invention, the preparation of tumor antigen nanoparticles can be carried out using existing technologies. The type and processing method of the tumor antigen are not limited; it can be whole-cell tumor antigen, tumor-specific antigen, tumor-associated antigen, differentiation antigen, embryonic antigen, etc. If it is a whole-cell tumor antigen, it can be obtained from tumor cells through various treatment methods such as freeze-thaw cycles, ultraviolet radiation, or photothermal ablation.

[0014] In one or more embodiments of the present invention, in step (1), the method for preparing the tumor antigen nanoparticles includes the following steps: obtaining monodisperse tumor cells from tumor tissue, collecting the supernatant by centrifugation after freeze-thaw cycles, and obtaining tumor antigen nanoparticles by extrusion using a liposome extruder.

[0015] In step (2) of this invention, the cationic liposomes contain one of the following: dioleoyltrimethylammonium propane (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), dioctadecyldimethylammonium bromide (DODAB), and cholesterol derivative cationic lipid (DC-Chol). Additionally, neutral accessory lipids (DOPE) and cholesterol are also included. The azido-containing metabolic glycoprotein labeling reagent is one of the following: tetraacetylated N-azidoacetylmannosamine (Ac4ManNAz), tetraacetylated N-azidoacetylgalactosamine (Ac4GalNAz), N-azidoacetylglucosamine (GlcNAz), azido-modified fucose analogue (FucAz), and 6-azidosialic acid (6-azidosialic acid).

[0016] In one or more embodiments of the present invention, in step (2), the cationic liposome is a mixture of DOTAP, DOPE and cholesterol, and the azide-containing metabolic glycoprotein labeling reagent is tetraacetyl N-azidoacetylmannosamine Ac4ManNAz. Preferably, the mass ratio of DOTAP, DOPE, cholesterol and Ac4ManNAz is (5~8):(15~20):(1~5):(0.5~2).

[0017] Specifically, the preparation method of azide nanoparticles includes the following steps: dissolving a mixture containing cationic lipids, DOPE, cholesterol and azide-containing metabolic glycoprotein labeling reagents in an organic solvent, evaporating under reduced pressure to form a lipid membrane; hydrating the dried membrane with PBS and then incubating it to obtain azide nanoparticles.

[0018] In step (3) of the present invention, the mass ratio of the tumor antigen nanoparticles to the azide nanoparticles is (1~5):(1~5).

[0019] In one or more embodiments of the present invention, in step (3), specifically, the method for preparing fused nanoparticles includes the following steps: using a liposome extruder, a mixture of antigen nanoparticles and azide nanoparticles is sequentially passed through polycarbonate films with pore sizes of 400 nm and 200 nm to co-extrude and prepare fused nanoparticles.

[0020] In step (4) of the present invention, the ratio of the fused nanoparticles to DC is (1~50) μg: 2×10 5 Each cell.

[0021] In one or more embodiments of the present invention, in step (4), the fused nanoparticles are co-incubated with DC for 18-36 h.

[0022] In one or more 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.

[0023] In step (5) of this invention, the ratio of dibenzocyclooctyn-T cell-specific antibody to mature DCs is (0.1~1) μg: 1×10 6 Each cell.

[0024] In one or more embodiments of the present invention, in step (5), mature DCs are co-incubated with DBCO-T cell-specific antibodies for (1~4) h.

[0025] In a second aspect of the invention, an engineered, potent dendritic cell vaccine prepared using the above method is provided.

[0026] In a third aspect of the invention, the use of the engineered potent dendritic cell vaccine in the preparation of antitumor drugs is provided.

[0027] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0028] (1) The present invention combines the cross-presentation of tumor antigens in DCs with the synergistic effect of DC-T cell contact mediated by T cell-specific antibodies to obtain a potent DC vaccine, which can improve DC-induced anti-tumor immune response, enhance the tumor killing effect mediated by CD8+ T cells, and reduce immune tolerance caused by Tregs cells.

[0029] (2) This invention significantly reduces the risk of tumor recurrence after surgery and prolongs survival. Compared with traditional DC vaccines, the tumor inhibition rate is increased by 41.4%.

[0030] (3) The potent DC vaccine obtained by the present invention has good activity. Based on the fine adjustment, its effectiveness is significantly increased, and its cell viability is comparable to that of the traditional DC vaccine obtained by the existing classic preparation process.

[0031] (4) The drugs and materials involved in this invention have good safety. The proportions and concentrations are data optimized for DC cells. They are safe, non-toxic, biodegradable, and have good biocompatibility.

[0032] (5) The fusion nanoparticles prepared by this invention have a suitable particle size (about 200 nm), the process is stable, simple and feasible, and easy to industrialize. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 : Flowchart of the preparation process of this invention.

[0035] Figure 2 Electron micrographs and particle size distributions of antigen nanoparticles, labeled nanoparticles, and fused nanoparticles.

[0036] Figure 3 The triple signaling pathway of mature dendritic cells (DCs): antigen presentation (MHC I), co-stimulatory molecules (CD80 and CD86), and cytokines (TNF-α and IL-6).

[0037] Figure 4 Flow cytometry characterizes the conjugation of CD3 antibody on the surface of mature dendritic cells (DCs).

[0038] Figure 5 Characterization of the lymph node accumulation capacity of potent DC vaccines.

[0039] Figure 6 Characterization of the ability of potent DC vaccines to activate T cells.

[0040] Figure 7 A potent DC vaccine inhibited postoperative tumor recurrence and prolonged the survival of mice. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the 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 art to which this invention pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0043] This invention designs a DC engineering platform that combines enhanced antigen cross-presentation with T-cell-specific antibody-mediated DC-T cell contact to develop a next-generation potent DC vaccine with enhanced immunotherapeutic efficacy.

[0044] First, tumor antigens are co-extruded with cationic liposomes to form fused nanoparticles. These fused nanoparticles are internalized into dendritic cells (DCs) into lysosomes. 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, thus enabling DCs to activate cellular immune responses against tumors. The cationic liposomes are then co-coated with a glycoprotein labeling reagent containing an azide group, allowing the azide group to be metabolically bound to the DC surface glycans during antigen processing. Subsequently, T-cell-specific antibodies are modified with dibenzocyclooctyl groups, achieving site-specific antibody conjugation via a cycloaddition reaction between azide and alkyne, effectively linking the T-cell-specific antibodies to the DC surface.

[0045] Following subcutaneous administration to the groin area of ​​mice, the engineered DC vaccine migrated to the lymph nodes, where it enhanced 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. In summary, this invention establishes a novel DC vaccine preparation strategy that combines nanoscale antigen delivery with synthetic immunology methods, providing a translational platform to overcome the limitations of current cancer immunotherapy.

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0047] Example 1

[0048] This embodiment designs and prepares a drug delivery system and drug combination for obtaining a potent DC vaccine. First, the tumor antigen is processed. Tumor antigens are obtained from excised tumor tissue, and after freeze-thaw cycles, the supernatant is collected by centrifugation and extruded using a liposome extruder to obtain nanoantigens. Azide nanoparticles are obtained by encapsulating the metabolic glycoprotein labeling reagent Ac4ManNAz with cationic lipids. The antigen nanoparticles and azide nanoparticles are co-extruded using a liposome extruder to obtain fused nanoparticles. Next, the DCs are functionalized. Co-incubating the fused nanoparticles with DCs stimulates DC maturation and, with the help of cationic lipids, disrupts the stability of the lysosomal membrane, allowing some antigens to escape into the cytoplasm for cross-presentation via the MHC I pathway. Simultaneously, the azide groups are metabolically bound to the DC surface during antigen processing. Subsequently, the obtained DC-N3 is co-incubated with a DBCO-CD3 antibody, and the CD3 antibody is coupled to the DC surface through a cycloaddition reaction, resulting in a potent DC vaccine.

[0049] The specific preparation method is as follows, and the preparation process is as follows: Figure 1 :

[0050] (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 mm², 3 At that 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 cultured at 2.5 × 10⁻⁶ cells per cell line. 7 Cells were resuspended at a concentration of [number] cells / mL in pre-chilled phosphate-buffered saline (PBS, pH 7.4). The cell suspension was then subjected to repeated freeze-thaw cycles (rapid freezing in liquid nitrogen followed by thawing at 37°C), and the supernatant was collected by centrifugation at 3000g for 30 min. The supernatant containing tumor antigen was extruded using a liposome extruder and passed through polycarbonate membranes with different pore sizes (400 nm and 200 nm) to obtain antigen nanoparticles.

[0051] (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.

[0052] (3) Preparation of fused nanoparticles: The tumor antigen nanoparticles and azide nanoparticles obtained above 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) to obtain fused nanoparticles. The morphological characteristics of the nanoparticles were observed using transmission electron microscopy (TEM), and the hydrodynamic diameter of the nanoparticles was measured using a Zatasizer Nano ZS analyzer.

[0053] (4) Obtaining DC cells: 6-8 week old C57BL / 6 mice were euthanized under aseptic conditions, and the femur and tibia were harvested, removing attached muscle tissue. The bone ends were transversely cut with a scalpel, and the bone marrow was washed with pre-cooled PBS into culture dishes. The resulting bone marrow suspension was filtered through a 200-mesh nylon mesh to remove bone fragments. Red blood cell lysis buffer was added and stored at 4°C for 5 min. 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 cultured at 1×10⁻⁶ cells / mL. 6 Cells were seeded at a concentration of [number] cells / mL in 6-well plates, and the medium was partially replaced with fresh medium containing cytokines every 48 hours. Immature bone marrow-derived dendritic cells (BMDCs) were harvested on day 8.

[0054] (5) Acquisition of mature DC (FmDC): BMDC is converted to 5×10 5 Cells were seeded at a density of [number] cells / well in 12-well plates and pre-cultured for 12 h. Then, normal saline (NS), antigen nanoparticles, or fusion nanoparticles (10 μg / well based on antigen content) were added, and the cells were co-incubated at 37°C and 5% CO2 for 24 h to obtain immature dendritic cells (DC group), antigen-activated mature DCs (mDC group), and fusion nanoparticle-activated mature DCs (FmDC group). Cells were collected, stained with fluorescently labeled anti-CD11c, anti-CD80, anti-CD86, and anti-MHC I antibodies, and analyzed by flow cytometry. Simultaneously, the culture supernatant was collected, and TNF-α and IL-6 levels were quantitatively detected using an ELISA kit.

[0055] (6) Obtaining a potent DC vaccine (FmDC-CD3): To obtain a potent DC vaccine, mature DCs activated by fusion nanoparticles (FmDC group, with azide groups -N3 on the surface) were further reacted with DBCO-CD3 antibody (1 μg antibody / 10 6 (100 cells) were co-incubated for 2 hours, and CD3 antibodies were coupled to the surface of FmDCs via an azide-alkyne cycloaddition reaction to obtain FmDC-CD3, which is a potent DC vaccine. The coupling of CD3 antibodies to the surface of FmDCs was observed using confocal microscopy.

[0056] The relevant characterization results are as follows:

[0057] (1) Characterization of nanoparticle morphology and particle size: The physicochemical properties of antigen nanoparticles, azide nanoparticles, and fused nanoparticles were characterized. For example... Figure 2 TEM images show that the three types of nanoparticles are uniformly distributed at the nanoscale. Particle size measurements show that the hydrodynamic sizes of the antigen nanoparticles, azide nanoparticles, and fusion nanoparticles are 227.8 ± 10.99 nm, 154.8 ± 8.00 nm, and 211.4 ± 9.38 nm, respectively.

[0058] (2) Characterization of mature DCs: such as Figure 3 Flow cytometry analysis of BMDC molecular expression showed that the MHC I in the FmDC group (91.1 ± 3.42%) was significantly higher than that in the mDC group (49.5 ± 4.60%). p <0.001), indicating that the presence of cationic lipids can promote antigen cross-presentation 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), and compared with the mDC group, the levels of TNF-α and IL-6 in the culture supernatant of the mDC, FmDC and FmDC-CD3 groups were also increased. These results indicate that MHC I-mediated antigen presentation does not affect DC maturation.

[0059] (3) Characterization of CD3 antibody conjugation on DC surface: Click chemistry causes DC-N3 to covalently conjugate with DBCO-CD3 antibody. To visualize this interaction, DBCO-CD3 antibody is conjugated with Cy5 fluorescent dye. Figure 4 Confocal fluorescence imaging results showed that after co-incubation of DC-N3 with DBCO-CD3-Cy5, the surface of DC exhibited obvious red fluorescence, indicating that the CD3 antibody was successfully modified on the surface of DC.

[0060] (4) Characterization of lymph node accumulation with potent DC vaccines: Different DC vaccine formulations labeled with the near-infrared lipophilic dye DiR were subcutaneously injected into the groin of C57BL / 6 mice. For example... Figure 5 Quantitative fluorescence imaging of isolated lymph nodes showed that the signal intensity reached its peak within 24-48 hours. Among them, the lymph node accumulation of FmDC-CD3 was the highest, followed by FmDC and mDC groups, all of which significantly exceeded the DC control group, indicating that the potent DC vaccine conjugated with CD3 antibody has excellent lymph node accumulation ability.

[0061] (5) Characterization of T cell activation by potent DC vaccine: Further, flow cytometry analysis was performed on lymph node T cells, such as... Figure 6 Compared with the DC group, the proportions of CD4+ T cells and CD8+ T cells were significantly increased in both the mDC group and the FmDC group, with the mDC group showing a greater increase in CD4+ T cells. p <0.05), while the proportion of CD8+ T cells increased more in the FmDC group ( p <0.05. The FmDC-CD3 group had the highest CD8+ T cell level (53.5 ± 4.30%), which was significantly higher than other groups, indicating that the potent DC vaccine can effectively activate cellular immune responses mainly composed of CD8+ T cells.

[0062] (6) Pharmacodynamic characterization: High-potency DC vaccines can significantly inhibit postoperative tumor recurrence. Combining them with PD-1 monoclonal antibodies can further enhance their effects, such as... Figure 7 This invention significantly reduces the risk of tumor recurrence after surgery and prolongs survival. Compared with traditional DC vaccines, the tumor inhibition rate is increased by 41.4%.

[0063] 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of preparing an engineered potent dendritic cell vaccine, characterized in that, The method comprises the following steps: (1) preparation of tumor antigen nanoparticles; The preparation method of the tumor antigen nanoparticles comprises the following steps: obtaining monodisperse tumor cells from tumor tissues, collecting the supernatant after freeze-thaw cycle and centrifugation, and obtaining tumor antigen nanoparticles after extrusion by a liposome extrusion instrument; (2) preparation of azido nanoparticles: azido nanoparticles are obtained by using cationic liposomes to encapsulate a metabolic glycoprotein labeling reagent containing an azido group; the cationic lipids are a mixture of DOTAP, DOPE and cholesterol; the metabolic glycoprotein labeling reagent containing an azido 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); The preparation method of the azido nanoparticles comprises the following steps: dissolving a mixture containing cationic lipids, DOPE, cholesterol and a metabolic glycoprotein labeling reagent containing an azido group in an organic solvent, evaporating under reduced pressure to form a lipid film; hydrating the dried film with PBS, and then incubating to obtain azido nanoparticles; (3) preparation of fusion nanoparticles: the tumor antigen nanoparticles in step (1) and the azido 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 surface of the DCs carries N3 groups; (5) preparation of a potent DC vaccine: the mature DCs in step (4) are co-incubated with dibenzocyclooctyne-T cell-specific antibodies to couple the T cell-specific antibodies to the surface of the mature DCs through an azido-alkyne cycloaddition reaction, thereby obtaining a potent DC vaccine; The T cell specific antibody is one of an anti-CD3 monoclonal antibody, an anti-CD4 monoclonal antibody, an anti-CD8 monoclonal antibody and an anti-PD-1 monoclonal antibody; the ratio of the dibenzocyclooctyne-T cell specific antibody to the mature DC is (0.1-1) μg:1×10 6 6 cells; the mature DC and the dibenzocyclooctyne-T cell specific antibody are co-incubated for (1-4) h.

2. The engineered potent dendritic cell vaccine preparation method of claim 1, wherein, In step (3), the mass ratio of the tumor antigen nanoparticles to the azido nanoparticles is (1-5):(1-5).

3. The method for preparing an engineered, potent dendritic cell vaccine as described in claim 1, characterized in that, In step (4), the ratio of the fusion nanoparticle and the DCs is (1-50) pg: 2 x 10 5 cells; the fusion nanoparticle and the DCs are co-incubated for 18-36 h.

4. An engineered potent dendritic cell vaccine obtained by the method of any one of claims 1-3.

5. Use of the engineered potent dendritic cell vaccine of claim 4 in the preparation of an antitumor drug.

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