Dendritic cell-derived drug-loaded vesicle capable of improving lymph nodes in targeted manner and enhancing anti-tumor treatment effect and preparation method of dendritic cell-derived drug-loaded vesicle

Abstract

The invention belongs to the technical field of drug targeting carriers, and more specifically relates to a dendritic cell-derived drug-loaded vesicle capable of improving lymph nodes in a targeting manner and enhancing the curative effect of anti-tumor treatment and a preparation method of the dendritic cell-derived drug-loaded vesicle. Comprising extracellular vesicles generated by dendritic cells overexpressing tumor antigens, and small molecule drugs wrapped by the extracellular vesicles, the small molecule medicine is an activated state fibroblast reticular cell deactivator. The dendritic cell-derived drug-loaded vesicles have remarkable lymph node homing ability, can directly activate CD8 + T cells and regulate functions of lymph node fibroblast reticulate cells, synergistically optimize the survival environment of the CD8 + T cells, promote recruitment, activation and proliferation of the CD8 + T cells and drive the CD8 + T cells to differentiate into dry memory cell subpopulations, and through multi-dimensional synergistic interaction, the dendritic cell-derived drug-loaded vesicles can be used for preparing the dry memory cell subpopulations. The antigen presentation is enhanced, the T cell activation and memory formation are promoted, the lymph node microenvironment and the tumor microenvironment are improved, and the tumor growth is effectively inhibited.

Description

Technical Field

[0001] This application belongs to the technical field of drug targeting carriers, and more specifically, relates to a dendritic cell-derived drug-loaded vesicle for targeted modification of lymph nodes to enhance the efficacy of anti-tumor therapy and a preparation method thereof. Background Art

[0002] As a major breakthrough in the field of cancer treatment, tumor immunotherapy involves multiple pathways such as antigen presentation mediated by dendritic cells, T cell activation, and immune microenvironment regulation. Among them, the strategy of enhancing anti-tumor T cell responses by optimizing the antigen presentation function of dendritic cells has become one of the key research directions in this field.

[0003] However, the clinical efficacy of existing dendritic cell vaccines is still limited by the following bottlenecks: Although traditional dendritic cell vaccines can present antigens, they are limited by problems such as short in vivo survival, low lymph node homing efficiency, and insufficient co-stimulatory signals, making it difficult to effectively activate naive T cells. In addition, under the tumor-bearing background, activated fibroblastic reticular cells (FRCs) in the lymph node microenvironment abnormally secrete extracellular matrix proteins (such as α-SMA and fibronectin), promoting their transformation into a fibrotic cell-like phenotype and forming a physical barrier that hinders drug penetration and immune cell infiltration and migration. At the same time, the expression of chemokines (such as CCL19, CCL21) and cytokines (such as IL-7) is dysregulated, inhibiting T cell recruitment, survival, and maintenance of the stem cell-like memory phenotype, and affecting the formation of long-term anti-tumor immune memory.

[0004] Therefore, the research and development of anti-tumor drugs based on the regulation of the lymph node microenvironment, by specifically regulating lymph node-activated fibroblastic reticular cells to synergistically enhance T cell-mediated immune responses, is of great significance for effectively inhibiting tumor growth. Summary of the Invention

[0005] Aiming at the defects of the existing technology, the purpose of this application is to provide a dendritic cell-derived drug-loaded vesicle for targeted modification of lymph nodes to enhance the efficacy of anti-tumor therapy and a preparation method thereof, aiming to solve key problems such as low lymph node targeting efficiency of existing drugs, insufficient activation of naive T cells, limited ability of effector T cells to recruit-activate-amplify, and insufficient function of stem cell-like memory T cells, while enhancing the regulatory ability of lymph nodes and the tumor microenvironment.

[0006] To achieve the above object, in a first aspect, the present application provides a dendritic cell-derived drug-loaded vesicle for targeted modification of lymph nodes to enhance the efficacy of anti-tumor therapy, which includes extracellular vesicles produced by dendritic cells overexpressing tumor antigens, and small molecule drugs encapsulated by the extracellular vesicles; the small molecule drugs are activatable fibroblastic reticular cell deactivators.

[0007] Preferably, the mass ratio of the extracellular vesicles to the small molecule drugs is 1000:(1~50).

[0008] Preferably, the dendritic cells overexpressing tumor antigens are obtained by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing.

[0009] Preferably, the tumor antigens include one or more of liver cancer antigen AFP, carcinoembryonic antigen CEA, prostate specific antigen PSA, squamous cell carcinoma antigen SCCA, ovarian cancer antigen CA125, pancreatic cancer antigen CA19-9, melanoma antigen, cancer-testis antigen CTAs and tumor neoantigens.

[0010] Preferably, the tumor specific antigens include one or more of Actn4, Adpgk, Ap3d1, Tubb3, Dag1, Eef2, Tnpo3, Tubb3, Reps1, Cpne1 and Cpsf3l.

[0011] Preferably, the small molecule drugs include one or more of calcipotriol, all-trans retinoic acid and pirfenidone.

[0012] Preferably, the average particle size of the dendritic cell-derived drug-loaded vesicles is 100 nm~600 nm.

[0013] In a second aspect, the present application provides a method for preparing the above dendritic cell-derived drug-loaded vesicles, which includes the following steps: S1. Construct dendritic cells stably overexpressing the above tumor antigens by genetic engineering transformation; S2. Subject the dendritic cells stably overexpressing the above tumor antigens to pressure treatment, and collect the extracellular vesicles produced by the dendritic cells overexpressing tumor antigens; S3. Co-incubate the extracellular vesicles and the small molecule drugs, and collect the dendritic cell-derived drug-loaded vesicles.

[0014] Preferably, in step S1, the dendritic cells stably overexpressing the above tumor antigens are prepared by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing.

[0015] Preferably, in step S2, the above pressure treatment methods include one or more of ultraviolet irradiation, starvation, and heat stimulation.

[0016] Preferably, in steps S2 and S3, the above collection conditions are: collecting at a centrifugal force of 500 - 20,000 g under the condition of 4°C.

[0017] In a third aspect, the present application provides an anti-tumor drug, which includes the above dendritic cell-derived drug-loaded vesicles.

[0018] Generally speaking, compared with the prior art through the above technical solutions conceived by the present application, the following technical advantages are mainly achieved: (1) A dendritic cell-derived drug-loaded vesicle for targeted modification of lymph nodes to enhance the efficacy of anti-tumor treatment provided by the present application includes extracellular vesicles produced by dendritic cells overexpressing tumor antigens, and small molecule drugs encapsulated by the extracellular vesicles; the above small molecule drugs are activatable fibroblastic reticular cell deactivators. This dendritic cell-derived drug-loaded vesicle has significant lymph node homing ability, can directly activate CD8 + T cells and regulate the functions of lymph node fibroblastic reticular cells, synergistically optimize the survival environment of CD8+ T cells, and promote the recruitment, activation, and proliferation of CD8 + T cells, and drive CD8 + T cells to differentiate into a stem cell-like memory cell subset, break through the dilemma of T cell exhaustion in traditional therapies, enhance antigen presentation through multi-dimensional synergistic effects, promote T cell activation and memory formation, improve the lymph node microenvironment and tumor microenvironment, and effectively inhibit tumor growth.

[0019] (2) The dendritic cell-derived drug-loaded vesicle provided by the present application can also reshape the lymph node chemokine network and degrade the extracellular matrix by regulating the functions of lymph node fibroblastic reticular cells, form a positive feedback loop to guide the dendritic cell-derived drug-loaded vesicles and T cells to chemotax to the lymph nodes, enhance lymph node targeting, and promote their infiltration into the lymph nodes.

[0020] (3) The dendritic cell-derived drug-loaded vesicle provided by the present application can effectively enhance the effector ability, proliferation ability, and memory of CD8 + T cells in the lymph node region, further enhance the infiltration and activation of stem cell-like memory T cells, central memory T cells, and effector memory T cells in the tumor tissue, facilitate the formation of long-term anti-tumor immune memory, provide a new solution for overcoming tumor immunotherapy drug resistance, and have important scientific value and clinical transformation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Western blot analysis of AFP expression; Content A shows the expression of AFP in dendritic cells transfected with an AFP-expressing lentivirus, and Content B shows the expression of AFP in extracellular vesicles derived from dendritic cells that overexpress AFP. Figure 2 Characterization of immune activation molecules in extracellular vesicles derived from dendritic cells overexpressing AFP; Content A is Western blot detection of AFP expression in extracellular vesicles derived from dendritic cells overexpressing AFP, and Content B, Content C, and Content D are the expression levels of MHC-I, MHC-II, CD80, and CD86, respectively; Figure 3 Characterization of Cal-loaded vesicles derived from dendritic cells overexpressing AFP; Content A, Content B, and Content C are the particle size distribution, Zeta potential, and TEM imaging, respectively; Figure 4 Western blot was used to detect the expression of CCR7 in Cal-loaded vesicles derived from dendritic cells overexpressing AFP; Figure 5 Figure 4: Distribution of Californium-loaded vesicles derived from AFP-overexpressing dendritic cells in lymph nodes. Figures A and B show the fluorescence imaging and ICG fluorescence intensity quantification of Californium-loaded vesicles derived from dendritic cells in lymph nodes (ALN, ILN, and MLN), respectively. Figures C and D show the Image J quantification and ICG fluorescence distribution images of Californium-loaded vesicles derived from dendritic cells in ILN sections, respectively. Figure 6 After the activation of FRCs was regulated by Cal-loaded vesicles from dendritic cells overexpressing AFP, the expression levels of various genes in FRCs changed; Content A, Content B, Content C, and Content D are Acta2 、 Pdpn 、 Ccl19 and Ccl21 mRNA expression level; Figure 7 After activated FRCs were regulated by Cal-loaded vesicles derived from AFP-overexpressing dendritic cells, the extracellular matrix deposition and chemokine expression levels of FRCs were changed; Figure 8Dendritic cell-derived Cal-loaded vesicles overexpressing AFP target lymph nodes via the CCR7-CCL19 / CCL21 pathway; wherein Content A and Content B are the fluorescence imaging results and ICG fluorescence intensity quantification results of dendritic cell-derived Cal-loaded vesicles in lymph nodes (ALN, ILN, MLN) after blocking CCL19 or CCL21, respectively, and Content C and Content D are the ICG fluorescence distribution images and Image J quantification results of dendritic cell-derived Cal-loaded vesicles in ILN sections after blocking CCL19 or CCL21, respectively; Figure 9 The direct activation effect of dendritic cell-derived Cal-loaded vesicles overexpressing AFP on CD8 + T cells; wherein Content A and Content B are the positive proportions of IFN-γ and GzmB in CD8 + T cells after co-incubation of CD8 + T cells and dendritic cell-derived Cal-loaded vesicles; Figure 10 The chemotactic effect of FRCs on CD8 + T cells after dendritic cell-derived Cal-loaded vesicles overexpressing AFP regulate activated FRCs; wherein Content A is the recruitment of CD8 + T cells by the cell supernatant collected after dendritic cell-derived Cal-loaded vesicles overexpressing AFP regulate activated FRCs, and Content B is the recruitment of CD8 + T cells by the cell supernatant collected after dendritic cell-derived Cal-loaded vesicles overexpressing AFP regulate activated FRCs and blocking CCL19 or CCL21; Figure 11 The changes in cytokine expression levels and the effect on the survival of CD8 + T cells after dendritic cell-derived Cal-loaded vesicles overexpressing AFP regulate activated FRCs; wherein Content A is the mRNA expression level of Il-7 , and Content B is the survival of CD8 + T cells; Figure 12 The promotion of memory CD8 + T cell production by FRCs after dendritic cell-derived Cal-loaded vesicles overexpressing AFP regulate activated FRCs; wherein Content A, Content B, Content C, Content D, and Content E are T + in CD8 naive (CD44 - CD62L + ), T naive proliferation, T SCM (Sca1 + CD44 - CD62L + ), TSCM (CD122 + CD44 - CD62L + ) and T CM (CD44 + CD62L + ) ratio; Figure 13 After the overexpressed AFP dendritic cell-derived ATRA-loaded vesicles regulate the activated FRCs, the changes in the extracellular matrix deposition and chemokine expression levels of FRCs; Figure 14 After the overexpressed Adpgk dendritic cell-derived Cal-loaded vesicles regulate the activated FRCs, promoting the recruitment of CD8 + T cells and the generation of memory phenotypes, where content A is the recruitment effect of the cell supernatant collected after the dendritic cell-derived Cal-loaded vesicles regulate the activated FRCs on CD8 + T cells, and contents B, C, and D are the effects of FRCs on CD8 + T cells, where T SCM (Sca1 + CD44 - CD62L + ), T SCM (CD122 + CD44 - CD62L + ) and T CM (CD44 + CD62L + ) ratio; Figure 15 For the in-situ hepatocellular carcinoma mice intravenously administered with the overexpressed AFP dendritic cell-derived Cal-loaded vesicles, the picture of the tumor-bearing liver after the treatment, and the part circled by the circle is the tumor; Figure 16 The anti-tumor effect of the overexpressed AFP dendritic cell-derived Cal-loaded vesicles; where content A is the tumor weight of the tumor-bearing mice, and content B is the survival period of the tumor-bearing mice; Figure 17 The influence of the overexpressed AFP dendritic cell-derived Cal-loaded vesicles on the lymph node microenvironment; where contents A, B, C, D, E, F, and G are CD8 in the lymph nodes + T, CD8 + IFN-γ + T, CD8 + GzmB + T, CD8 + Ki67 + T, CD8 + TSCM (Sca1 + CD44 - CD62L + ), CD8 + T SCM (CD122 + CD44 - CD62L + ), and CD8 + T CM cell numbers; Figure 18 is the effect of AFP-overexpressing dendritic cell-derived Cal-loaded vesicles on the tumor microenvironment; where Contents A, B, C, D, E, F, G, and H are respectively CD8 + T, CD8 + T SCM (Sca1 + CD44 - CD62L + ), CD8 + T SCM (CD122 + CD44 - CD62L + ), CD8 + T CM (CD44 + CD62L + ), CD8 + T EM (CD44 + CD62L - ), CD8 + IFN-γ + T, CD8 + GzmB + T, and CD8 + Ki67 + T cell numbers; Figure 19 is the in vivo biosafety of AFP-overexpressing dendritic cell-derived Cal-loaded vesicles; where Contents A, B, C, D, E, and F are respectively the level changes of AST, ALT, LDH, BUN, CREA, and CK in mouse serum after treatment; In all the figures, the same reference numerals are used to indicate the same significance level, where: represents p < 0.05, represents p < 0.01, represents p < 0.001. Specific implementation manners

[0022] To make the objectives, technical solutions, and advantages of this application clearer, the following further elaborates on this application in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0023] In the description of the embodiments of this application, words such as "exemplary" or "for example" are used to represent examples, illustrations, or explanations. Any embodiment or design solution described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or having more advantages than other embodiments or design solutions. Rather, the use of words such as "exemplary" or "for example" is intended to present relevant concepts in a specific manner.

[0024] In the description of the embodiments of this application, unless otherwise specified, the meaning of "a plurality" refers to two or more.

[0025] This application provides a dendritic cell-derived drug-loaded vesicle for targeted modification of lymph nodes to enhance the efficacy of anti-tumor therapy, which includes extracellular vesicles produced by dendritic cells overexpressing tumor antigens, and small molecule drugs encapsulated by the extracellular vesicles; the above small molecule drugs are lymph node fibroblastic reticular cell deactivators.

[0026] In some embodiments, the mass ratio of the above extracellular vesicles to the above small molecule drugs is 1000:(1 - 50).

[0027] In some embodiments, the above dendritic cells overexpressing tumor antigens are obtained by lentiviral transfection, adenoviral transfection, plasmid transfection, or gene editing.

[0028] The tumor antigens in this application can be tumor-associated antigens and tumor-specific antigens recognized in medicine or identifiable through whole-genome sequencing and exome sequencing. In some embodiments, the above tumor-associated antigens are one or more of liver cancer antigen AFP, carcinoembryonic antigen CEA, prostate-specific antigen PSA, squamous cell carcinoma antigen SCCA, ovarian cancer antigen CA125, pancreatic cancer antigen CA19-9, melanoma antigens (including but not limited to Tyrosinase, MAGE family, MART-1, gp100), and cancer-testis antigens CTAs (including but not limited to NY-ESO-1, PRAME). In some embodiments, the above tumor antigens further include tumor neoantigens, where tumor neoantigens are antigenic peptides generated by tumor cell gene mutations that can be identified by genomic sequencing technology, including one or more of Actn4, Adpgk, Ap3d1, Tubb3, Dag1, Eef2, Tnpo3, Tubb3, Reps1, Cpne1, and Cpsf3l.

[0029] In this application, the above-mentioned drug is a small molecule drug and an activator of activated fibroblastic reticular cells. It can deactivate activated lymph node fibroblastic reticular cells, down-regulate extracellular matrix proteins such as α-SMA and Fibronectin, and up-regulate chemokines CCL19 and CCL21. In some embodiments, the above-mentioned drug includes one or more of calcipotriol (Cal), all-trans retinoic acid (ATRA), and pirfenidone (PFD). Additionally, it is not yet known whether the deactivation effect of the above-mentioned small molecule drug on activated fibroblastic reticular cells can be enhanced by combining with extracellular vesicles of dendritic cells overexpressing tumor antigens. Therefore, the synergistic effect of cancer treatment by combining a small molecule drug that deactivates activated fibroblastic reticular cells and extracellular vesicles derived from dendritic cells overexpressing tumor antigens is also one of the technical features of this application.

[0030] In some embodiments, the average particle size of the above-mentioned dendritic cell-derived drug-loaded vesicles is 100 nm to 600 nm.

[0031] On the other hand, this application also provides a method for preparing the above-mentioned dendritic cell-derived drug-loaded vesicles, including the following steps: S1. Construct dendritic cells that stably overexpress the above-mentioned tumor antigen through genetic engineering transformation; S2. Subject the above-mentioned dendritic cells that stably overexpress the above-mentioned tumor antigen to pressure treatment, and collect the extracellular vesicles produced by the above-mentioned dendritic cells overexpressing the tumor antigen; S3. Co-incubate the above-mentioned extracellular vesicles and the above-mentioned small molecule drug, and collect the above-mentioned dendritic cell-derived drug-loaded vesicles.

[0032] It can be understood that this application does not limit the preparation method of the above-mentioned dendritic cells overexpressing the tumor antigen in step S1, which can be but is not limited to lentiviral transfection, adenoviral transfection, plasmid transfection, or gene editing, etc.

[0033] As an alternative, in the method for preparing the above-mentioned dendritic cells overexpressing the tumor antigen by lentiviral transfection, step S1 includes the following steps: S1-1. Transfect 293T cells with a lentiviral packaging plasmid, a lentiviral envelope plasmid, and a vector plasmid carrying a tumor antigen gene as the target gene, and collect the lentivirus that stably overexpresses the tumor antigen; S1-2. Transfect the above-mentioned lentivirus that stably overexpresses the tumor antigen into dendritic cells, and screen to obtain dendritic cells that stably overexpress the tumor antigen.

[0034] In some embodiments, in step S1-1, the above-mentioned lentiviral packaging plasmid is one or more of psPAX2, pMDLg / pRRE, and pRSV / Rev.

[0035] In some embodiments, in step S1-1, the lentiviral envelope plasmid is one or more of pMD2.G-VSVG and pCMV-VSV-G.

[0036] In some embodiments, in step S1-1, the mass ratio of the lentiviral packaging plasmid, the lentiviral envelope plasmid, and the vector plasmid carrying the tumor antigen gene as the target gene is 3:1:4, and a lentivirus with high titer overexpressing the tumor antigen can be obtained.

[0037] In some embodiments, in step S1-2, the specific operation of the transfection is as follows: The dendritic cells and the lentivirus overexpressing the tumor antigen are statically cultured in a serum-free medium to maintain cell growth; according to the cell growth conditions, the medium type is changed, and finally the medium is changed to a complete medium containing a screening reagent, and dendritic cells stably overexpressing the tumor antigen are obtained by culturing.

[0038] In some embodiments, in step S1-2, the ratio of the lentivirus overexpressing the tumor antigen to the dendritic cells is (4-6)×10 6 PFU:5×10 5 cells.

[0039] In some embodiments, in step S1-2, the screening reagent is puromycin.

[0040] In some embodiments, in step S2, the methods of the pressure treatment include but are not limited to one or more of ultraviolet irradiation, starvation, and heat stimulation.

[0041] In some embodiments, the specific operation of the ultraviolet irradiation is as follows: The dendritic cells overexpressing the tumor antigen are irradiated under an ultraviolet lamp of 300 J / m 2 for 1 h to induce the dendritic cells to produce extracellular vesicles.

[0042] In some embodiments, the conditions for the collection in step S2 are as follows: Centrifuge at 600 g for 10 min to collect the supernatant, then centrifuge the supernatant at 18,000 g for 2 min to remove cell debris, and centrifuge the supernatant at 18,000 g for 30 min again to collect the precipitate, which is the extracellular vesicles produced by the dendritic cells overexpressing the tumor antigen.

[0043] In some embodiments, in step S3, the mass ratio of the extracellular vesicles to the small molecule drug is 1000:(1-200).

[0044] In some embodiments, the conditions for the collection in step S3 are as follows: Centrifuge at 18,000 g for 30 min to collect the precipitate, which is the drug-loaded vesicles derived from the dendritic cells.

[0045] The dendritic cell-derived drug-loaded vesicles provided in this application can target lymph nodes by directly activating CD8 + T cells and regulate the function of lymph node fibroblast reticular cells, synergistically optimize the survival environment of CD8+ T cells, and promote CD8 + T cell recruitment, activation, and proliferation, and drives CD8 + The differentiation of T cells into stem cell subsets overcomes the dilemma of T cell exhaustion in traditional therapies. Through multi-dimensional synergistic enhancement, it enhances antigen presentation, promotes T cell activation and memory formation, improves the lymph node microenvironment and tumor microenvironment, and effectively inhibits tumor growth. Based on this, this application provides the use of the above-mentioned dendritic cell-derived drug-loaded vesicles in the preparation of anti-tumor drugs.

[0046] On the other hand, the present application also provides an anti-tumor drug, which includes the above-mentioned dendritic cell-derived drug-loaded vesicles.

[0047] In some embodiments, the anti-tumor drug may comprise only dendritic cell-derived drug-loaded vesicles, or may comprise dendritic cell-derived drug-loaded vesicles and a chemotherapeutic drug, or may be formulated into a suitable form together with a pharmaceutically acceptable carrier and further including an excipient or diluent. In some embodiments, the carrier includes all types of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes.

[0048] In some embodiments, the anti-tumor drug is administered intravenously or intratumorally, and its dosage form includes injection or freeze-dried powder.

[0049] In some embodiments, the dendritic cell-derived drug-loaded vesicles and the chemotherapeutic drug can be co-administered simultaneously, sequentially, or separately. "Simultaneously" refers to the simultaneous administration of both drugs, while "sequentially" refers to the administration of one drug within 5 minutes, 10 minutes, or several hours of the other. Furthermore, simultaneous, sequential, or separate administration is not limited to a single administration, and these methods of administration can be repeated or combined.

[0050] The total effective dose of the anti-tumor drug provided in this application can be administered to the patient in a single dose, or according to a fractionated treatment regimen, can be administered to the patient in long-term multiple doses. The content of the active ingredient in the anti-tumor drug can vary according to the severity of the disease. Those skilled in the art can determine the appropriate effective dose of the anti-tumor drug by comprehensively considering various factors, including the patient's age, weight, health status and gender, the severity of the disease, diet and excretion rate, as well as the formulation method, route of administration and number of treatments, and therefore the total dose of the anti-tumor drug is not limited. The above-mentioned anti-tumor drug of this application is not particularly limited to the formulation, route of administration and method of administration, as long as the effect of this application is shown.

[0051] It should be understood that materials with the same or similar types, models, qualities, properties or functions as the reagents and instruments used in the following examples can be used to implement this application. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial sources.

[0052] The following are the examples: The sources of the main reagents and materials used in the following examples are as follows: 293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences' Committee for the Preservation of Type Cultures; DC2.4 cells were purchased from Hunan Fenghui Biotechnology Co., Ltd.; Hepa1-6 cells were purchased from Wuhan Boster Biological Engineering Co., Ltd. pCDH-CMV-MCS-EF1-Puro-AFP and pCDH-Adpgk-Zeocin were constructed by conventional molecular biology methods; calcipotriol, all-trans retinoic acid, and IR780 were all purchased from Sigma; C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; Mojosort™ Mouse CD8 + T cell sorting kit was purchased from Biolegend; Annexin-V / PI kit was purchased from Yeasen Biotech.

[0053] Example 1 Preparation of Drug-loaded Vesicles Derived from Dendritic Cells Overexpressing Antigens 1. Experimental steps (1) Construction of the expression vector pCDH-CMV-MCS-EF1-Puro-AFP for AFP Extract the RNA of MEF cells and reverse transcribe it to obtain cDNA; amplify the CDS fragment by PCR using the restriction enzyme primers of AFP; perform double digestion on the CDS fragment and the expression vector pCDH-CMV-MCS-EF1-Puro; ligate the double-digested products with DNA ligase, and screen for the AFP expression vector pCDH-CMV-MCS-EF1-Puro-AFP using the puromycin resistance of the pCDH-CMV-MCS-EF1-Puro vector and identify it by sequencing.

[0054] (2) Transfect 293T cells Precisely mix plasmid psPAX2, pM2.GVSVG and pCDH-CMV-MCS-EF1-Puro-AFP (total amount 16 μg) according to the mass ratio of 3:1:4. Subsequently, evenly disperse the mixed plasmid into an EP tube containing 1 mL of opti-MEM medium (labeled as tube A), and incubate at 37 °C for 5 min; add 20 μL of PEI and 1 mL of opti-MEM medium into another EP tube (labeled as tube B), and incubate at 37 °C for 5 min; mix the contents of tubes A and B, continue to incubate at 37 °C for 15 min, then add it to the 293T cell culture system, gently shake the culture dish to ensure mixing, and finally place it in a sterile incubator at 37 °C and 5% CO2 for 72 h.

[0055] (3)Obtain and purify AFP lentivirus Collect the supernatant of the transfected 293T cells, and remove impurities and cell debris through a sterile filter with a pore size of 0.45 μm. Centrifuge the filtered supernatant at 8×10 4 g at 4 °C for 2 h, and collect the precipitate, which is the lentivirus expressing AFP. Resuspend the lentivirus with PBS and store it at -80 °C for later use.

[0056] (4)Construct a dendritic cell line stably expressing AFP Add serum-free medium containing 5×10 6 PFU of AFP lentivirus to DC2.4 cells, statically culture for 2 h, and then supplement serum to maintain cell growth. After culturing for 24 h, change to fresh complete medium to resume normal cell culture and passage. At the same time, add puromycin with a concentration of 2 μg / mL during the culture process to screen out the cell line DC AFP that stably expresses AFP. Digest Hepa1-6 cell samples (positive control), DC2.4 cell samples, DC2.4 cell samples transfected with lentivirus expressing empty plasmid (DC vec ), and DC AFP with trypsin respectively, centrifuge to obtain cell precipitates, lyse the cells with RIPA, centrifuge at 1×10 4 rpm for 10 min, take the supernatant to obtain protein precipitates, boil the samples at 100 °C for 10 min, add 5× protein loading buffer, and detect the expression of AFP by western blot.

[0057] (5)Prepare dendritic cell-derived extracellular vesicles overexpressing tumor antigens Resuspend DC AFP with serum-free medium, irradiate it under ultraviolet light (300 J / m 2 ) for 1 h, collect the cell suspension, centrifuge at 600 g for 10 min to collect the supernatant, and the supernatant is centrifuged at 18000g Centrifuge for 2 min to remove debris, collect the supernatant again, centrifuge at 18,000 g for 30 min to collect the precipitate, and wash it twice with PBS to obtain extracellular vesicles derived from dendritic cells overexpressing tumor antigen, abbreviated as DEV AFP . According to step (4), detect the expression of AFP protein in Hepa1-6 EV (positive control), DEV, DEV vec , DEV AFP . At the same time, use flow cytometry to detect the expression of major histocompatibility complex (MHC-I, MHC-II) and costimulatory molecules (CD80, CD86) in DEV, DEV vec , DEV AFP .

[0058] (6) Preparation of Cal-loaded vesicles derived from dendritic cells overexpressing tumor antigen AFP Incubate the above DEV AFP with calcipotriol (Cal) at a final concentration of 40 µg / mL at 37 °C for 4 h, centrifuge at 18,000 g for 30 min to collect the precipitate, and wash it three times with PBS to obtain Cal-loaded vesicles derived from dendritic cells overexpressing tumor antigen AFP, abbreviated as Cal@DEV AFP. . According to similar operating steps, collect DEV and Cal@DEV derived from DC2.4 cells, DEV vec derived from DC vec cells and Cal@DEV vec , and Hepa1-6 EV derived from Hepa1-6 cells. Resuspend the above materials with PBS and store them at -80 °C for later use. Dissolve the vesicles in PBS, and use a DLS nanoparticle size analyzer to detect their particle size and surface potential, and observe their microscopic morphology

[0059] 2. Experimental results The western blot detection results showed that DC AFP successfully expressed AFP antigen ( Figure 1 Content A); DEV AFP had obvious AFP bands ( Figure 1 Content B), indicating that DC AFP cells could secrete DEV expressing AFP protein

[0060] The flow cytometry detection results showed that compared with DEV and DEV vec , the expression levels of major histocompatibility complex MHC-I, MHC-II, and costimulatory molecules CD80 and CD86 in DEV AFP were significantly up-regulated, indicating that DEV AFPHave the potential to present antigens and activate T cells ( Figure 2 Content A~ Figure 2 Content D).

[0061] The average particle size of various extracellular vesicles is 100 - 600 nm ( Figure 3 Content A), and the zeta potential is about -14 mV ( Figure 3 Content B). There is no significant difference among groups, indicating that extracellular vesicles derived from dendritic cells carrying antigens and loaded with drugs basically do not change the particle size and Zeta potential of extracellular vesicles. The TEM detection results show ( Figure 3 Content C) that various extracellular vesicles are monodisperse and irregular spherical structures, with membranous vesicular structures conforming to the characteristics of extracellular vesicles.

[0062] Example 2 Lymph node targeting of drug-loaded vesicles derived from dendritic cells overexpressing tumor antigens 1. Experimental procedures (1) Detect the expression of CCR7 in DEV, Cal@DEV, DEV AFP , Cal@DEV AFP. by western blot according to the steps of Example 1 (4).

[0063] (2) Establish a mouse Hepa1-6 orthotopic liver cancer tumor model Inoculate 3×10 6 Hepa1-6 cells at the axillary part of C57BL / 6 mice to establish a subcutaneous liver cancer model. Wait until the tumor volume grows to about 500 mm 3 . Dissect the tumor and cut it into tumor pieces of about 1 mm×1 mm×1 mm in size. Inoculate 3 - 4 tumor pieces at the liver part of C57BL / 6 mice to establish a mouse Hepa1-6 orthotopic liver cancer tumor model.

[0064] (3) On the 16th day after modeling, randomly divide the orthotopic liver cancer mice into four groups, and inject IR780-labeled DEV, DEV AFP , Cal@DEV, Cal@DEV AFP(Protein amount was 7.5 mg / kg, and the amount of Cal was 134 μg / kg). After 48 h of administration, the mice were sacrificed by cervical dislocation. The axillary lymph nodes (ALN), inguinal lymph nodes (ILN), and mesenteric lymph nodes (MLN) of the mice were surgically dissected, and the IR780 fluorescence intensity of each lymph node was detected using a small animal in vivo imager. Using the immunofluorescence section method, the inguinal lymph nodes of mice in different treatment groups were fixed overnight in 4% paraformaldehyde, dehydrated with 10%, 20%, and 30% sucrose solutions in gradient, and then the lymph nodes were embedded with OCT frozen section embedding agent. After being cut into 20-μm-thick slices using a cryostat, they were transferred onto glass slides, covered with coverslips, and the distribution of the material in the lymph nodes was detected using an FV3000 laser confocal microscope.

[0065] 2. Experimental results The Western blot results showed that ( Figure 4 ) both DEV AFP and Cal@DEV AFP could express high levels of CCR7, indicating that Cal@DEV AFP had the potential for lymph node homing.

[0066] Compared with the DEV, DEV AFP , and Cal@DEV groups, the fluorescence intensity in the ALN, ILN, and MLN of the mice in the Cal@DEV AFP group was the highest ( Figure 5 Content A and Figure 5 Content B). The immunofluorescence section results also showed that, compared with other treatment groups, the distribution amount of Cal@DEV AFP group in the paracortical area of the ILN was the highest ( Figure 5 Content C and Figure 5 Content D). It indicated that Cal@DEV AFP could effectively target lymph nodes.

[0067] Example 3 Regulation effect of Cal-loaded vesicles derived from dendritic cells overexpressing tumor antigen AFP on FRCs 1. Experimental procedures The activated lymph node FRCs were obtained by pretreating lymph node FRCs with the supernatant of Hepa1-6 cells, and equal volumes of PBS, DEV, DEV AFP , Cal, Cal@DEV, Cal@DEV AFPCo - incubate with activated FRCs for 48 h (protein concentration is 3.35 μg / mL, Cal concentration is 60 ng / mL), and use qRT - PCR to detect the changes in the expression levels of FRCs - related genes ( Acta2 , Pdpn , Ccl19 , Ccl21 ) in different treatment groups, and use immunofluorescence to detect the changes in the extracellular matrix proteins (α - smooth muscle actin α - SMA; fibronectin) and chemokines (CCL19 and CCL21) of FRCs after treatment in different treatment groups.

[0068] 2. Experimental results The qRT - PCR results showed that, compared with other groups, the mRNA expression levels of AFP and Acta2 in FRCs treated with Cal@DEV or Cal@DEV Pdpn were significantly down - regulated, and the mRNA expression levels of Ccl19 and Ccl21 were significantly up - regulated ( Figure 6 Content A~ Figure 6 Content D).

[0069] The immunofluorescence detection results showed ( Figure 7 ) that the Cal@DEV group and the Cal@DEV AFP group could significantly reduce the expression of extracellular matrix proteins (α - SMA and fibronectin) of FRCs and significantly up - regulate the expression of chemokines (CCL19 and CCL21) of FRCs. In summary, the drug encapsulated in dendritic cell - derived drug - loaded vesicles can regulate the function of fibroblastic reticular cells in lymph nodes, reshape the lymph chemokine network and degrade the extracellular matrix.

[0070] Example 4 Dendritic cell - derived Cal - loaded vesicles overexpressing tumor antigen AFP target lymph nodes via the CCR7 - CCL19 / CCL21 pathway 1. Experimental procedures Construct a mouse Hepa1 - 6 orthotopic liver cancer tumor model according to the steps (2) of Example 2. Intraperitoneally inject equal volumes of PBS, αCCL19 (10 μg), and αCCL21 (10 μg) (administer the drug every two days). 6 h before the second injection, intravenously inject IR780 - labeled Cal@DEV AFP (protein amount is 7.5 mg / kg, Cal amount is 134 μg / kg). After 48 h of drug administration, use a small animal in - vivo imager and immunofluorescence sectioning to detect the distribution of Cal@DEV AFP in lymph nodes.

[0071] 2. Experimental Results The results showed that blocking either CCL19 or CCL21 significantly reduced the distribution of Cal@DEV AFP in ALN and ILN, and blocking CCL21 also reduced the distribution of Cal@DEV AFP in MLN ( Figure 8 Content A and Figure 8 Content B). The results of immunofluorescence detection further confirmed that blocking either CCL19 or CCL21 significantly reduced the distribution of Cal@DEV AFP in the paracortical area of ILN ( Figure 8 Content C and Figure 8 Content D). It indicated that Cal@DEV AFP could increase lymph node targeting by upregulating the CCR7-CCL19 / CCL21 signaling pathway.

[0072] Example 5 Activation of CD8 + T Cells by Dendritic Cell-Derived Cal-Encapsulating Vesicles Overexpressing Tumor Antigen AFP 1. Experimental Procedures Use the Mojosort™ Mouse CD8 + T Cell Isolation Kit and Mojosort magnetic poles to isolate CD8 + T cells, and co-incubate them with an equal volume of PBS, Cal, DEV, Cal@DEV, DEV AFP , Cal@DEV AFP (protein concentration 3.35 μg / mL, Cal concentration 60 ng / mL) for 96 h. Use a flow cytometer to detect the expression of IFN-γ and GzmB in CD8 + T cells to evaluate the activation degree of CD8 + T cells.

[0073] 2. Experimental Results The results showed that compared with other treatment groups, the DEV AFP group and the Cal@DEV AFP group could significantly upregulate the positive ratios of IFN-γ ( Figure 9 Content A) and GzmB ( Figure 9 Content B) in CD8+ T cells, indicating that both DEV AFP and Cal@DEV AFP could directly activate CD8 + T cells.

[0074] Example 6 Regulation of Activated FRCs by Dendritic Cell-Derived Cal-Encapsulating Vesicles Overexpressing Tumor Antigen AFP on CD8 +Role of T cell recruitment, survival, and phenotypic changes 1. Experimental procedures (1) Pretreat lymph node FRCs with the supernatant of Hepa1-6 cells to obtain activated lymph node FRCs, and add equal volumes of PBS, DEV, DEV AFP , Cal, Cal@DEV, Cal@DEV AFP (protein concentration: 3.35 μg / mL, Cal concentration: 60 ng / mL) and co-incubate with the activated FRCs for 96 h. Collect the FRCs cell supernatant. Place the FRCs cell supernatant in the lower layer of a Transwell (5 μm pore size) chamber, and place CD8 + T cells in the upper layer of the Transwell chamber. After co-incubation for 8 h, detect the number of CD8 + T cells in the lower layer by flow cytometry. Additionally, place the FRCs cell supernatant treated with equal volumes of PBS, DEV AFP , Cal@DEV AFP (protein concentration: 3.35 μg / mL, Cal concentration: 60 ng / mL) for 96 h in the lower layer of a Transwell (5 μm pore size) chamber, and add PBS, αCCL19 (5 μg / mL), or αCCL21 (5 μg / mL) to the FRCs cell supernatant treated with Cal@DEV AFP for pre-treatment for 2 h. Place CD8 + T cells in the upper layer of the Transwell chamber. After co-incubation for 8 h, detect the number of CD8 + T cells in the lower layer by flow cytometry, that is, the recruitment of CD8 + T cells.

[0075] (2) Use qRT-PCR to detect the Il-7 mRNA expression level of FRCs in the treatment group in step (1). Then collect the FRCs cell supernatant and co-culture it with CD8 + T cells for 72 h. Use the Annexin-V / PI kit to detect the survival of CD8 + T cells after co-culture.

[0076] (3) Co-incubate CD8 + T cells with equal volumes of PBS, DEV, DEV AFP , Cal, Cal@DEV, Cal@DEV AFP (protein concentration: 3.35 μg / mL, Cal concentration: 60 ng / mL) for 96 h. Then co-culture the treated CD8 + T cells with the FRCs cell supernatant collected after the same treatment in step (1) for 72 h, and detect CD8 by flow cytometry.+ Phenotypic changes of T cells. Sca1 + CD122 + CD44 - CD62L + is stem cell-like memory T cell CD8 + T SCM (T cells with T naive phenotype and highly expressing Sca1 and CD122); CD44 + CD62L + is central memory T cell CD8 + T CM ; CD44 + CD62L - is effector memory T cell CD8 + T EM .

[0077] 2. Experimental results The results showed that, compared with other groups, the supernatant of FRCs cells collected after treatment with Cal@DEV and Cal@DEV AFP significantly upregulated the recruitment of CD8 + T cells ( Figure 10 Content A). When the supernatant of FRCs cells collected after Cal@DEV AFP regulated FRCs was pretreated with αCCL19 or αCCL21 to neutralize the chemokines CCL19 or CCL21 in the supernatant of FRCs cells, the recruitment of CD8 + T cells was significantly reduced ( Figure 10 Content B). In summary, it was shown that Cal@DEV AFP could increase the recruitment of CD8 + T cells by regulating activated FRCs, and this effect was dependent on the upregulation of chemokines CCL19 and CCL21.

[0078] Compared with other groups, the mRNA expression level of AFP of FRCs after treatment with the Cal@DEV group and the Cal@DEV Il-7 group was the highest ( Figure 11 Content A). The survival ratio of CD8 AFP T cells co-cultured with the supernatant of FRCs cells collected after treatment with Cal@DEV or Cal@DEV + was significantly increased ( Figure 11 Content B), indicating that Cal@DEV AFP regulated activated FRCs was beneficial to the survival of CD8 + T cells.

[0079] The results showed that CD8 +T cells are pretreated with Cal@DEV AFP and then co-cultured with the supernatant of FRCs cells collected after being treated with Cal@DEV AFP to increase the proportion of CD44 + CD62L - in CD8 + T cells ( Figure 12 Content A), and at the same time, the proliferation proportion of CD44 - CD62L + in CD8 Figure 12 T cells can be increased ( AFP Content B). The results of flow cytometry detection show that, compared with other groups, the Cal@DEV + group can significantly increase the proportion of T SCM in CD8 Figure 12 T cells ( Figure 12 Content C and AFP Content D). At the same time, after treatment with Cal@DEV + the proportion of T CM in CD8 Figure 12 T cells can be significantly increased ( AFP Content E). In summary, Cal@DEV + regulates FRCs, which is beneficial to the generation of memory CD8

[0080] Example 7 Regulation of FRCs by Dendritic Cell-derived ATRA-loaded Vesicles Overexpressing Tumor Antigen AFP 1. Experimental procedures (1) Replace calcipotriol (Cal) with all-trans retinoic acid (ATRA), and collect dendritic cell-derived ATRA-loaded vesicles overexpressing tumor antigen AFP according to the method of Example 1.

[0081] (2) Construct activated lymph node FRCs according to the method of Example 3, add equal volumes of PBS, DEV, DEV AFP , ATRA, ATRA@DEV, ATRA@DEV AFP (protein concentration is 3.35 μg / mL, ATRA concentration is 60 ng / mL) and co-incubate with activated FRCs for 48 h, and use qRT-PCR to detect the changes in the expression levels of FRCs-related genes ( Acta2 , Pdpn , Ccl19 , Ccl21 ) in different treatment groups.

[0082] 2. Experimental results Compared with other groups, the FRCs treated with ATRA@DEV or ATRA@DEV AFP ...Acta2 and Pdpn showed significantly down - regulated mRNA expression levels ( Figure 13 Content A and Figure 13 Content B), Ccl19 and Ccl21 showed significantly up - regulated mRNA expression levels ( Figure 13 Content C and Figure 13 Content D).

[0083] In summary, it is shown that the extracellular vesicles derived from dendritic cells overexpressing tumor antigens provided in this application can carry various activated fibroblastic reticular cell deactivators to achieve functional regulation of FRCs, degrade the extracellular matrix and reshape the lymph chemokine network.

[0084] Example 8 Effect of Dendritic Cell - derived Cal - loaded Vesicles Overexpressing Tumor Neoantigen Adpgk on the Recruitment and Phenotypic Changes of CD8 + T Cells 1. Experimental Procedures (1) Preparation of Dendritic Cell - derived Cal - loaded Vesicles Stably Expressing Adpgk Based on the amino acid sequence of antigen peptide Adpgk, the DNA sequence of antigen peptide Adpgk was obtained, primers were designed for circular PCR, and the DNA sequence of Adpgk was integrated into the expression vector pCDH - Zeocin to obtain the expression vector pCDH - Adpgk - Zeocin, which was sequenced and identified for expression in 293T cells. According to the method of Example 1, the expression vector pCDH - Adpgk - Zeocin was transfected into 293T cells, and a dendritic cell line (DC Adpgk ) stably expressing the neoantigen Adpgk was screened using bleomycin at a final concentration of 100 μg / mL. Then, dendritic cell - derived Cal - loaded vesicles overexpressing tumor neoantigen Adpgk (Cal@DEV Adpgk ) were prepared.

[0085] (2) According to the method of step (1) in Example 6, different treatment groups (PBS, DEV, DC Adpgk , Cal, Cal@DEV, Cal@DEV Adpgk ) were used for treatment, and the recruitment of CD8 + T cells and the phenotypic changes of CD8 + T cells were detected.

[0086] 2. Experimental Results Compared with other groups, the FRCs cell supernatant collected after treatment with Cal@DEV, Cal@DEV Adpgk showed significantly up - regulated recruitment of CD8 + T cells ( Figure 14Content A). Meanwhile, CD8 + T cells were pretreated with Cal@DEV Adpgk and co-cultured with the supernatant of FRCs cells collected after being treated with Cal@DEV Adpgk to increase the proportion of T + in CD8 SCM ( Figure 14 Content B and Figure 14 Content C) and T CM ( Figure 14 Content D).

[0087] In summary, the drug-loaded vesicles provided in this application can express different types of tumor antigens, effectively regulate activated fibroblastic reticular cells, and promote the recruitment of CD8 + T cells and the generation of a stem cell memory phenotype.

[0088] Example 9 Antitumor effect of dendritic cell-derived drug-loaded vesicles overexpressing tumor antigens 1. Experimental procedures According to the steps (2) of Example 2, a mouse orthotopic liver cancer tumor model of Hepa1-6 was constructed. On the 7th day of modeling, an equal volume of PBS, Cal, DEV, Cal@DEV, DEV AFP , Cal@DEV AFP (protein amount was 7.5 mg / kg, Cal amount was 134 μg / kg) were injected via the tail vein respectively. Record the day before administration as day 0, then administer the drug once a week for a total of 3 times. On the 17th day of the experiment, collect the mouse serum, sacrifice the mice, and dissect the mouse lymph nodes and tumor tissues.

[0089] 2. Experimental results From Figure 15 , Figure 16 Content A, it can be seen that the tumor tissues and average tumor weights of the mice in the Cal@DEV AFP treatment group were significantly smaller than those of other treatment groups. In addition, the survival period of the mice in the Cal@DEV AFP treatment group was significantly prolonged ( Figure 16 Content B), significantly better than other treatment groups. In summary, it shows that Cal@DEV AFP treatment can significantly inhibit the growth of orthotopic liver cancer tumors and prolong the survival period of tumor-bearing mice.

[0090] Example 10 Regulation of the immune microenvironment by dendritic cell-derived drug-loaded vesicles overexpressing tumor antigens 1. Experimental procedures The mouse lymph nodes and tumor tissues dissected in Example 9 were made into single-cell suspensions, labeled with fluorescent antibodies, and the changes in immune cells in the lymph nodes and tumor tissues were detected using a flow cytometer. CD8 + T cells activated at the lymph node site need to be recruited and infiltrated into the tumor tissue to exert anti-tumor efficacy. The flow cytometer was used to further analyze the effects of Cal@DEV AFP on the changes in CD8 + T cells and their subsets in the tumor tissue. CD8 + IFN-γ + T cells are specific CD8 + T cells capable of secreting interferon-γ (IFN-γ). CD8 + GzmB + T cells are T cells that secrete granzyme. CD8 + Ki67 + T cells are CD8 + T cells that highly express the Ki-67 protein. Sca1 + CD122 + CD44 - CD62L + are stem cell-like memory T cell CD8 + T SCM (T cells with a T naive phenotype and highly expressing Sca1 and CD122); CD44 + CD62L + are central memory T cell CD8 + T CM ; CD44 + CD62L - are effector memory T cell CD8 + T EM .

[0091] 2. Experimental results The results showed that compared with other treatment groups, the Cal@DEV AFP treatment group could significantly increase the number of CD8 + T ([[]] Figure 17 Content A), effector CD8 + IFN-γ + T cells and effector CD8 + GzmB + T cells ([[]] Figure 17 Content B and Figure 17 Content C) and proliferating CD8 + Ki67 + T cells ([[]] Figure 17 Content D), indicating that Cal@DEV AFPThe treatment can effectively enhance the effector function and proliferative ability of CD8 + T cells in the lymph node. Meanwhile, Cal@DEV AFP treatment can significantly increase the number of CD8 + T SCM ( Figure 17 Content E and Figure 17 Content F) and CD8 + T CM ( Figure 17 Content G) cells in the lymph node, indicating that Cal@DEV AFP treatment can effectively enhance the memory of CD8 + T cells in the lymph node. In summary, it shows that Cal@DEV AFP can activate the CD8 + T cell-mediated immune response in the lymph nodes of orthotopic liver cancer-bearing mice.

[0092] Compared with other treatment groups, the number of CD8 AFP T cells in the tumor tissue of the Cal@DEV + treatment group mice increased significantly ( Figure 18 Content A). Meanwhile, the number of CD8 AFP T cells in the tumor tissue of the Cal@DEV + T SCM ( Figure 18 Content B and Figure 18 Content C), CD8 + T CM ( Figure 18 Content D) and CD8 + T EM ( Figure 18 Content E) in the Cal@DEV AFP treatment group was significantly higher than that in other groups, indicating that Cal@DEV + treatment can significantly increase the number of memory CD8 + T cells in the tumor tissue. Under the chronic antigen stimulation in the tumor microenvironment, memory CD8 AFP T cells can rapidly differentiate into effector T cells. Compared with other treatment groups, the number of activated effector CD8 + T cells (CD8 + IFN-γ + 、CD8 + GzmB + ) in the tumor tissue of the Cal@DEV Figure 18 treatment group increased significantly ( Figure 18 Content F and AFP After Cal@DEV treatment, the proliferating CD8 + T cells (CD8 +Ki67 + also showed a significant increase in the number of Figure 18 Content H). In summary, Cal@DEV AFP can significantly increase the memory CD8 + T cells and effector CD8 + T cell subsets in the matrix-rich Hepa1-6 primary liver cancer, and activate the anti-tumor immune response mediated by CD8 + T cells.

[0093] Example 11 Biosafety of Drug-loaded Vesicles Derived from Dendritic Cells Overexpressing Tumor Antigens 1. Experimental Procedures The sera of the mice in each treatment group collected in Example 9 were tested for the contents of the blood biochemical indexes AST, ALT, LDH, BUN, CREA, and CK.

[0094] 2. Experimental Results The results showed that there were no significant changes in the contents of AST, ALT, LDH, BUN, CREA, and CK in the sera of the mice in different treatment groups ( Figure 19 Content A~ Figure 19 Content F), indicating that Cal@DEV AFP has no toxic or side effects on the body and has good biosafety.

[0095] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A dendritic cell-derived drug-loaded vesicle for targeted modification of lymph nodes to enhance the efficacy of anti-tumor therapy, characterized in that, It includes extracellular vesicles produced by dendritic cells overexpressing tumor antigens, and small molecule drugs encapsulated by the extracellular vesicles; the small molecule drug is an activator fibroblast reticular cell deactivator.

2. The dendritic cell-derived drug-loaded vesicles according to claim 1, wherein The mass ratio of the extracellular vesicles to the small molecule drug is 1000:(1 - 50).

3. The dendritic cell-derived drug-loaded vesicles according to claim 1, wherein The dendritic cells overexpressing tumor antigens are obtained by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing.

4. The dendritic cell-derived drug-loaded vesicles according to claim 1 or 3, characterized in that, The tumor antigens include one or more of liver cancer antigen AFP, carcinoembryonic antigen CEA, prostate specific antigen PSA, squamous cell carcinoma antigen SCCA, ovarian cancer antigen CA125, pancreatic cancer antigen CA19 - 9, melanoma antigen, cancer - testis antigen CTAs and tumor neoantigens; The tumor - specific antigens include one or more of Actn4, Adpgk, Ap3d1, Tubb3, Dag1, Eef2, Tnpo3, Tubb3, Reps1, Cpne1 and Cpsf3l.

5. The dendritic cell-derived drug-loaded vesicles according to claim 1 or 2, characterized in that, The small molecule drugs include one or more of calcipotriol, all - trans retinoic acid and pirfenidone.

6. The dendritic cell-derived drug-loaded vesicles according to claim 1, characterized in that, The average particle size of the dendritic cell - derived drug - loaded vesicles is 100 nm - 600 nm.

7. A method for preparing dendritic cell-derived drug-loaded vesicles according to any one of claims 1 to 6, characterized in that, It includes the following steps: S1. Construct dendritic cells stably overexpressing the tumor antigen by genetic engineering modification; S2. Subject the dendritic cells stably overexpressing the tumor antigen to pressure treatment, and collect the extracellular vesicles produced by the dendritic cells overexpressing the tumor antigen; S3. Co - incubate the extracellular vesicles and the small molecule drugs, and collect the dendritic cell - derived drug - loaded vesicles.

8. The preparation method according to claim 7, wherein In step S1, the dendritic cells overexpressing the tumor antigen are prepared by lentiviral transfection, adenoviral transfection, plasmid transfection or gene editing; and / or, In step S2, the pressure treatment methods include one or more of ultraviolet irradiation, starvation, heat stimulation.

9. The preparation method according to claim 7, wherein In steps S2 and S3, the collection conditions are: collecting at 4°C with a centrifugal force of 500 - 20,000g.

10. An anti-tumor drug, characterized in that, It includes the dendritic cell - derived drug - loaded vesicles according to any one of claims 1 to 6.

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