CN114807048A

CN114807048A - Genetically engineered antigen-presenting extracellular vesicle and preparation method and application thereof

Info

Publication number: CN114807048A
Application number: CN202210545450.3A
Authority: CN
Inventors: 汪超; 许方
Original assignee: Suzhou University
Current assignee: Xili Biotechnology Suzhou Co ltd
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2022-07-29

Abstract

The invention discloses a genetically engineered antigen-presenting extracellular vesicle, which carries tumor-associated antigen, overexpresses an immunoregulatory molecule and a single-chain antibody, and prepares a vaccine preparation for tumor treatment based on the antigen-presenting extracellular vesicle, wherein the vaccine preparation is especially prominent in tumor metastasis inhibition and has great potential as a new-generation drug-carrying platform. The genetically engineered extracellular vesicle constructed by the invention not only maintains antigen presenting capability, but also targets tumor antigen, and adjusts solid tumor immune microenvironment, thereby effectively activating intratumoral T cells to exert antitumor activity, and finally realizing the functions of tumor killing and metastasis inhibition.

Description

Genetically engineered antigen-presenting extracellular vesicle and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a genetically engineered antigen-presenting extracellular vesicle and a preparation method and application thereof.

Background

Immune system-targeted cancer immunotherapy has achieved clinical success in a variety of cancers. However, the relatively low response rate of immunotherapy remains a challenge for solid tumors, e.g., Chimeric Antigen Receptor (CAR) T cells, which, due to their relatively large size, are not effective in penetrating tumor tissue through the vascular endothelium and thus are not effective in treating solid tumors. Immune Checkpoint Blockade (ICB) antibodies lack tumor accumulation due to their relatively low tumor targeting and have a relatively low response rate in some solid tumors.

Nanoscale Extracellular Vesicles (EVs) are membrane vesicles secreted by cells and released to Extracellular matrix, have the diameter of 30-100nm, can reach other cells and tissues through a circulating system, are a novel cell-free technology, generate a remote control effect, have the advantages of high physicochemical stability, high biocompatibility, biodegradability, low toxicity, non-immunogenicity and the like, can be used as excellent carriers of various bioactive substances, and become a replacement choice for cell therapy in the fields of regenerative medicine, drug delivery, tumor immunotherapy and the like. However, in the treatment of cancer, natural EVs are easy to be trapped in non-specific tissues (particularly liver and lung), so that the in vivo targeting is insufficient, and the clinical treatment effect is greatly reduced due to the problems of low drug loading rate and the like. Therefore, the EVs are engineered, the problems of complicated drug loading process, low delivery efficiency and insufficient targeting capability are solved, and a new treatment strategy is provided.

Dendritic cells play a role in tumor immunotherapy, while extracellular vesicles play an important role in antigen presentation of dendritic cells, and the extracellular vesicles produced by dendritic cells can carry Major Histocompatibility Complex (MHC) -I, MHC-II and T cell costimulatory molecules, activating anti-tumor immune responses. (Yantsunami et al. role of extracellular vesicles of dendritic cells in antigen presentation and tumor immunotherapy research progress [ J ]. International journal of immunology 2020,43(4):406-412.DOI:10.3760/cma.j. issn.1673-4394.2020.04.010.). There are currently no reports of dendritic cells in combination with CAR-T therapy and immune checkpoint blockade therapy (ICB) to achieve dendritic cell-derived EVs in tumor-targeted immunotherapy.

Disclosure of Invention

In order to solve the technical problems, the invention discloses a genetically engineered antigen-presenting extracellular vesicle and provides a drug delivery system for treating solid tumors and lung metastasis by using the extracellular vesicle. The preparation has good biocompatibility and simple and mature preparation process, can effectively act on lymph nodes and tumor parts, and shows remarkable curative effect in tumor treatment by activating the activity of lymphocyte T cells and remodeling the tumor immunosuppressive microenvironment.

It is a first object of the present invention to provide a genetically engineered antigen-presenting extracellular vesicle that presents a tumor-associated antigen, while overexpressing an immunomodulatory molecule and a Single-chain antibody (scfv); the immune regulatory molecule is selected from one or more of programmed death receptor 1(PD-1), tumor necrosis factor receptor superfamily member 9 receptor ligand (4-1BBL), tumor necrosis factor receptor superfamily member 4 ligand (OX40L), Glucocorticoid-induced tumor necrosis factor receptor ligand (glucocorticoids-induced TNF receptor ligand, GITRL) and T-cell immunoglobulin domain mucin domain protein-3 (T-cell immunoglobulin domain 3, TIM3), and the single chain antibody is selected from HER2-targeted scFv (single chain antibody variable region targeting proto-oncogene human epidermal growth factor receptor 2), CD19-targeted scFv (single chain antibody variable region targeting leukocyte differentiation antigen 19), MSLN-targeted scFv (single chain antibody variable region targeting mesothelin), GD2-targeted scFv (single chain antibody variable region targeting double ganglioside variable region), One or more of CEA-targeted scFv (Single chain antibody variable region targeting carcinoembryonic antigen), EGFRvIII-targeted scFv (Single chain antibody variable region targeting epidermal growth factor receptor type III mutant antibody), and VEGFR2-targeted scFv (Single chain antibody variable region targeting vascular endothelial growth factor receptor 2).

Further, the antigen-presenting extracellular vesicles also reduce the expression of the immunomodulatory molecular ligand, wherein reducing expression includes knock-down and knock-out conditions.

Further, immunomodulatory molecular ligands include, but are not limited to, one or more of Programmed cell death ligand-1 (PD-L1), Programmed cell death ligand-2 (PD-L2), and Galectin-9 (Galectin-9, Gal-9).

Further, expression of immunomodulatory molecular ligands was knocked down by siRNA technology.

Furthermore, the load is that the tumor vaccine and the antigen presenting cells are incubated together, after the tumor vaccine is phagocytized by the antigen presenting cells, the antigen presenting cells can be used for antigen presenting and activating, then the prepared nano vesicles contain tumor-related antigens, and the tumor-specific T cells can be activated by the loaded antigens after the nano vesicles are infused into the body.

Furthermore, immune adjuvants such as CpG, poly (I: C), poly ICLC and the like can be added in the incubation process, and when the immune adjuvants and the antigen are loaded on the tumor vaccine together, the tumor vaccine can be better activated after being phagocytized by antigen presenting cells.

Further, the antigen-presenting cells include Dendritic Cells (DCs), macrophages, B lymphocytes, and the like.

Further, the DC cells can be derived from any cell from which isolated dendritic cells can be prepared, including but not limited to, stem cells, cell lines, bone marrow cells, peripheral immune cells, and the like.

Furthermore, the particle diameter of the genetic engineering antigen presenting extracellular vesicle is 30-200nm, and the potential is 0-40 mV.

The present invention is based on the advantages of CAR-T therapy and immune checkpoint blockade therapy (ICB) in cancer treatment, and develops a new therapeutic EVs by genetically modifying EVs-producing cells, with two functional features: one is a single chain antibody (scFv) capable of expressing a tumor targeting molecule, specifically targeting a tumor antigen, similar to the CAR-T tumor targeting strategy; and secondly, the tumor immune regulatory molecule can be expressed, can be competitively combined with and block the immune regulatory molecule in the tumor environment, is similar to an ICB strategy, and can remodel the immunosuppressive tumor microenvironment. In addition, the prepared genetically engineered antigen-presenting extracellular vesicle overcomes the defects of the traditional DC cell therapy, and the antigen-presenting cell has a costimulatory molecule, so that the costimulatory molecule does not need to be expressed like a CAR-T cell, and the problem of complicated construction process of the CAR-T cell is solved. Therefore, the invention combines the advantages of CAR-T and ICB in cancer treatment and the advantages of EVs derived from antigen presenting cells, carries out engineering transformation on the EVs, becomes a new treatment strategy, solves the problems of complicated drug loading process, low delivery efficiency, insufficient targeting capability and the like, and obviously improves the treatment effect of solid tumors.

The second purpose of the invention is to provide a preparation method of the genetically engineered antigen-presenting extracellular vesicle, which comprises the following steps:

(1) overexpressing genes encoding an immunomodulatory molecule and a single-chain antibody in an Antigen Presenting Cell (APC), and optionally knocking down the expression of an immunomodulatory molecule ligand in the antigen presenting cell to obtain a genetically engineered antigen presenting cell (eAPC);

(2) co-incubating a tumor vaccine containing a tumor-associated antigen with a genetically engineered antigen presenting cell (eAPC) to obtain the genetically engineered antigen presenting cell loaded with the tumor-associated antigen;

(3) preparing the genetic engineering antigen presenting cells loaded with the tumor-associated antigens obtained in the step (2) into vesicles to obtain the genetic engineering antigen presenting extracellular vesicles (eAPC-EVs).

Further, genes encoding immune modulatory molecules and single chain antibodies are overexpressed by lentiviral transfection, plasmid transfection, or gene editing techniques (e.g., gene insertion). In one embodiment of the invention, BMDCs (Bone-marrow dendritic cells) are made to highly express immune regulatory molecules and single-chain antibody variable region gene segments capable of targeting tumor surface antigen molecules by using a lentivirus transfection technology.

Further, Lipofectamine3000 transfection reagent mediated siRNA technology was used to knock down the expression of immunomodulatory molecular ligands in antigen presenting cells.

Furthermore, the tumor-associated antigen can be a whole-cell antigen prepared from tumor cells or tumor tissues, or can be an antigen polypeptide associated with tumor.

In the invention, firstly, the high expression of one or more scFv is used to enhance the extracellular vesicle targeting to the tumor with high expression and specific binding of scFv to antigen molecules; secondly, the immunoregulation function is achieved through the high-expression immunoregulation molecules, and the immunoregulation molecule function is further enhanced through knocking down or knocking out the expression of the immunoregulation molecule ligand, so that the tumor immune microenvironment is regulated; finally, according to the antigen delivery characteristics of the antigen presenting cells, the antitumor effect is generated by enhancing the lymphocyte activation function of extracellular vesicles through loading tumor-associated antigens. In conclusion, the genetically engineered antigen-presenting extracellular vesicles achieve tumor-targeted immunotherapy through multiple functions.

The third purpose of the invention is to provide the application of the genetically engineered antigen presenting extracellular vesicle in preparing antitumor drugs.

Further, the tumor includes solid tumors including, but not limited to, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, skin cancer, breast cancer, cervical cancer, bone cancer, and the like.

Further, the antitumor drug can be used for the prevention and treatment of cancer, such as the prevention and treatment of tumor metastasis and recurrence. In the treatment of tumor metastasis, the antitumor drug can block the establishment of a pre-metastatic microenvironment induced by tumor extracellular vesicles, thereby inhibiting tumor metastasis.

Further, the administration mode of the medicine is intravenous injection.

In order to further improve the anti-tumor effect of the DC extracellular vesicle, the invention combines the antigen presentation function with the immune checkpoint blockade therapy and the CAR-T therapy, enhances the positioning of the nano preparation on lymph nodes and tumors, generates high-efficiency anti-tumor immune activation efficacy and lasting immune memory effect, and realizes tumor killing and the inhibition of the recurrence and metastasis of cancers.

The fourth purpose of the invention is to provide a drug delivery system, which comprises the genetically engineered antigen-presenting extracellular vesicles. The extracellular vesicle can be independently used as a drug loading platform, so that the problem of low drug loading rate is solved, and drugs can be modified on the basis of the extracellular vesicle to serve as the drug loading platform, such as endogenous expression proteins or RNA and other drugs for realizing internal drug loading or external drug loading through mechanical, chemical and other modes.

By means of the scheme, the invention at least has the following advantages:

(1) the antigen-presenting extracellular vesicle capable of simultaneously highly expressing the immunoregulatory molecule and the single-chain antibody provided by the invention has high biological safety and a simple preparation process, and when the antigen-presenting extracellular vesicle is used for treating tumors, on one hand, lymph nodes are targeted to induce tumor specific T cells to generate, on the other hand, the tumor immune microenvironment is locally improved by targeting tumors to improve the tumor immunotherapy effect, so that a new strategy is provided for anti-tumor immunotherapy.

(2) Because the tumor cells release the extracellular vesicles expressing PD-L1 to create a good pre-tumor metastasis microenvironment for a far-end tissue or promote tumor progression through immunosuppression of the whole body, the invention blocks the function of the extracellular vesicles of the tumor cells and inhibits tumor metastasis by utilizing the function of the genetically engineered APC extracellular vesicles targeting tumor surface immunoregulation molecular ligands and tumor surface antigens.

(3) The gene engineering antigen-presenting extracellular vesicle constructed by the invention has stability and targeted accumulation in vivo, overcomes the defects of low response rate, low treatment efficiency and the like of the traditional DC vaccine, can be used as a high-efficiency and safe cell-free treatment system, is beneficial to clinical transformation, and has important significance for further enhancing the immunotherapy of tumors.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a diagram of immunofluorescence analysis of mouse bone marrow-derived dendritic cells (BMDCs) of the present invention on cells with PD-1 and anti-CD19scFv after lentiviral transfection;

FIG. 2 is a graph showing the flow results of the expression of PD-1 and anti-CD19scFv in cells after lentivirus transfection of BMDCs according to the present invention;

FIG. 3 is a graph of immunofluorescence analysis of PD-L1 on cells of BMDCs of the present invention after transfection with PD-L1 siRNA;

FIG. 4 is a graph showing the flow results of PD-L1 on cells after BMDCs of the present invention were transfected with PD-L1 siRNA;

FIG. 5 is a graph showing the flow-through results of the expression levels of MHC-peptide and co-stimulatory molecules in BMDCs of the present invention after loading with tumor associated antigens;

FIG. 6 shows the size and shape of the extracellular vesicles (eDC-EVs) derived from genetically engineered BMDCs according to the present invention by transmission electron microscopy;

FIG. 7 is a graph showing the particle size distribution of eDC-EVs according to the present invention;

FIG. 8 is a graph of Zeta potential results for eDC-EVs according to the present invention;

FIG. 9 shows the expression of PD-1, PD-L1, anti-CD19scFv, MHCII, CD80 by immunoblot analysis eDC-EVs in accordance with the present invention;

FIG. 10 is a graph showing the flow results of 4-1BBL, TIM3, aHER2scFv, aVEGFR 2scFv expressed on cells after lentivirus transfection of BMDCs according to the present invention;

FIG. 11 shows eDC-EVs and PD-L1 in the present invention ⁺ Immunofluorescence analysis of cellular interactions;

FIG. 12 is a graph of immunofluorescence analysis of the interaction of eDC-EVs with CD19 on CT26-huCD19 cells in accordance with the present invention;

FIG. 13 shows the activation and proliferation of lymph node T cells by eDC-EVs according to the present invention;

FIG. 14 is a graph showing that eDC-EVs enhance the killing effect of T cells on tumor cells in the present invention;

FIG. 15 is a diagram of an Elisa analysis of the interaction of eDC-EVs with tumor extracellular vesicles PD-L1 according to the present invention;

FIG. 16 shows that eDC-EVs block the inhibition effect of tumor extracellular vesicle PD-L1 on T cell activation and proliferation;

FIG. 17 shows the effect of eDC-EVs in tumor burden (CT26-huCD19) in tumor and lymph node enrichment in mice of the present invention;

FIG. 18 shows the enrichment of eDC cells in tumor-negative (CT26-huCD19) mice and lymph nodes:

FIG. 19 is a graph showing the interaction of eDC-EVs of the present invention with PD-L1 in tumors and with CD3 in lymph nodes ⁺ Immunofluorescence analysis of T-interactions;

FIG. 20 is a graph showing the inhibition of tumor growth and survival rate of tumor-bearing mice by eDC-EVs of the present invention;

FIG. 21 is a graph of the effect of eDC cells of the invention on tumor growth in tumor-bearing mice and survival of the mice;

FIG. 22 shows eDC-EVs versus tumor-bearing mouse lymph node CD8 ⁺ Activation of T cells;

FIG. 23 shows the potentiating effect of eDC-EVs on anti-tumor immunity in tumor-bearing mice in accordance with the present invention;

FIG. 24 shows the effect of eDC cells of the invention on lymph node T cells from tumor-bearing mice:

FIG. 25 shows the effect of eDC cells on tumor immune cells of tumor-bearing mice:

FIG. 26 is a graph showing the inhibitory effect of eDC-EVs of the present invention on the pre-metastatic microenvironment of lungs constructed from the outer tumor vesicle;

FIG. 27 shows the inhibitory effect of eDC-EVs on lung metastasis caused by extracellular vesicles of tumor cells in the present invention.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The material sources in the embodiments of the invention are:

lentiviruses encoding the PD-1 gene were purchased from Heyu Biotechnology, Inc., and lentiviruses encoding the anti-CD19scFv gene were gifted by professor Campylons of Suzhou university; PD-L1 siRNA (SEQ ID NO: 5'-UGAGAAAACAAGUGAGAAUTT-3' and 5'-AUUCUCACUUGUUUUCUCATT-3') was purchased from general biosystems, Inc.; mouse granulocyte-macrophage colony stimulating factor GM-CSF (315-03-50) and interferon IFN gamma (315-05) were purchased from PeproTech; lipopolysaccharide LPS (L2630) was purchased from Sigma; PD-L1 antibody was purchased from Proteitech; the immunological adjuvant CpG-1826(5'-TCC ATG ACG TTC CTG ACG TT-3', tlrl-1826-5) was purchased from Invivogen; mouse Anti-CD3(100202) and Anti-CD28(16-0281038) were purchased from BioLegend; PD-L1 Elisa kit (RDR-PDCD1LG1-Mu) was purchased from Reddot Bio; cy5.5 fluorescent dye (HY-D0924) is available from MedChemexpress. PD-1 antibody (84651) was purchased from Cell signaling technology; PD-L1 antibody (25229) was purchased from Proteintech;

MHCII antibody (70257) was purchased from Abclonal; the CD80 antibody (15416) was purchased from Cell signaling technology.

Dendritic cells were extracted from mouse bone marrow and differentiated using 20ng/mL GM-CSF and 10ng/mL IL-4.

Female C57BL/6 mice, 6-8 weeks old, were purchased from Calvens laboratory animals, Inc., Changzhou. Mice were treated according to the protocols of the institute for Biochemical and cellular laboratory animal Care (IACUC).

EXAMPLE 1 construction of dendritic cell tumor vaccine (Engineered DCs, eDCs) with high expression of anti-CD19scFv and PD-1 and knockdown of PD-L1

(1) Isolation and culture of mouse Bone Marrow Dendritic Cells (BMDCs): bone marrow cells of tibia and femur of a mouse are separated, 20ng/mL GM-CSF and 10ng/mL IL-4 are added into RPMI 1640 culture medium containing 10% fetal bovine serum to be cultured for 6-7 days, and then dendritic cells are obtained.

(2) Constructing dendritic cells highly expressing anti-CD19scFv (HM852952) and PD-1(NM _ 008798.3): after mouse bone marrow-derived dendritic cells (BMDCs) are formed, the BMDCs are highly expressed with anti-CD19scFv and PD-1 by using a lentivirus-mediated gene transfection mode, and the results of immunofluorescence (figure 1) and flow cytometry (figure 2) show that the anti-CD19scFv and the PD-1 are highly expressed on the BMDCs, which indicates that the BMDCs highly expressed with the anti-CD19scFv and the PD-1 are successfully established.

(3) Constructing a dendritic cell with low expression of PD-L1: dendritic cells highly expressing anti-CD19scFv and PD-1 knock down the expression of PD-L1 by using siRNA, and immunofluorescence (figure 3) and flow (figure 4) results show that the expression of PD-L1 in BMDCs is remarkably reduced.

(4) Preparing a tumor-associated antigen: under the aseptic condition, fresh tumor tissues cut off from a tumor mouse are prepared into tumor cell single-cell suspension, the suspension is centrifuged at 1800rpm for 3min, the suspension is washed for 3 times by sterile PBS, the single-cell suspension is quickly frozen at minus 80 ℃ for 30min, then the suspension is quickly put into a water bath at 37 ℃ to be thawed for 10min, the suspension is repeatedly frozen and thawed for 4 to 5 times, the suspension is centrifuged at 3000rpm for 10min, and the supernatant is collected to obtain the tumor-associated antigen.

(5) Preparing a DC tumor vaccine: mu.g CpG and the obtained tumor associated antigen were incubated with genetically engineered BMDCs for 48h to load the BMDCs with tumor associated antigen, 10ng/mL LPS was stimulated for 24h and then flow cytometry was used to detect the expression of CD80, CD86, MHCI and MHCII, and flow cytometry results (FIG. 5) showed successful construction of a DC tumor vaccine carrying MHC-peptide and co-stimulatory molecules.

Example 2 extraction and characterization of Engineered DC-derived extracellular vesicles (eDC-EVs)

(1) The BMDCs which are loaded with tumor-associated antigens, highly express anti-CD19scFv and PD-1 and knock down PD-L1 are replaced by RPMI 1640 culture medium without exosome serum for continuous culture for 48h, and culture medium supernatant is collected;

(2) centrifuging culture medium supernatant at 4 deg.C for 10min at 300g, removing precipitate, centrifuging for 10min at 1000g, removing precipitate, centrifuging for 20min at 2000g, removing precipitate, centrifuging for 30min at 10000g, removing precipitate, centrifuging for 70min at 100000g, discarding supernatant, and adding PBS for suspension precipitation to obtain genetically engineered DC-derived extracellular vesicles (eDC-EVs);

(3) analyzing the shape and the particle size of the extracellular vesicles obtained in the step (2) by using a transmission electron microscope and DLS (figure 6, figure 7), wherein the Zeta potential is about-20 mV (figure 8); the expression of anti-CD19scFv, PD-1, PD-L1, CD80, MHCII on extracellular vesicles was analyzed by immunoblotting (FIG. 9).

In addition, the invention also successfully constructs BMDCs (figure 10) highly expressing other tumor antigen targeting molecules and immune regulatory molecules, and shows that a plurality of genetically engineered therapeutic extracellular vesicles can be constructed by using lentivirus transfection.

Example 3eDC-EVs Effect on the ability to interact with CD19 on PD-L1 and CT26 cells in vitro and on the anti-tumor efficiency of T cells

(1) eDC-EVs and PD-L1 ⁺ Cell interaction: CT26 colon cancer cells, bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) express PD-L1 under the induction of 20ng/mL IFN-gamma, Cy5.5 marker eDC-EVs and cells expressing PD-L1 are incubated for 24h, and Confocal Microcopy observes the co-localization of Cy5.5 and PD-L1, and the result shows (figure 11) that PD-1 on extracellular vesicles and PD-L1 have strong interaction.

(2) eDC-EVs interact with human-CD19 expressed on CT26 colon cancer cells: CT26 colon cancer cells highly express human-CD19 under the mediation of lentivirus encoding human-CD19 gene, then are incubated with Cy5.5 labeled eDC-EVs for 24 hours, and Confocal Microcopy observes the co-localization of Cy5.5 and CD19, and the result shows (figure 12) that anti-CD19scFv on extracellular vesicles has stronger interaction with CD 19.

(3) eDC-activation of lymph node T cells by EVs: and (3) taking the inguinal lymph node cells of the mice, incubating the inguinal lymph node cells with eDC-EVs and CT26-CD19 tumor cells for 48 hours, and detecting the activation and proliferation of T cells by flow cytometry. Flow cytometry results showed (FIG. 13), eDC-EVs promoted CD8 ⁺ T cell activation (IFN γ, GzmB) and proliferation (CFSE, Ki 67).

(4) eDC killing ability of EVs against tumor cells: eDC-EVs were incubated with lymph node cells and CT26-CD19 tumor cells for 48h, and the viability of the tumor cells was assessed by measuring the activity of medium supernatant Lactate Dehydrogenase (LDH). The results show (fig. 14), that eDC-EVs significantly enhanced LDH release by tumor cells, suggesting promotion of killing of tumors by T cells.

Example 4eDC-EVs blocking the inhibitory Effect of tumor extracellular vesicles (T-EVs) PD-L1 on T cell function

(1) eDC-EVs interact with T-EVs with PD-L1: stimulating CT26 colon cancer cells for 24h in 50ng/mL IFN-gamma, performing differential speed-ultra high speed centrifugation to obtain extracellular vesicles in a culture medium, detecting the expression of PD-L1 by Western blot, and detecting the interaction capacity of PD-L1 and eDC-EVs of t-EVs by using an Elisa kit of PD-L1. The results show (FIG. 15) that eDC-EVs strongly interacted with PD-L1 of t-EVs, indicating that eDC-EVs can block PD-L1 function of t-EVs.

(2) eDC-EVs block the effect of PD-L1 of T-EVs on the proliferation and activation of T cells: eDC-EVs, T-EVs and lymphocytes are incubated for 48h, and the proliferation and activation of the T cells are detected by flow cytometry, and the result shows (figure 16) that eDC-EVs block the inhibition effect of PD-L1 of the T-EVs on the proliferation and activation of the T cells.

Example 5eDC biodistribution of EVs in tumor mice

(1) eDC-biodistribution of EVs in tumor-bearing mice: after eDC-EVs are marked by Cy5.5, the tail vein is injected into mice inoculated with CT26-CD19 colon cancer cells, and after 12 hours, a small animal imaging system observes the distribution of Cy5.5 in tumors and lymph nodes. The results show (FIG. 17) that both genetically modified and non-genetically modified DC extracellular vesicles have a large distribution in lymph nodes, and that high expression of anti-CD19scFv enhances the distribution of vesicles in tumors compared to extracellular vesicles that are not genetically modified or only highly express PD-1. Meanwhile, the invention further inspects the biological distribution of the genetically modified DC cells in tumor-bearing mice, and the result shows that (figure 18), because the cell size is larger, after intravenous injection, the genetically modified DC cells are mostly blocked in the lung, and no significant enrichment in lymph nodes and tumor parts is found.

(2) Confocal Microcopy observed the interaction of Cy5.5-labeled eDC-EVs with PD-L1 locally in tumors and with T cells in lymph nodes. The results show (FIG. 19) that eDC-EVs interacted with PD-L1 at the tumor site and bound to CD 3T cells in the lymph nodes.

Example 6 establishment of mouse tumor model to evaluate eDC-EVs for anti-tumor Effect

(1) Constructing a mouse colon cancer model: a mouse colon cancer model is established by subcutaneously inoculating CT26-CD19 cells, PBS, DC-EVs and eDC-EVs are respectively added after 7 days, and the influence of eDC-EVs on the weight, the size and the weight of a tumor of a mouse and whether the survival rate of the mouse has a relieving effect are examined. The results show (figure 20) that the modification significantly inhibited tumor growth and extended mouse survival compared to DC extracellular vesicles that were not genetically modified. Meanwhile, no improvement effect of the genetically modified DC cells on the growth and survival rate of tumors was found (fig. 21).

(2) eDC-EVs promote lymph node cell activation and remodeling of the tumor immune microenvironment: after different treatment treatments, flow cytometry is used for detecting lymph node T cell activation and the proportion of tumor microenvironment immune cells, and the result shows that eDC-EVs remarkably enhance lymph node CD8+ T cell activation (figure 22), inhibit tumor immunosuppressive cells and promote antitumor immune response (figure 23). Meanwhile, no significant promotion effect of the DC cells with the gene modification on the lymph node CD8+ T cell activation (figure 24) and the tumor immune response (figure 25) is found.

(3) Constructing a mouse lung metastasis model: IFN-gamma treated CT26-CD19 cell-derived vesicles and DC extracellular vesicles are injected into tail vein, and after 14 days, CT26-CD19 cells are inoculated into vein, and whether eDC-EVs can block the establishment of microenvironment before lung metastasis by the CT26-CD19 extracellular vesicles is examined. The results show that eDC-EVs significantly inhibited the formation of a pre-metastatic microenvironment (immunosuppression, inflammatory response, angiogenesis, etc.) of the lung (fig. 26), and lung metastasis of tumors (fig. 27), compared to unmodified DC extracellular vesicles.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A genetically engineered antigen-presenting extracellular vesicle, comprising: the antigen presenting extracellular vesicle is loaded with tumor-associated antigen, and simultaneously over-expresses immune regulatory molecules and single-chain antibodies;

the immune regulatory molecule is selected from one or more of programmed death receptor 1, tumor necrosis factor receptor superfamily member 9 receptor ligand, tumor necrosis factor receptor superfamily member 4 ligand, glucocorticoid-induced tumor necrosis factor receptor ligand and T cell immunoglobulin domain mucin domain protein-3,

the single-chain antibody is selected from one or more of HER2-targeted scFv, CD19-targeted scFv, MSLN-targeted scFv, GD2-targeted scFv, CEA-targeted scFv, EGFRvIII-targeted scFv and VEGFR2-targeted scFv.

2. The antigen-presenting extracellular vesicle of claim 1, wherein: the antigen-presenting extracellular vesicles also reduce the expression of immunomodulatory molecular ligands.

3. The antigen-presenting extracellular vesicle of claim 2, wherein: the immunoregulation molecule ligand is selected from one or more of programmed death ligand-1, programmed death ligand-2 and galectin-9.

4. The antigen-presenting extracellular vesicle of claim 1, wherein: the load is that the tumor-associated antigen and the antigen presenting cell are incubated together before the antigen presenting cell is prepared into the vesicle.

5. The antigen-presenting extracellular vesicle of claim 1, wherein: the particle size of the antigen presenting extracellular vesicle is 30-200nm, and the potential is 0-40 mV.

6. The method for producing antigen-presenting extracellular vesicles according to any one of claims 1 to 5, comprising the steps of:

(1) overexpressing genes encoding the immunoregulatory molecule and the single-chain antibody in an antigen presenting cell to obtain a genetically engineered antigen presenting cell;

(2) co-incubating a tumor vaccine containing a tumor-associated antigen with the genetically engineered antigen-presenting cell to obtain a genetically engineered antigen-presenting cell loaded with the tumor-associated antigen;

(3) preparing the genetic engineering antigen presenting cell loaded with the tumor-associated antigen into a vesicle to obtain the genetic engineering antigen presenting extracellular vesicle.

7. The method of claim 6, wherein: in step (1), further comprising the step of reducing the expression of an immunomodulatory molecule ligand in said antigen presenting cell.

8. Use of the antigen-presenting extracellular vesicles according to any one of claims 1 to 5 in the preparation of an anti-tumor medicament.

9. Use according to claim 8, characterized in that: the antitumor drug is used for inhibiting tumor metastasis.

10. A drug delivery system, characterized by: the drug delivery system comprises the antigen-presenting extracellular vesicle of any one of claims 1-5.