1. An antibody functionalized exosome preparation is characterized in that the exosome preparation is prepared by modifying anti-CD 3 antibody aCD3 and anti-EGFR antibody aEGFR to the surface of exosome Exo-Ag by using exosome Exo-Ag of dendritic cells activated by specific tumor antigen Ag as a carrier and using phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS as a cross-linking agent.

2. Exosome preparation according to claim 1, characterized in that the dendritic cells are at least one of bone marrow derived dendritic cells, DC2.4 cell line, peripheral blood dendritic cells.

3. Exosome preparation according to claim 1, characterised in that the specific tumour antigen Ag is at least one of an OVA antigen, a melanoma-associated antigen MAGE and a melanoma-specific antigen extracted by cell or tissue disruption.

4. A method of preparing an exosome formulation according to claim 1, characterised in that it comprises the steps of:

(a) culturing the dendritic cells: the preserved dendritic cells were activated and seeded in culture dishes in 5% CO₂Culturing in a cell culture box at 37 ℃;

(b) and (3) extracting exosomes: incubating specific tumor antigen Ag and dendritic cells together, collecting cell culture supernatant after incubation is finished, removing cell debris and large-particle protein impurities by using a gradient centrifugation method, and then concentrating and removing impurities by using an ultrafiltration centrifugal tube to obtain exosome solution Exo-Ag;

(c) antibody-modified Exo-Ag: dissolving phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS in DMSO, respectively mixing washed antibody aCD3 and antibody aEGFR with the DSPE-PEG-NHS solution, wherein the molar ratio of the antibody aCD3 or antibody aEGFR to the DSPE-PEG-NHS is 1: 1-3, and continuously stirring and reacting for 20-28h at 2-4 ℃; after the reaction is finished, removing unreacted DSPE-PEG-NHS by centrifugation to respectively obtain a DSPE-PEG-NHS-aCD3 solution and a DSPE-PEG-NHS-aEGFR solution; and (3) reacting the DSPE-PEG-NHS-aCD3 solution and the DSPE-PEG-NHS-aEGFR solution with Exo-Ag at the temperature of 2-4 ℃ for 2-4h, so that the DSPE ends of the DSPE-PEG-NHS-aCD3 and the DSPE-PEG-NHS-aEGFR are inserted into a membrane phospholipid bilayer of an exosome, and thus obtaining an exosome preparation Exo-Ag-aCD3/aEGFR successfully modifying aCD3 and aEGFR.

5. The method according to claim 4, wherein in the step (a), the frozen dendritic cells are taken out, thawed in a 37 ℃ water bath, transferred to an EP tube, added with a preheated 1640 complete medium, uniformly blown, centrifuged, the supernatant is discarded, the 1640 complete medium is added, and the supernatant is uniformly blown and inoculated into a petri dish for culture.

6. The method according to claim 4, wherein in the step (b), 0.1-0.5 mg/mL of the tumor antigen Ag is incubated with the dendritic cells for 12-24 hours, and then the culture medium is replaced with fresh medium for 48 hours, and then the cell culture supernatant is collected and filtered.

7. The method of claim 4, wherein in step (c), the excess DSPE-PEG-NHS is removed by using a 10 kDa ultrafiltration centrifuge tube, and the solution of antibody aCD3 and antibody aEGFR is ultracentrifuged to remove the solvent, washed with PBS and dissolved, and then recovered for use.

8. Use of an exosome formulation according to any one of claims 1-3 in a targeted antineoplastic medicament.

9. The use according to claim 8, wherein the medicament is an injection and the medicament comprises the exosome formulation Exo-Ag-aCD 3/aigfr.

Background

CAR-T cell therapy, also called chimeric antigen receptor T cell immunotherapy, is one of the adoptive T cell transfer. The CAR-T cell therapy is used as a novel precise targeted therapy for treating tumors, can be used for precisely, quickly and efficiently treating cancers, has possibility of curing the cancers, and is a novel promising tumor immunotherapy method. However, extensive research has revealed limitations in CAR-T cell immunotherapy.

The principle of CAR-T cell therapy is to use the patient's own immune cells to achieve the elimination of cancer cells, the main purpose of which is to enhance the inherent anti-cancer capacity of the immune system. CAR-T cell therapy is the extraction of T cells from a patient's blood and genetically modifying these cells in vitro to load them with "chimeric antigen receptors" (CARs) that recognize cancer cell surface antigens. Subsequently, the modified cells are subjected to mass expansion and then are infused back into the body of a patient, so that the treatment effect of recognizing and killing cancer cells is achieved. The Chimeric Antigen Receptor (CAR) of CAR-T cells is composed of an extracellular antigen binding domain, a hinge region, a transmembrane region, and an intracellular domain. When the recoded T cells are infused back into the patient, the chimeric antigen receptor can specifically track and identify and guide the T cells to kill tumor cells like a positioning and navigation device.

To date, CAR-T cell development is still not complete and there are still a number of problems to be solved. First, the resistance effects resulting from the absence of tumor surface antigens and the poor persistence of CAR-T function may still lead to disease progression. Secondly, after a large dose of CAR-T cells are infused, severe side effects such as cytokine release syndrome and the like can be caused, and the CAR-T technology is also one of the most important adverse reactions in clinical application. Furthermore, the laborious and costly procedures of in vitro engineering, expanding and reinfusing patient T cells is also one of the limitations of application of CAR-T therapy. And predicting optimal numbers of infused cells is also a challenge due to differences in T cell responses, persistence, and side effects from patient to patient. More notably, CAR-T cell therapy is not ideal for the treatment of solid tumors due to the tumor desmoplastic properties and immunosuppressive tumor microenvironment, which makes CAR-T cells generally less efficient to infiltrate in tumors. Therefore, design strategies that can overcome the application hurdles in CAR-T cell therapy are of great interest to enhance their clinical value.

Disclosure of Invention

The invention aims to provide an antibody functionalized exosome preparation, and a preparation method and application thereof, so as to solve the problems of complicated operation, low storage/transportation stability, off-target, low retention rate and the like of in-vitro modification and expansion of T cells in the conventional CAR-T cell therapy.

The purpose of the invention is realized as follows: an antibody functionalized exosome preparation is an exosome Exo-Ag-aCD3/aEGFR, wherein exosome Exo-Ag of dendritic cells activated by specific tumor antigen Ag is used as a carrier, phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS is used as a cross-linking agent, and an anti-CD 3 antibody aCD3 and an anti-EGFR antibody aEGFR are modified on the surface of the exosome Exo-Ag.

The dendritic cell is at least one of a bone marrow-derived dendritic cell, a DC2.4 cell line and a peripheral blood dendritic cell.

The specific tumor antigen Ag is at least one of OVA antigen, melanoma related antigen MAGE and melanoma specific antigen extracted by cell or tissue disruption.

The preparation method of the exosome preparation comprises the following steps:

(a) culturing the dendritic cells: the preserved dendritic cells were activated and seeded in culture dishes in 5% CO₂Culturing in a cell culture box at 37 ℃;

(b) and (3) extracting exosomes: incubating specific tumor antigen Ag and dendritic cells together, collecting cell culture supernatant after incubation is finished, removing cell debris and large-particle protein impurities by using a gradient centrifugation method, and then concentrating and removing impurities by using an ultrafiltration centrifugal tube to obtain exosome solution Exo-Ag;

(c) antibody-modified Exo-Ag: dissolving phospholipid-polyethylene glycol-succinimidyl ester DSPE-PEG-NHS in DMSO, respectively mixing washed antibody aCD3 and antibody aEGFR with the DSPE-PEG-NHS solution, wherein the molar ratio of the antibody aCD3 or antibody aEGFR to the DSPE-PEG-NHS is 1: 1-3, and continuously stirring and reacting for 20-28h at 2-4 ℃; after the reaction is finished, removing unreacted DSPE-PEG-NHS by centrifugation to respectively obtain a DSPE-PEG-NHS-aCD3 solution and a DSPE-PEG-NHS-aEGFR solution; and (3) reacting the DSPE-PEG-NHS-aCD3 solution and the DSPE-PEG-NHS-aEGFR solution with Exo-Ag at the temperature of 2-4 ℃ for 2-4h, so that the DSPE ends of the DSPE-PEG-NHS-aCD3 and the DSPE-PEG-NHS-aEGFR are inserted into a membrane phospholipid bilayer of an exosome, and thus obtaining an exosome preparation Exo-Ag-aCD3/aEGFR successfully modifying aCD3 and aEGFR.

In the step (a), the frozen dendritic cells are taken out, melted in a water bath kettle at 37 ℃, transferred into an EP tube, added with preheated 1640 complete culture medium, evenly blown, centrifuged, discarded, added with the 1640 complete culture medium, evenly blown, inoculated in a culture dish and cultured.

In the step (b), 0.1-0.5 mg/mL of tumor antigen Ag and dendritic cells are incubated for 12-24h, and then fresh culture medium is replaced for 48 h, and cell culture supernatant is collected and filtered.

In step (c), excess DSPE-PEG-NHS was removed using a 10 KDa ultrafiltration centrifuge tube, and then the solution of antibody aCD3 and antibody aigfr was ultracentrifuged to remove the solvent, washed with PBS and dissolved for later use.

The exosome preparation is applied to targeted antitumor drugs.

The medicament is an injection and comprises an exosome preparation Exo-Ag-aCD 3/aEGFR.

According to the effects of intracellular and extracellular domains of the CAR-T cells, the functionalized dendritic cell exosomes are used as intermediates, and the CAR-T cell mimics with tumor specific antigen activation and targeting properties are constructed, so that the resistance effect caused by the deletion of tumor surface antigens in CAR-T therapy is overcome, and the problems of time and cost caused by in-vitro modification and T cell amplification in the CAR-T therapy are solved.

The invention combines aCD3 and aEGFR, and enhances infiltration of T cells to tumor tissues and anchorage to the surface of cancer cells by using an exosome with double targets as an intermediate bridge. Experiments demonstrated that after tail vein injection of Exo-OVA-aCD3/aEGFR (OVA is a widely used antigen model) into tumor-bearing mice, Exo-OVA-aCD3/aEGFR accumulated into lymphoid tissues and attached to the T cell surface based on homing by DC2.4 exosomes. MHC II molecules on the surface of Exo-OVA-aCD3/aEGFR present carried antigen peptides to T cells to stimulate the activation of the T cells. Subsequently, during the blood circulation, the activated T cells infiltrate to the tumor tissue under the dual action of the aiegfr targeting and finally anchor to the cancer cells to release cytotoxins to kill the cancer cells. MHC II molecules serve as the intracellular domain of CAR-T cells, promoting activation of T cells by presenting OVA antigens, while the combined use of aCD3 and aiegfr serves as the extracellular domain of CAR-T cells as a "homing device" for T cells. By the mode, the CAR-T cell simulacrum which is activated by the tumor specific antigen and has targeting property is constructed in vivo and has obvious immunotherapy effect.

The preparation method is simple, and the aCD3 and the aEGFR modified Exo-Ag are obtained through the specific cross-linking agent, so that the preparation cost is reduced, and the toxic and side effects are reduced.

Drawings

FIG. 1 is a schematic diagram of the preparation of Exo-OVA-aCD3/aEGFR of the present invention and a comparison of conventional CAR-T and acellular CAR-T.

FIG. 2 is an electron micrograph of Exo-OVA.

FIG. 3 is a gel electrophoresis image during Exo-OVA-aCD3/aEGFR synthesis.

FIG. 4 is a Western blot analysis of Exo-OVA-aCD 3/aEGFR.

FIG. 5 is a nano-flow analysis chart of Exo-OVA-aCD 3/aEGFR.

FIG. 6 is a particle size distribution diagram of Exo-OVA-aCD 3/aEGFR.

FIG. 7 is a flow cytometer analyzing Exo-OVA-aCD3/aEGFR immune activation capacity in vitro.

FIG. 8 is a confocal demonstration of Exo-OVA-aCD3/aEGFR enhancing T cell anchorage to cancer cell surface.

FIG. 9 shows the inhibition of tumor growth by Exo-OVA-aCD 3/aEGFR.

FIG. 10 is the survival rate of tumor-bearing mice after Exo-OVA-aCD3/aEGFR treatment.

FIG. 11 is a pathological analysis of organs and tumor tissues.

FIG. 12 is flow cytometry analysis of tumor local T cell infiltration following Exo-OVA-aCD3/aEGFR treatment.

FIG. 13 is an assay of relevant immunocytokines in mouse serum after treatment.

FIG. 14 is a mouse tumor recurrence assay.

FIG. 15 is a tumor-bearing mouse lung tissue metastasis nodule assay.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples. The test conditions and procedures not mentioned in the examples of the present invention were carried out according to the conventional methods in the art or the conditions suggested by the manufacturer.

Example 1:

the following details the preparation of Exo-OVA-aCD3/aEGFR using a widely used antigen model OVA as an example, as shown in FIG. 1, comprising the following steps:

(1) culturing dendritic cells (DC 2.4)

Frozen DC2.4 cells were removed from liquid nitrogen and thawed by constant shaking in a 37 ℃ water bath. Transfer into 10 mL EP tube, add 10 mL pre-heated 1640 complete medium, gently blow, 2000 rpm centrifugation for 5 min, discard the supernatant. Adding 10 mL 1640 complete medium, gently blowing, inoculating in a culture dish, adding 5% CO₂At 37 ℃ in a cell culture chamber.

(2) Extraction of exosomes

After 0.1 mg/mL OVA and DC2.4 are incubated for 24h, the fresh culture medium is replaced for 48 h, and cell culture supernatant is collected, and impurities such as cell debris, large granular protein and the like are removed by a gradient centrifugation method. And then centrifuging for 5 min at 4000 g by using a 100 KD ultrafiltration centrifugal tube, and removing small-particle impurities in the culture solution while concentrating. And obtaining the exosome solution (Exo-OVA) with better purity after concentration. Can be stored in a refrigerator at-80 ℃ for a long time or at 4 ℃ for at most one week.

(3) anti-CD 3 and anti-EGFR antibodies modified Exo-OVA

Exo-OVA was purified and surface modified with anti-CD 3 antibody (aCD 3) and anti-EGFR antibody (aEGFR). In order to prevent the solvents of aCD3 and aEGFR from interfering the binding reaction of the antibody and the linking agent, a 10 KDa ultrafiltration centrifugal tube is used for centrifuging at 2000 g for 5 min to remove the solvents, and PBS is used for washing once and is recovered for standby. Phospholipid-polyethylene glycol-succinimidyl ester (DSPE-PEG-NHS) is selected as a cross-linking agent. The DSPE-PEG-NHS is dissolved in DMSO, is mixed with the centrifuged aCD3 and aEGFR respectively in a ratio of 2:1, and is continuously stirred and reacted for 24 hours at the temperature of 4 ℃, and the aCD3 and the aEGFR are connected with the DSPE-PEG-NHS through amidation reaction. 4500 g of the mixture is ultrafiltered and centrifuged for 5 min to remove unreacted DSPE-PEG-NHS, the upper solution DSPE-PEG-NHS-aCD3 or DSPE-PEG-NHS-aEGFR is recovered and then reacts with purified Exo-OVA for 3 h under the condition of 4 ℃, the DSPE end of the DSPE-PEG-NHS-aCD3 or DSPE-PEG-NHS-aEGFR is inserted into a membrane phospholipid bilayer of an exosome, and aCD3 and aEGFR are successfully modified to obtain Exo-OVA-aCD 3/aEGFR.

The principle of Exo-OVA-aCD3/aEGFR production is shown in FIG. 1A, and the principle of conventional CAR-T versus acellular CAR-T is shown in FIG. 1B. Exo-OVA-aCD3/aEGFR was characterized and shown in FIGS. 2-6.

FIG. 2 shows that exosomes (Exo-OVA) of the extracted DC2.4 cells are spherical hollow vesicles of about 100nm as observed by a biomicroscope.

FIG. 3 shows gel electrophoresis results showing that the electrophoresis bands of aCD3 and aEGFR linked with DSPE-PEG-NHS (Exo-OVA-aCD 3 and Exo-OVA-aEGFR) are positioned on the pure aCD3 and aEGFR bands, which shows that the molecular weight of aCD3 and aEGFR linked with a linker becomes larger and the bands are dispersed, and that aCD3 and aEGFR linked with DSPE-PEG-NHS successfully through amidation reaction is proved.

FIG. 4 shows that Western blot qualitative analysis results of Exo-OVA-aCD3/aEGFR show that the extracted vesicles express marker proteins such as CD63, CD81 and the like, and the extracted extracellular vesicles are proved to belong to exosomes. And the exosomes secreted by DC2.4 successfully express OVA antigen after OVA stimulation, which indicates that Exo-OVA-aCD3/aEGFR has the function of presenting antigen activated T cells.

FIG. 5 nanometer flow meter results show that compared with the control group, the exosomes of the Exo-OVA-aCD3/aEGFR group detected the signal of fluorescent antibodies (aCD 3-PE/Cy5, aEGFR-FITC), and that aCD3 and aEGFR were modified to the surface of the exosomes.

FIG. 6 particle size of Exo-OVA-aCD3/aEGFR was 102.9 nm and concentration was 7.3X 10 by nano particle tracking Analyzer (NTA)⁷one/mL.

Example 2

The anti-tumor effect of Exo-OVA-aCD3/aEGFR was studied, and the results were as follows:

(1) Exo-OVA-aCD3/aEGFR in vitro T cell activation capacity evaluation

The ability of Exo-OVA-aCD3/aEGFR to activate T cells in vitro was verified by examining the differentiation of T cells. After incubation of T cells with Exo, Exo-OVA-aCD3/aEGFR, the PBS was centrifuged to resuspend the cells and the T cells were labeled with fluorescent antibodies CD4, CD 8. The results of flow cytometry analysis are shown in FIG. 7, and Exo is substantially the same as the control group, and has no ability to activate T cells. And Exo-OVA-aCD3/aEGFR can activate T cells to different degrees and promote the proliferation and differentiation of the T cells. In-vitro T cell differentiation experiments show that the exosome carrying the OVA antigen can effectively promote the activation of T cells.

(2) Evaluation of Exo-OVA-aCD3/aEGFR ability to enhance anchoring of T cells to cancer cells

The ability of Exo-OVA-aCD 3/aigfr to enhance T cell anchorage to cancer cells was confirmed by confocal observation. DiO-stained T cells were incubated with Exo-OVA-aCD3, Exo-OVA-aEGFR, Exo-OVA-aCD3/aEGFR for 30 min, and then DiD-stained B16-OVA cells were added for 1 h of incubation. The cross-linking of Exo-OVA-aCD3/aEGFR mediated T cells with B16-OVA cells was observed by confocal observation, and the results are shown in FIG. 8, compared with Exo-OVA-aCD3 and Exo-OVA-aEGFR, Exo-OVA-aCD3/aEGFR could effectively mediate the anchoring of T cells to cancer cells.

(3) Exo-OVA-aCD3/aEGFR in vivo tumor suppression level evaluation

To verify whether the CAR-T-like immunotherapy platform constructed in vivo using Exo-OVA-aCD3/aEGFR could enhance the tumor growth-inhibiting effect, different materials PBS (G1), Exo (G2), Exo-OVA (G3), Exo-OVA-a were usedCD3（G4）、Exo-OVA-aEGFR（G5）、Exo-OVA-aCD3/aEGFR（G6）（Exo 10¹⁰aPD-L140 mug/mouse) was administered to the tail vein of B16-OVA tumor-bearing mice. Meanwhile, the tumor size of each group of mice is measured every two days, and the tumor growth condition is detected. As shown in fig. 9, the inhibition of tumor growth was most significant for the Exo-OVA-aCD 3/aigfr treatment group, with a very significant difference compared to the PBS control group. And the treatment groups such as Exo, Exo-OVA-aCD3, Exo-OVA-aEGFR and the like have limited inhibition effect on tumor growth, wherein the tumor volumes of the Exo-OVA-aCD3/aEGFR group and the Exo-OVA, Exo-OVA-aCD3 and Exo-OVA-aEGFR group are obviously different. The combined use of aCD3 and aEGFR was shown to increase anchoring of T cells to the surface of cancer cells, contributing to the immunotherapy of tumors. In addition, the tumor volume growth rate of the Exo-OVA-aCD3/aEGFR treated group was still slow until 40 days after treatment, showing a significant antitumor effect.

The results in FIG. 10 show that treatment with Exo-OVA-aCD3/aEGFR effectively extended the mean survival time of mice, with 6 mice surviving until the end of the 40-day experiment.

FIG. 11 shows H & E stained heart, liver, spleen, lung, kidney, and no apparent damage was observed to the organs of each group. The tumor tissue staining results show that the tumor tissue treated by Exo-OVA-aCD3/aEGFR is obviously necrotic.

Tumor samples from tumor-bearing mice were collected 21 days after treatment and analyzed by flow cytometry. As shown in FIG. 12, infiltration of CD4 + T cells and CD8+ T cells was significantly increased in the Exo-OVA-aCD3/aEGFR group compared to the untreated group. Notably, the Exo-OVA-aCD3/aEGFR group is more favorable for infiltration of immune cells than the Exo-OVA, Exo-OVA-aCD3 and Exo-OVA-aEGFR groups, because the aEGFR can serve as a target molecule of T cells to increase anchoring of the target molecule to the surface of the cancer cells after the Exo-OVA-aCD3/aEGFR is bound to the surface of the T cells through aCD 3. The infiltration of local immune cells of the tumor is increased, the immunosuppressive microenvironment of the local tumor can be effectively improved, and the tumor immunotherapy effect is obviously enhanced. Studies have shown that an increased proportion of Tumour Infiltrating Lymphocytes (TILs) can effectively reverse the inhibitory microenvironment.

Serum was analyzed for relevant immunocytokines during treatment. As shown in FIG. 13, the increase of cytokine secretion, including interferon- γ, tumor necrosis factor- α, interleukin-6, etc., further confirmed that Exo-OVA-aCD3/aEGFR could effectively activate innate immunity and adaptive immune response, resulting in effective killing.

(4) Evaluation of Exo-OVA-aCD3/aEGFR inhibitory Effect on tumor metastasis and recurrence

To test the therapeutic potential of Exo-OVA-aCD3/aEGFR, a model of relapsing metastases was developed and tested. Based on the tumor recurrence volume after mouse surgery, Exo-OVA-aCD 3/aigfr treatment was significantly effective in preventing local tumor recurrence compared to the control group, as shown in fig. 14.

In the mouse tumor recurrence and metastasis model, metastatic lung tissues are collected, each group of lung tissues is analyzed to count the number of lung nodules, and a blank group is found to have obvious metastasis points, while a group treated by Exo-OVA-aCD3/aEGFR has obviously reduced average number of lung metastasis and has obvious difference with the blank group, and the result can be shown in FIG. 15.