Chimeric antigen receptor targeting EGFRvIII, engineered exosome containing chimeric antigen receptor and application of engineered exosome

Abstract

The invention discloses a chimeric antigen receptor targeting EGFRvIII, an engineered exosome containing the chimeric antigen receptor and application of the engineered exosome. The chimeric antigen receptor comprises an antigen binding structural domain, wherein the antigen binding structural domain comprises a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region comprises an HCDR1, an HCDR2 and an HCDR3, the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are respectively shown as SEQ ID NO: 3-5, and the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3, the amino acid sequences of the LCDR1, the LCDR2 and the LCDR3 are respectively shown as SEQ ID NO: 6-8. The chimeric antigen receptor is combined with an engineered exosome delivery system, so that auxiliary function enhancement and in-vivo preparation of CAR-T cells are realized, the EGFRvIII target modified CAR-T cells which can be prepared in vivo and can be specifically combined with EGFRvIII antigens are provided, and the effect of killing malignant tumor cells can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a chimeric antigen receptor targeting EGFRvIII, an engineered exosome containing the same and application thereof. BACKGROUND

[0002] Glioma is the most common primary intracranial malignant tumor, and glioblastoma (GBM) is the most common malignant primary brain tumor, which is one of the types with the strongest lethality and the highest recurrence rate, accounting for about 57% of all gliomas and 48% of all primary malignant central nervous system tumors. It has poor prognosis, high recurrence rate, low survival rate, and the median survival time is less than 2 years, and the 5-year survival rate is 4%-5%.

[0003] The related molecular markers of glioblastoma include IDH mutation, H3 mutation, EGFR gene amplification or mutation, TERT promoter mutation, chromosome 7 increase and chromosome 10 loss.

[0004] At present, the effective treatment method for GBM is still surgical resection, and postoperative adjuvant radiotherapy, chemotherapy, immunotherapy, photodynamic therapy, electric field therapy, etc., and the effect is not ideal. In view of the current effective treatment methods, targeted therapy can be one of the effective treatment strategies for GBM. At present, by understanding the mechanism of EGFR / EGFRvIII in GBM, EGFR / EGFRvIII has been used as an ideal target, and various treatment methods have been developed for EGFR / EGFRvIII target, including EGFR / EGFRvIII small molecule inhibitor treatment, EGFR / EGFRvIII antibody treatment, EGFR / EGFRvIII mediated related vaccine treatment. Chimeric antigen receptor T-cell immunotherapy (CAR-T) treatment has gradually become a research hotspot, although it shows potential in some patients, but the overall efficacy still needs further verification. Therefore, developing more effective and less side effect treatment methods is an important direction of current GBM research.

[0005] CAR-T cell therapy has a significant effect on leukemia and lymphoma, but at present, solid tumors are not sensitive to CAR-T cell therapy, which is mainly because CAR-T cells are difficult to target and transport to tumor tissues and cannot effectively proliferate in the host body for a long time. For glioblastoma, CAR-T cells are more difficult to transport to tumor tissues due to the blood-brain barrier. The traditional CAR-T construction strategy needs to go through many steps such as blood collection of patients, T cell extraction, virus transfection, in vitro CAR-T cell culture and expansion, etc., before it can be used for patients. It can also cause serious side effects such as cytokine release syndrome (CRS), and due to the problem of target selectivity, it can cause damage to normal tissues. For each patient, the same process needs to be customized, which is difficult to be universal, and the whole process is cumbersome, long cycle and expensive, only a small number of patients have the opportunity to receive effective treatment, and it is difficult to benefit a large number of tumor patients. In view of the above clinical problems, it is necessary to construct a new method of universal in vivo and directional transfection of T cells to construct CAR-T cells for the treatment of GBM. SUMMARY

[0006] In order to provide a universal in vivo and directional transfection of T cells to construct CAR-T cells, the application discloses a chimeric antigen receptor targeting EGFRvIII, an engineered exosome containing the same and application thereof.

[0007] The application solves the above technical problems by the following technical means:

[0008] The application provides a chimeric antigen receptor targeting EGFRvIII in a first aspect, wherein the chimeric antigen receptor comprises an antigen binding domain, and the antigen binding domain comprises a heavy chain variable region and a light chain variable region.

[0009] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NO: 3-5, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively as shown in SEQ ID NO: 6-8.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 9 or has at least 80% sequence identity with SEQ ID NO: 9.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 10 or has at least 80% sequence identity with SEQ ID NO: 10.

[0012] In some preferred embodiments, the heavy chain variable region and the light chain variable region are connected by a linker.

[0013] In some embodiments, the linker has an amino acid sequence as set forth in SEQ ID NO: 11.

[0014] In some embodiments, the antigen binding domain comprises, in N-terminal to C-terminal order, a heavy chain variable region, a linker, and a light chain variable region, or the antigen binding domain comprises, in N-terminal to C-terminal order, a light chain variable region, a linker, and a heavy chain variable region.

[0015] In some embodiments, the antigen binding domain comprises, in N-terminal to C-terminal order, a heavy chain variable region, a linker, and a light chain variable region.

[0016] In some preferred embodiments, the antigen binding domain has an amino acid sequence as set forth in SEQ ID NO: 2 or at least 80% sequence identity to SEQ ID NO: 2.

[0017] In some embodiments, the chimeric antigen receptor further comprises a transmembrane domain and an intracellular domain, the intracellular domain containing a signaling domain and a costimulatory domain in tandem; optionally, further comprising a signal peptide.

[0018] In some preferred embodiments, the transmembrane domain is a transmembrane domain of CD28.

[0019] In some preferred embodiments, the signaling domain is a signaling domain of CD3 zeta.

[0020] In some preferred embodiments, the costimulatory domain is a costimulatory domain of 4-1BB.

[0021] In some preferred embodiments, the signal peptide is N6 (SP).

[0022] The present application realizes the enhancement of CAR-T cell auxiliary function and its in vivo preparation by optimizing the design of anti-EGFRvIII CAR molecule combined with the engineered exosome delivery system, and provides a CAR-T cell modified with EGFRvIII target point which can be prepared in vivo and specifically combined with EGFRvIII antigen. In the cell killing test of glioblastoma cell line, the in vivo construction of CAR-T cell modified with EGFRvIII target point can realize the killing of malignant tumor cells, and realize the effect of in vivo formation of CAR-T.

[0023] The second aspect of the present application provides an isolated nucleic acid encoding the chimeric antigen receptor as described in the first aspect of the present application.

[0024] In some preferred embodiments, the sequence of the nucleic acid encoding the antigen binding domain is as set forth in SEQ ID NO: 1.

[0025] In some preferred embodiments, the nucleic acid is DNA and / or RNA.

[0026] In the present application, the RNA is for example mRNA.

[0027] In some preferred embodiments, when the nucleic acid is mRNA, the mRNA comprises a 5’ UTR, a 3’ UTR and a RNA recruiting element, for example a C / D box.

[0028] The third aspect of the present application provides an engineered exosome, the engineered exosome targeting a T cell and encapsulating a nucleic acid as described in the second aspect of the present application.

[0029] The engineered exosome is used for delivering the nucleic acid to a T cell.

[0030] In some embodiments, the T cell is a CD8+ T cell.

[0031] In some embodiments, the surface of the engineered exosome has a polypeptide targeting a T cell, whereby the engineered exosome targets the T cell.

[0032] In some embodiments, the polypeptide is an anti-CD8 antibody.

[0033] In some embodiments, the polypeptide is a nanobody.

[0034] In some preferred embodiments, the polypeptide is an anti-CD8 nanobody

[0035] The engineered exosome has a targeting peptide and is loaded with a CAR structure targeting EGFRvIII. A CAR frame plasmid is constructed using EGFRvIII as a target, and a T cell-targeted modified exosome is used as a carrier to encapsulate the CAR frame plasmid, so as to improve the specificity of the carrier in binding to T cells, to transfect T cells in vivo and convert them into CAR-T cells, while reducing off-target effects.

[0036] The fourth aspect of the present application provides a packaging system of the engineered exosome as described in the third aspect of the present application, the packaging system comprising a nucleic acid as described in the second aspect of the present application and a RNA packaging device.

[0037] In some embodiments, the RNA packaging device comprises a cell surface protein and an RNA binding protein; the cell surface protein and the RNA binding protein form a fusion protein; the RNA binding protein can specifically bind to the RNA recruiting element; thereby enriching the target nucleic acid in the engineered exosome.

[0038] In some preferred embodiments, the cell surface protein is CD63; the RNA binding protein is L7Ae.

[0039] In some specific embodiments, the RNA packaging device is pcDNA3.1-CD63-L7Ae.

[0040] In some embodiments, the packaging system further comprises a targeted delivery device, the targeted delivery device comprising a polypeptide targeting T cells and an exosome surface protein.

[0041] In some preferred embodiments, the exosome surface protein is exosome surface protein Lamp2b; the polypeptide targeting T cells is expressed in fusion with the exosome surface protein, enabling the engineered exosome to target the T cells.

[0042] In some specific embodiments, the targeted delivery device is BRD-PTK-CD8+-Lamp2b.

[0043] The fifth aspect of the present application provides a delivery system comprising the chimeric antigen receptor of the first aspect of the present application, the nucleic acid of the second aspect of the present application, or the engineered exosome of the third aspect of the present application.

[0044] The sixth aspect of the present application provides a method for preparing the engineered exosome of the third aspect of the present application, the method comprising: transfecting human cells with the packaging system of the fourth aspect of the present application, and obtaining the supernatant containing the engineered exosome after culturing.

[0045] In some embodiments, the engineered exosome of the third aspect of the present application is obtained after purifying the supernatant.

[0046] In some more preferred embodiments of the present application, the human cells are 293F cells.

[0047] The seventh aspect of the present application provides a pharmaceutical composition comprising the chimeric antigen receptor of the first aspect of the present application, the nucleic acid of the second aspect of the present application, the engineered exosome of the third aspect of the present application, the packaging system of the fourth aspect of the present application, or the delivery system of the fifth aspect of the present application, and a pharmaceutically acceptable carrier.

[0048] The eighth aspect of the present application provides a use of the chimeric antigen receptor according to the first aspect of the present application, the nucleic acid according to the second aspect of the present application, the engineered exosome according to the third aspect of the present application, the packaging system according to the fourth aspect of the present application or the delivery system according to the fifth aspect of the present application in the preparation of a medicament for targeting and / or treating a tumor.

[0049] In some preferred embodiments of the present application, the tumor is glioblastoma.

[0050] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred embodiments of the present application.

[0051] The reagents and raw materials used in the present application are commercially available.

[0052] The positive progress effect of the present application is that the innovation of the present application lies in generating a CAR molecule using a nanobody against EGFRvIII and combining an exosome delivery system to deliver CAR-mRNA to T cells and convert CD8+ cells into CAR-T cells, and the advantages are that 1. blood of infected persons does not need to be extracted; 2. CAR molecules against malignant cells in the body can be prepared in vivo; 3. no chemotherapy or radiotherapy is needed, and there is no side effect. 4. it has universality. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 For the construction of PLV-Luc-GFP and lentivirus packaging. (A) Schematic diagram of PLV-Luc-GFP plasmid structure; (B) Lentivirus titer rapid detection card detects lentivirus vector packaging efficiency; (C) Lentivirus transfection of 293T cells, 48 h later, the fluorescence intensity is detected using a fluorescence microscope.

[0054] Figure 2 For the detection of GFP and Luc in GFP and Luc double-labeled cells. (A) Flow cytometry detects the expression of GFP on the surface of U251 double-labeled cells; (B) Fluorescence microscope observation of the expression of green fluorescent protein fluorescence intensity of U251 double-labeled cells; (C) In vivo imaging instrument detects the fluorescence intensity of luciferase gene expressed by U251 double-labeled cells.

[0055] Figure 3 For the schematic diagram of engineered EXO mRNA targeted delivery.

[0056] Figure 4 For the isolation and characterization of CAR-engineered EXO derived from 293F cells. (A) Schematic diagram of engineered EXO isolation experiment workflow; (B) TEM image of the morphology of engineered EXO; (C) Particle size distribution and concentration analysis (NTA) analysis of engineered EXO; (D) Analysis of specific markers on the surface of engineered EXO.

[0057] Figure 5 The expression of CD63, CD81, and CD9 on the engineered EXO surface.

[0058] Figure 6 To investigate the expression of the Strep2 tag and VHH nanobody on the surface of engineered EXO.

[0059] Figure 7 To analyze the positive expression of CAR-T cells in CD8+ T cells at different doses and time points after incubation with EGFRvIII-engineered EXO.

[0060] Figure 8 Quantitative statistical curves of the proportion of EGFRvIII CAR+ T cells targeted under two dosage conditions. (A) 10 5 Analysis of CAR-T cell positivity rate under Particles / cell dosage conditions; (B) 10 6 Analysis of CAR-T cell positivity rate under Particles / cell dosage conditions.

[0061] Figure 9 Cytotoxicity analysis of CD8+ T cells to generate CAR-T cells using EGFRvIII-engineered EXO.

[0062] Figure 10 To detect the proportion of human immune cells in humanized mice using flow cytometry.

[0063] Figure 11 To establish glioma xenograft animals and evaluate the in vivo antitumor activity of engineered EXO targeting EGFRvⅢ. (A) Flowchart of mouse experiment; (B) Fluorescence analysis of in vivo imaging of mice in each group before and after treatment with the two engineered EXOs; (C) Comprehensive analysis of luciferase expression changes in each group of mice before and after treatment; (D) Body weight changes in each group of mice; (E) Survival curves of each group of mice; (F) Growth curves of subcutaneous xenograft volume in each group of mice.

[0064] Figure 12 Analysis of the CAR-T cell positivity rate after co-incubation of PBMC cells derived in vivo with EGFRvIII-targeted engineered EXO. (A) After incubating mouse PBMCs with the above two engineered EXOs for 24 h, the expression level of Strep2 in CD8+ T cells was detected by flow cytometry; (B) Statistical analysis of the proportion of CAR-positive T cells (n=3, ****p<0.0001). Detailed Implementation

[0065] The application will be further described in the following by way of examples without limiting the application to the described examples. The experimental methods in the following examples, if not specified, are selected according to the conventional methods and conditions, or according to the commercial instructions.

[0066] Example 1, Construction of a Targeting EGFRvIII CAR Functional Plasmid

[0067] 1. The anti-EGFRvIII nanobody sequence fragment targeting EGFRvIII was synthesized by GenScript Biotech Corporation, and the synthesized sequence was cloned into a pUC57 vector.

[0068] The nucleic acid sequence of the EGFRvIII single-chain antibody (SEQ ID NO: 1):

[0069] CGGCCCGAGATTCAGCTCGTGCAATCGGGAGCGGAAGTCAAGAAGCCAGGAGAGTCCTTGCGGATCTCATGCAAGGGTAGCGGCTTTAACATCGAGGATTACTACATCCACTGGGTGAGGCAGATGCCGGGGAAGGGACTCGAATGGATGGGACGGATCGACCCAGAAAACGACGAAACTAAGTACGGTCCGATCTTCCAAGGCCATGTGACTATTAGCGCCGATACTTCAATCAATACCGTGTATCTGCAATGGTCCTCATTGAAAGCCTCAGATACCGCGATGTACTACTGTGCTTTCAGAGGAGGGGTCTACTGGGGACAGGGAACTACCGTGACTGTCTCGTCCGGCGGAGGCGGGTCAGGAGGTGGCGGCAGCGGAGGAGGAGGGTCCGGCGGAGGTGGGTCCGACGTCGTGATGACCCAGAGCCCTGACAGCCTGGCAGTGAGCCTGGGCGAAAGAGCTACCATTAACTGCAAATCGTCGCAGAGCCTGCTGGACTCGGACGGAAAAACGTACCTCAATTGGCTGCAGCAAAAGCCTGGCCAGCCACCGAAGCGCCTTATCTCACTGGTGTCGAAGCTGGATTCGGGAGTGCCCGATCGCTTCTCCGGCTCGGGATCGGGTACTGACTTCACCCTCACTATCTCCTCGCTTCAAGCAGAGGACGTGGCCGTCTACTACTGCTGGCAGGGAACCCACTTTCCGGGAACCTTCGGCGGAGGGACGAAAGTGGAGATCAAG

[0070] Amino acid sequence of EGFRvIII single chain antibody (SEQ ID NO: 2):

[0071] RPEIQLVQSGAEVKKPGESLRISCKGSGFNIEDYYIHWVRQMPGKGLEWMGRIDPENDETKYGPIFQGHVTISADTSINTVYLQWSSLKASDTAMYYCAFRGGVYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDVVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIK

[0072] The CDRs of the EGFRvIII single chain antibody variable region are numbered by the Kabat numbering system:

[0073] HCDR1 (SEQ ID NO: 3): DYYIH

[0074] HCDR2 (SEQ ID NO: 4): RIDPENDETKYGPIFQG

[0075] HCDR3 (SEQ ID NO: 5): RGGVY

[0076] LCDR1 (SEQ ID NO: 6): KSSQSLLDSDGKTYLN

[0077] LCDR2 (SEQ ID NO: 7): LVSKLDS

[0078] LCDR3 (SEQ ID NO: 8): WQGTHFPGT

[0079] VH chain (SEQ ID NO: 9): RPEIQLVQSGAEVKKPGESLRISCKGSGFNIEDYYIHWVRQMPGKGLEWMGRIDPENDETKYGPIFQGHVTISADTSINTVYLQWSSLKASDTAMYYCAFRGGVYWGQGTTVTVSS

[0080] VL chain (SEQ ID NO: 10): DVVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIK

[0081] Linker (SEQ ID NO: 11): GGGGSGGGGSGGGGSGGGGS

[0082] 2, Using human cDNA library as template, primers were designed to amplify fragments N6-SP, anti-EGFRvIII, G4S-Strep tag II-CD8 hinge by PCR. Overlap PCR technique was used to sequentially amplify anti-EGFRvIII, G4S-Strep tag II-CD8 hinge, N6-SP into a fragment with enzyme cutting sites Nhe I and SgrA I.

[0083] 3, The plasmid pcDNA 3.1 N6(SP)-T20-VRC C / D box was double enzyme cut using Nhe I and SgrA I restriction endonuclease, the product was subjected to 0.8% agarose gel electrophoresis, and the corresponding fragment was recovered in an Eppendorf tube using the agarose gel recovery kit from Kangweishijie Company, and the purity and concentration of the product were determined.

[0084] 4, The above vector recovery fragment and N6-SP-anti-EGFRvIII-G4S-Strep tag II-CD8 hinge fragment were added to an Eppendorf tube with Exnase II ligase (Vazyme) and homologous recombinase 5x CE II buffer at a molar ratio of 1:2, and reacted at 37°C for 0.5 hours; 10 μL of the ligation solution was taken out and added to 100 μL of DH5α competent cells, which were incubated in an ice bath for 30 min and then heated at 42°C for 90 s; after completion, 500 μL of SOC medium was added and incubated at 37°C, 220 rpm for 2 hours; after 2 hours, the Eppendorf tube was centrifuged at 4000g for 1 min to remove 400 μL of excess liquid. The remaining liquid was spread on LB plates containing kanamycin and incubated at 37°C for 12 hours; single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium, which was incubated at 37°C, 220 rpm for 12 hours.

[0085] 5, The plasmid was extracted using the Kangweishijie miniprep kit, and the plasmid pcDNA3.1-N6(SP)-anti-EGFRvIII-T20-C / D box was obtained and sent to Shengong Biotechnology (Shanghai) Co., Ltd. for first-generation sequencing to verify that there was no error, and then the DH5α strain containing the plasmid pcDNA3.1-N6(SP)-anti-EGFRvIII-T20-C / D box was preserved.

[0086] Example 2, PLV-Luc-GFP lentivirus packaging

[0087] PLV-Luc-GFP lentivirus expression vector was constructed by our laboratory, which structure was shown in Fig. Figure 1 A, the vector co-expressed Luciferase and GFP (green fluorescent protein) driven by EF-1α promoter, and connected by Internal Ribosoe Entry Site (IRES) sequence to ensure stable co-expression of fluorescent marker and resistance screening. Lentivirus packaging and titer detection results showed that 48 h after co-transfection of 293T cells with PLV-Luc-GFP plasmid and auxiliary packaging plasmid, the culture supernatant was collected, and the virus particles were concentrated. The lentivirus titer rapid detection card (Fig. Figure 1 B) was used to verify the virus packaging efficiency, and the detection band was clear, indicating that the lentivirus was successfully packaged. Lentivirus infection efficiency was further verified by fluorescence microscope detection of GFP expression (Fig. Figure 1 C). At the same time, the cells were observed under bright field microscope, and the state was good, with no obvious apoptosis or toxic effect.

[0088] Example 3, Construction of GFP and Luc double-labeled target cells

[0089] 1. Infected U251 cells

[0090] 1) Take U251 cells in good growth state, adjust the density to 1×10 6 cells / mL, and inoculate 1×10 6 cells / well into a 6-well plate.

[0091] 2) Add 100 μL of concentrated PLV-GFP-Luc lentivirus solution to each well, and add Polybrene at a final concentration of 8 μg / mL to enhance virus infection efficiency. After incubation at 37℃, 5% CO2 for 4-6 h, discard the virus culture medium, and add preheated RPMI-1640 fresh complete culture medium for continuous culture.

[0092] 2. Resistance screening of U251 double-labeled target cells

[0093] 1) After 24-48 h of infection, add puromycin at a final concentration of 4 μg / mL for resistance screening. During the screening process, observe the cell morphology, survival rate and green fluorescent signal expression every day, and dynamically evaluate the screening process.

[0094] 2) When the cell density reaches 80-90% density, use 0.05% Trypsin-EDTA for digestion, and subculture at a ratio of 1:3-1:5 to maintain the proliferation activity and long-term stability of the cells. The screening lasted for about 2 weeks, and after the uninfected cells completely apoptosed, the puromycin concentration was reduced to 1 μg / mL to maintain stable screening.

[0095] 3. U251 double-labeled target cell phenotype and function identification

[0096] 1) The expression of GFP was observed and recorded by fluorescence microscopy, and the expression level of exogenous gene and the intensity of fluorescence signal were directly evaluated. The screened cells were washed twice with PBS and resuspended in flow detection buffer, then the proportion of GFP positive cells was detected by BD flow cytometry, and the data was analyzed by FlowJo-V10 software to quantify the proportion of GFP positive cells, analyze the purity and transfection efficiency of the cell population. The results showed that the GFP positive rate of U251-Luc-GFP cells was 99.6%, while the control cells (U251-Control) had no GFP signal. The results showed that the lentiviral vector successfully mediated the efficient transfection of GFP gene in U251 cells, and the purity of the screened cell population was high. Figure 2 A).

[0097] Further observation of GFP expression by fluorescence microscopy showed that U251-Luc-GFP cells showed strong green fluorescence signal under GFP channel, while the control group (U251-Control) had no GFP signal. This indicates that the expression of GFP after lentiviral transfection has stability and uniformity, and the expression level of GFP in the whole cell population is high, which meets the construction standard of stable cell lines. Figure 2 B).

[0098] 2) The luciferase activity of the cells was evaluated by small animal live imaging system (In Vivo Imaging System, IVIS).

[0099] The cells to be tested were collected, and the cell density was adjusted to 1×10 6 cells / mL, then the cells were evenly inoculated in a 6-well culture plate. After 24 h, the cell supernatant was aspirated, washed twice with PBS, then 1 mL of bioluminescent substrate solution (final concentration 150 ug / mL) was added to each well, and incubated at 37℃ for 5 min. After incubation, the 6-well plate was immediately placed in the IVIS for imaging, and the emission wavelength of Luciferase was selected as 560 nm for exposure imaging, and the background correction and signal normalization analysis were performed by 1.7.05_amiview.sav software, so as to quantitatively evaluate the luciferase expression of the target cells.

[0100] The results showed that U251-Luc-GFP cells exhibited significant luciferase activity with high luminescence intensity, while no obvious bioluminescence signal was detected in the control group cells (U251-Control). This further demonstrated the stable expression of the Luciferase gene in the target cells and the real-time detection by bioluminescence imaging. Figure 2 C).

[0101] Example 4, Construction of the engineered EXO targeted delivery system

[0102] To establish the L7Ae-C / D box specific packaging system, the C / D box was added to the 3'UTR region of CAR-mRNA, and the EXO specific four-transmembrane protein CD63 was fused with L7Ae to form a fusion protein. By utilizing the specific binding of the RNA recruiting element C / D box and the RNA binding protein L7Ae, CAR-mRNA was enriched in EXO, and the loading efficiency of CAR-mRNA in EXO was improved. By fusing the nanobody specifically targeting human CD8+ with the N-terminal of Lamp2b, it was displayed on the membrane surface of the engineered EXO to play the role of receptor recognition and mediate its recognition of specific receptor cells, so that the content mRNA in the target cells can be translated to express the target protein. Figure 3 The engineered EXO targeted delivery system was constructed by co-transfecting pcDNA3.1-N6 (SP)-T20-EGFRvIII-CD box; BRD-PTK-CD8+ (Flag)-Lamp2b, pcDNA3.1-CD63-L7Ae, and the above 3 plasmids.

[0103] Example 5, Preparation and characterization of exosomes

[0104] 1. 293F cell culture and exosome preparation

[0105] 293F cells were cultured in 293 CD05 medium without antibiotics and SMM293-TII medium (1:1 ratio) at 125 rpm, 37°C, 5% CO2. 60 μg of pcDNA3.1-N6(SP)-T20-EGFRvIII-CDbox plasmid, 20 μg of PTK-CD8+-Lamp2b, 20 μg of pcDNA3.1-CD63-L7Ae, and 4 mL of F complete medium were mixed, while 300 μL of PEI solution (1 mg / mL) was added to another 4 mL of F complete medium, and after 5 minutes of standing, the plasmid mixture was combined with the PEI mixture, and after 30 minutes of standing, 293F cells were added for co-transfection. After 24 hours, OPM-CHO PFF06 medium (1 / 20) and L-glutamine (1 / 50) were added, and after 72 hours, the cell supernatant was collected. The live cells were removed by centrifugation at 300 x g for 5 min at 4°C, and the dead cells and debris were removed by centrifugation at 3000 x g for 30 min, and finally the large extracellular vesicles were removed by centrifugation at 10,000 x g for 60 min. Universal Benzo nuclease (2 U) and magnesium chloride (2 μmol / L) were added to the pretreated supernatant, which was then filtered through a 0.22-μm filter cup overnight at 4°C, and the supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000 x g for 90 min using an Optima XE-100 ultracentrifuge. The supernatant was discarded, the pellet was resuspended in 20 mL of PBS, and centrifuged again at 100,000 x g for 90 min. Finally, the pellet was resuspended in 200 μL of PBS and stored at -80°C. Figure 4 A).

[0106] 2. Exosome characterization

[0107] (1) Transmission Electron Microscopy (TEM) detection of engineered EXO

[0108] TEM was used to observe the morphology, size, and integrity of engineered EXO to confirm the nanoscale features of the sample. In this experiment, the negative staining method was used to analyze the transmission electron microscopy of the isolated and purified engineered EXO, and the specific method was as follows:

[0109] 1) Sample preparation: Take an appropriate amount of engineered EXO suspension (concentration about 10 9 particles / mL), dilute with PBS as appropriate, and add 10 μL to a copper mesh, incubate at room temperature for 10 min to allow the engineered EXO to fully adsorb.

[0110] 2) Negative staining: Excess liquid was absorbed with filter paper, followed by adding 2% phosphotungstic acid or 2% uric acid solution for negative staining for 30-60 s, and then the staining solution was gently absorbed again with filter paper to avoid sample cracking.

[0111] 3) Drying and observation: After drying the copper grid at room temperature, TEM was used to image at 80-120 kV voltage to observe the morphology, size and integrity of engineered EXOs, and record the images.

[0112] TEM observation showed that engineered EXOs were about 100 nm, showing a typical "cup-shaped" vesicular structure (Fig. 1B). Figure 4 Fig. 1B.

[0113] (2) Nanoparticle Tracking Analysis (NTA)

[0114] NTA technology was used to analyze the particle size distribution and concentration of engineered EXOs, which was based on the principle of dynamic light scattering, calculating particle size and measuring particle concentration by tracking particle Brownian motion. The experiment was determined by Zeta View nanoparticle tracking analyzer, and the specific method was as follows:

[0115] 1) Sample preparation: Take an appropriate amount of engineered EXO suspension (about 10 9 particles / mL), dilute with sterile PBS to the instrument detection range (usually 10 7 particles / mL) as appropriate, and mix well for testing.

[0116] 2) Instrument calibration: Before the experiment, use standard nanoparticles to calibrate the instrument to ensure the accuracy of light source, focusing and particle concentration measurement.

[0117] 3) Detection process: Inject the sample into the detection pool, use laser scattering and Brownian motion analysis, record the particle size distribution data under multiple fields of view while the camera captures the trajectory of engineered EXO movement. The detection parameters are usually set as: temperature 25℃, detection time 60 s.

[0118] NTA results showed that the main peak particle size range of the collected engineered EXOs was 120-150 nm, and showed a single peak, which was consistent with the normal distribution (Fig. 1C), which was basically consistent with the particle size of engineered EXOs shown by TEM images. Figure 4

[0119] (3) Western Blot (WB)

[0120] ​1) 72 h after transfection, 1 ml of cell suspension was taken, centrifuged to discard the culture medium, and the cells were collected after gentle washing twice with pre-cooled PBS, and 200 μL of RIPA lysis buffer (containing 1% PMSF protease inhibitor) was added, and the cells were lysed on ice for 30 min. Then, centrifuged at 10000 x g at 4°C for 10 min, and the supernatant was collected;

[0121] 2) The protein content in the lysed cell suspension was determined using the Biyun Tian BCA protein quantification kit. The lysed cell suspension was mixed with 5x Loading buffer, heated at 100°C for 10 min, and then stored at -20°C for later use;

[0122] 3) SDS-PAGE electrophoresis was performed according to the loading amount of 20 μg protein per well, and the electrophoresis conditions were set as follows: constant voltage of 80 V for 20 min until the bromophenol blue entered the separation gel; then constant voltage of 120 V for 60-90 min until the bromophenol blue migrated to the bottom of the gel;

[0123] 4) The protein was transferred to the NC membrane at a rate of 1 kD protein / min using the wet transfer method, and then the NC membrane was placed in the protein-free rapid blocking solution and blocked at room temperature for 20 min;

[0124] 5) The primary antibody was diluted according to the corresponding proportion of the antibody instruction using TBST buffer, and incubated at 4°C overnight. Then, the NC membrane was washed 3 times with TBST buffer for 10 min each time to remove the unbound primary antibody;

[0125] 6) The secondary antibody was incubated by immersing the NC membrane in a 1:5000 diluted secondary antibody solution, and incubating in the dark for 1 h. Then, it was washed 3 times with TBST buffer for 10 min each time to remove the unbound secondary antibody;

[0126] 7) Finally, ECL luminescent liquid A and B were mixed at a ratio of 1:1 for development, and the expression of the target protein was detected using a gel imaging analysis system.

[0127] The WB results showed that the engineered EXO surface had CD63, CD81, and TSG10 typical EXO markers Figure 4 D).

[0128] (4) Nano-flow cytometry (Nano-flow Cytometry, nFCM)

[0129] The expression of CD63, CD81 and CD9 on the surface of the targeted engineered EXO and the unmodified blank EXO from 293F cells was detected by nano-flow cytometry. The results showed that the corresponding positive rates of CD63, CD81 and CD9 on the surface of CD8+ / EGFRvIII+CAR+EXO were 61.2%, 42.3% and 48.3%, respectively; while the expression rates of CD63, CD81 and CD9 on the unmodified EXO from 293F were 20.0%, 48.0% and 35.1%, respectively Figure 5 ); the results showed that the targeted engineered EXO stably expressed typical EXO surface proteins, and the expression of CD63 in the targeted delivery EXO was significantly up-regulated, much higher than that of the unmodified EXO from 293F. The above results showed that the targeted engineered EXO successfully enriched overexpressed CD63 during the packaging process.

[0130] (5) Verification of the expression of specific proteins on the surface of engineered EXO

[0131] To evaluate whether the specific labels Strep2 and nanobody sequences in the CAR+ plasmid were successfully expressed on the surface of engineered EXO, the unmodified EXO from 293F cells and the targeted engineered EXO were directly stained and detected by nFCM. FITC-labeled Strep2 antibody and PC5-labeled VHH antibody were used to stain the surface of EXO samples to identify whether the engineered EXO carried Strep tag (representing the expression of CAR structure) and nanobody structure (including VHH domain in CAR and nanobody targeting sequence of CD8+ T cells fused with N-terminal of Lamp2b).

[0132] The flow detection results showed that the positive rate of Strep2 expression on the surface of CD8+ / EGFRvIII+CAR+EXO engineered targeted EXO was 9.20%, which was significantly higher than that of 293F-EXO 0.59%, indicating that the CAR structure was successfully expressed and effectively loaded into the engineered EXO. The positive rate of VHH expression on the surface of CD8+ / EGFRvIII+CAR+EXO was 26.9%, while that of 293F-EXO was 0.16%, indicating that scFv / VHH, Strep2 structure and Lamp2b N-terminal fusion Targeting CD8+ T cell nanobody in CAR+ plasmid could be stably expressed and located on the surface of engineered EXO Figure 6 ). The above results comprehensively showed that 293F cells could effectively secrete and express engineered EXO with double specific functions by transfecting CAR structure and Lamp2b N-terminal fusion Targeting CD8+ T cell nanobody Figure 6 ).

[0133] Example 6: Preparation of CAR+ T cells by exosome incubation and detection of CD8+ T cell transduction efficiency

[0134] After peripheral blood sample collection, plasma was removed, and the resulting blood cell pellet was diluted twice its original volume with 0.9% sodium chloride injection. Then, 30 mL of the blood cell suspension was slowly spread onto 15 mL of Ficoll-Paque lymphocyte separation medium, ensuring a clear interface. The mixture was centrifuged at 800 g for 20 min at room temperature (centrifuge set to 1 step up, 1 step down). After centrifugation, the white membrane layer was carefully collected using a pipette and transferred to a new centrifuge tube. 0.9% sodium chloride injection was added to dilute the cell suspension at a volume ratio of ≥2:1. The mixture was centrifuged at 500 g for 7 min to remove residual lymphocyte separation medium. The supernatant was discarded, and the cells were resuspended in 10 mL of erythrocyte lysis buffer. After standing at room temperature for 10 min, three times the volume of 0.9% sodium chloride injection was added, and the mixture was centrifuged at 500 g for 7 min. The cell pellet was resuspended again in 0.9% sodium chloride injection and thoroughly mixed. 100 μL of the cell suspension was used for cell counting and viability testing. The remaining suspension was centrifuged at 500 × g for 10 min to obtain PBMCs. The drug was activated by adding anti-CD3 / CD28 activating magnetic beads (Miltenyi Biotec) and 1000 IU / mL recombinant human IL-2 for 24 h, and then a dose gradient of 10 was designed. 4 10 5 and 10 6 Particles / cells were collected and co-incubated with human PBMC cells. The cells were then analyzed by flow cytometry at 12 h, 24 h, 48 h, 72 h, Day 5, Day 9, and Day 12. Strep-II positive cells were identified as CAR-T cells.

[0135] The results show that ( Figure 7 In the CD8+ / EGFRvIII+CAR+EXO group, the proportion of positive expression of CAR-T cells in CD8+ T cells significantly increased with increasing engineered EXO dose. This was especially true in the high-dose group (10...). 6 The positivity rate of CAR+ T cells (particles / cells) peaked at 99.3% at 12 h, and remained high at 94.9%, 93.1%, and 91% at 24 h, 48 h, and 72 h, respectively. On Day 5, the positivity rate of CAR+ T cells was 86.5%, subsequently decreasing gradually to 17.9% on Day 9. A positivity rate of 3.11% was still detectable on Day 12, indicating a significant time-decrease trend in CAR mRNA expression within cells. In contrast, at 10 h... 5particles / cell conditions, the CAR+T cell expression positive rate can also reach 70.9% at 12 h, but is lower than 10 6 particles / cell conditions; it is indicated that the low-dose engineered EXO has a lower efficiency in transforming CD8+T cells into CAR-T cells. The CAR+T cell positive rate of the control group Flag / EGFRvIII+CAR+EXO at the same dose and time conditions is maintained at a very low level, with a maximum of not more than 2.04%, and the detection results at most time points are close to the background expression (<1%), which further proves that the non-targeted CD8+T cell engineered EXO lacks the ability to effectively target CD8+T cells and transform them into CAR-T cells.

[0136] 10 5 、10 6 particles / cell group were quantitatively analyzed, and the results showed that the CD8+ / EGFRvIII+CAR+EXO group had the strongest efficiency in transforming CD8+T cells into CAR-T cells under high-dose conditions, the longest expression duration, and obvious "dose-dependent" and "time-dependent" change rules. The CAR+T cell positive rate remained at a peak value for 12-72 h and then gradually attenuated, which is consistent with the kinetics of mRNA delivery and expression and degradation in cells Figure 8 A-B).

[0137] In summary, the EGFRvIII-targeted engineered EXO can efficiently deliver CAR mRNA to CD8+T cells in a dose-dependent manner, and induce the transformation of CD8+T cells into functional CAR-T cells in a short time. The CAR-mRNA expression is time-dependent, and the expression level starts to decrease after 5 days, significantly attenuates after 9 days, and is basically degraded after 12 days. It is indicated that this type of engineered EXO can realize the efficient, controllable, and short-cycle CAR-T cell generation process.

[0138] Example 7, in vitro killing of glioma cell lines by CAR-T cells targeting EGFRvIII:

[0139] To verify the ability of EGFRvIII-targeted engineered EXO to induce CD8+T cells to generate CAR-T cells in vitro and its tumor killing activity, the CD8+ / EGFRvIII+CAR+EXO and Flag / EGFRvIII+CAR+EXO two types of engineered EXO were respectively co-incubated with activated human PBMC for 24 hours. The flow cytometry detection results showed that Figure 9The proportion of CD8+ / Strep2+ double positive cells in the CD8+ / EGFRvIII+CAR+EXO group was as high as 94.6%, while the proportion of Flag group was only 1.83%, indicating that EGFRvIII-targeted engineered EXO can efficiently deliver CAR structure to CD8+ T cells and induce its expression, realizing the effective generation of CAR-T cells in vitro.

[0140] The luciferase activity was detected by a multifunctional enzyme label instrument. The results showed that under the condition of E:T=1:1, the anti-tumor activity of CAR-T cells in the CD8+ / EGFRvIII+CAR+EXO group was 34.7%, while the anti-tumor activity of CAR-T cells in the Flag / EGFRvIII+CAR+EXO group was 20.5%; when E:T=1:5, the anti-tumor activity of CAR-T cells in the CD8+ / EGFRvIII+CAR+EXO group increased to 69.4%, which was significantly higher than 48.3% of the Flag / EGFRvIII+CAR+EXO group; under the condition of E:T=1:10, the anti-tumor activity of CAR-T cells in the CD8+ / EGFRvIII+CAR+EXO group reached 92.5%, while the anti-tumor activity of CAR-T cells in the Flag / EGFRvIII+CAR+EXO group was 67.2% Figure 9 FIG. 8B shows that the CAR-T cells generated by the CD8+ / EGFRvIII+CAR+EXO have strong and specific cytotoxicity to BXPC-3 tumor cells.

[0141] In summary, EGFRvIII-targeted engineered EXO can not only efficiently convert CD8+ T cells into CAR-T cells in vitro, but also the generated CAR-T cells have strong specific anti-glioma tumor cell line activity.

[0142] Example 8, Establishment of a mouse humanization model

[0143] To evaluate the reconstitution of human immune system in mice, the chimeric level of human immune cells and T cell subpopulation composition in peripheral blood of humanized mice were analyzed by flow cytometry. After BV421-CD45, APC-Cy7-CD3, FITC-CD8 and APC-CD4 antibody combined staining, the expression proportion of human immune cells was detected. The detection results showed that the proportion of human CD45+ cells in the total lymphocytes in the peripheral blood of mice was 51.4%, and the proportion of CD3+ T cells in the CD45+ population was 97.4%, which showed that human T lymphocytes were effectively reconstructed in mice. Further analysis of CD3+ T cell subpopulation showed that the proportion of CD8+ T cells was 66.1%, the proportion of CD4+ T cells was 20.9%, and the proportion of CD4+CD8+ double positive cells was 9.12% (Fig. 1B). Figure 10 In summary, the humanized mouse model with high level of human T cell reconstitution was successfully constructed, which provided a reliable model basis for subsequent evaluation of CAR-T cell function in vivo.

[0144] Example 9, Establishment of Glioma Tumor Animal Model and Anti-tumor Effect of Exosomes in Vivo

[0145] The successfully humanized mice were subcutaneously injected with U251-GFP-Luc cells, and the injection dose was uniformly 1×10 7 cells / mouse, and the injection site was located in the right subcutaneous tissue. U251-GFP-Luc cells stably expressed GFP and luciferase double reporter genes, which could be used to monitor tumor formation and development in real time through in vitro and in vivo fluorescence signals. After one week of injection of U251-GFP-Luc cells, the mice were imaged in vivo, and when the tumor cell-related fluorescence signal in the mice reached 10 6 −10 8 photons / s / cm², it was proved that the tumor animal model was successfully established. The successfully modeled mice were randomly grouped at Day 0 and entered the subsequent engineered EXO treatment experiment (Fig. 2A). Figure 11

[0146] ​To further evaluate the antitumor activity of EGFRvIII-targeted engineered EXO against U251 cells in vivo, this study, based on the successful establishment of a humanized glioma animal model, randomly divided mice into three groups, which were injected via tail vein with CD8+EGFRvIII+CAR+EXO, Flag / EGFRvIII+CAR+EXO, and others, respectively. All mice underwent their first in vivo small animal imaging before treatment (Day 0) as baseline signal, and were immediately given their first engineered EXO injection at a uniform dose of 10¹² particles / mouse. Immediately after treatment, on Day 4, Day 7, Day 11, Day 14, and Day 18, imaging and repeated engineered EXO injections were performed. Changes in tumor burden were dynamically monitored, and quantitative analyses were conducted on changes in tumor burden, physiological status, and survival in the mice. Figure 11 A).

[0147] The results showed that from day 4 to day 7 of treatment, the tumor bioluminescence signal in the Flag / EGFRvIII+CAR+EXO group mice continued to increase, further increasing on days 11 and 14, with some mice dying due to excessive tumor burden. In contrast, the tumor burden in the CD8+ / EGFRvIII+CAR+EXO group mice showed significantly suppressed growth from day 4, remaining at a low level until day 18. Figure 11 (B and C). Further quantitative bioluminescence analysis showed that the signal in the Flag / EGFRvIII+CAR+EXO group increased rapidly from day 4, while the ROI in the CD8+ / EGFRvIII+CAR+EXO group remained at a low level throughout the observation period. Furthermore, mice in the Flag / EGFRvIII+CAR+EXO group died on day 11, while mice in the CD8+ / EGFRvIII+CAR+EXO group did not die on day 18. Figure 11 The results of weight change trends in the two groups of mice showed that the overall weight change trends of mice in the Flag / EGFRvIII+CAR+EXO group and the CD8+ / EGFRvIII+CAR+EXO group were consistent, and no obvious malignant emaciation or sudden weight loss was observed, indicating that the administration strategy of engineered EXO had good biosafety and tolerability within the dosage range of this experiment. Figure 11 The subcutaneous tumor volume of mice was measured every 7 days. The results showed that the tumor volume of mice in the CD8+ / EGFRvIII+CAR+EXO group was smaller than that in the Flag group, indicating that the targeted engineered EXO may have a certain inhibitory effect on tumors in the early stages of treatment. Figure 11 (F).

[0148] The above results show that CD8+ / EGFRvIII+CAR+EXO can effectively achieve the generation of functional CAR-T cells in vivo, and has therapeutic potential to inhibit and eliminate glioma cells.

[0149] Example 10, Targeting EGFRvIII engineered EXO in vivo to convert CD8+T cells into CAR-T cells

[0150] To verify whether the EGFRvIII-targeted engineered EXO can effectively deliver CAR mRNA and induce T cell conversion into CAR-T cells in vivo, we selected mice with successful humanized tumor model, collected peripheral blood by submandibular blood collection, and labeled the PBMC cells of mouse blood with Anti Human-CD8 flow cytometry antibody, and then incubated them with CD8+ / EGFRvIII+CAR+EXO and Flag / EGFRvIII+CAR+EXO in vitro, respectively. After 24 hours of incubation, flow cytometry was used to detect the expression level of Strep2 positive cells in CD8+T cell population to evaluate the delivery efficiency of CAR molecules and the conversion ability of T cells. The results showed that in the Flag / EGFRvIII+CAR+EXO group, only 0.46% of CD8+T cells were converted into CAR-T cells. While CD8+ / EGFRvIII+CAR+EXO converted 54.4% of CD8+T cells into CAR-T cells. Figure 12 A and B of FIG. 1.

[0151] The above results show that EGFRvIII-targeted EXO can effectively recognize CD8+T cells in vivo and efficiently deliver EGFRvIII-CAR mRNA, thereby achieving the conversion of CD8+T cells into CAR-T cells.

Claims

1. A chimeric antigen receptor targeting EGFRvlll, characterized in that, The chimeric antigen receptor comprises an antigen binding domain comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 3-5, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 6-8, respectively.

2. The chimeric antigen receptor of claim 1, wherein, The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9 or has at least 80% sequence identity to SEQ ID NO: 9; and / or, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 10 or has at least 80% sequence identity to SEQ ID NO: 10; Preferably, the heavy chain variable region and the light chain variable region are connected by a linker; More preferably, the linker has an amino acid sequence set forth in SEQ ID NO: 11; and / or, the antigen binding domain comprises, in order from N-terminus to C-terminus, a heavy chain variable region, a linker and a light chain variable region, or the antigen binding domain comprises, in order from N-terminus to C-terminus, a light chain variable region, a linker and a heavy chain variable region.

3. The chimeric antigen receptor of claim 2, wherein, The antigen binding domain comprises, in order from N-terminus to C-terminus, a heavy chain variable region, a linker and a light chain variable region; Preferably, the antigen binding domain has an amino acid sequence set forth in SEQ ID NO: 2 or has at least 80% sequence identity to SEQ ID NO:

2.

4. The chimeric antigen receptor of any one of claims 1-3, wherein, The chimeric antigen receptor further comprises a transmembrane domain and an intracellular domain, the intracellular domain containing a signaling domain and a costimulatory domain in tandem; optionally, further comprising a signal peptide; Preferably, the transmembrane domain is a transmembrane domain of CD28; and / or, the signaling domain is a signaling domain of CD3 zeta; and / or, the costimulatory domain is a costimulatory domain of 4-1BB; and / or, the signal peptide is N6 (SP).

5. An isolated nucleic acid, comprising, The nucleic acid encodes the chimeric antigen receptor of any one of claims 1-4; Preferably, the sequence of the nucleic acid encoding the antigen binding domain is set forth in SEQ ID NO: 1; and / or, the nucleic acid is DNA and / or RNA, for example, mRNA; More preferably, when the nucleic acid is mRNA, the mRNA comprises a 5' UTR, a 3' UTR and a RNA recruitment element, for example, a C / D box.

6. An engineered exosome, characterized in that, The engineered exosome targets a T cell and encapsulates the nucleic acid of claim 5; The engineered exosome is used for delivering the nucleic acid to a T cell; Preferably, the T cell is a CD8+ T cell.

7. The engineered exosome of claim 6, wherein, The surface of the engineered exosome has a polypeptide targeting a T cell, so that the engineered exosome targets the T cell; Preferably, the polypeptide is an anti-CD8+ antibody; and / or, the polypeptide is a nanobody.

8. A packaging system of the engineered exosome of claim 6 or 7, wherein, The packaging system comprises the nucleic acid of claim 5 and a RNA packaging device; Preferably, the RNA packaging device comprises a cell surface protein and an RNA binding protein; the cell surface protein and the RNA binding protein form a fusion protein; the RNA binding protein can specifically bind to the RNA recruiting element; so as to enrich the target nucleic acid in the engineered exosome; More preferably, the cell surface protein is CD63; the RNA binding protein is L7Ae; Further preferably, the RNA packaging device is pcDNA3.1-CD63-L7Ae.

9. The packaging system of claim 8, wherein, The packaging system further comprises a targeted delivery device, the targeted delivery device comprises a polypeptide targeting T cells and an exosome surface protein; Preferably, the exosome surface protein is exosome surface protein Lamp2b; the polypeptide targeting T cells is expressed in fusion with the exosome surface protein, so that the engineered exosome can target the T cells; More preferably, the targeted delivery device is BRD-PTK-CD8+-Lamp2b.

10. A delivery system characterized by, The delivery system comprises the chimeric antigen receptor of any one of claims 1-4, the nucleic acid of claim 5, or the engineered exosome of claim 6 or 7.

11. A method of making the engineered exosome of claim 6 or 7, characterized in that, The method comprises: transfecting human cells with the packaging system of claim 8 or 9, and obtaining the supernatant containing the engineered exosome by culturing; Preferably, the engineered exosome is obtained by purifying the supernatant; More preferably, the human cells are 293F cells.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the chimeric antigen receptor of any one of claims 1-4, the nucleic acid of claim 5, or the engineered exosome of claim 6 or 7, or the delivery system of claim 10, and a pharmaceutically acceptable carrier.

13. Use of the chimeric antigen receptor of any one of claims 1-4, the nucleic acid of claim 5, the engineered exosome of claim 6 or 7, the delivery system of claim 10, or the pharmaceutical composition of claim 12 in the preparation of a medicament for targeting and / or treating tumors; Preferably, the tumor is glioblastoma.

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