Chimeric antigen receptor exosome of targeted CD8 T cell, CAR-T cell and construction method and application of chimeric antigen receptor exosome

Abstract

The invention discloses a chimeric antigen receptor exosome targeting a CD8T cell, a CAR-T cell and a construction method and application of the chimeric antigen receptor exosome, and relates to the technical field of biomedicine, and the construction method comprises the following steps: co-transfecting three plasmids, namely BRD-PTK-CD8 < + >-Lamp2b, pcDNA3.1-CD63-L7Ae and pcDNA3.1-N6 (SP)-Nb60-T20-C / D box, into a 293F cell, incubating, and purifying to obtain the exosome capable of targeting the CD8T cell. The exosome can deliver CAR-mRNA to T cells and convert CD8 + cells into CAR-T cells, and blood of an infected person does not need to be drawn; cAR molecules aiming at in-vivo malignant cells can be prepared in vivo; chemotherapy and radiotherapy are not needed, and side effects are avoided; and universality is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a chimeric antigen receptor exosome targeting CD8 T cells, a CAR-T cell, and a construction method and application thereof. Background Art

[0002] Pancreatic cancer is a highly malignant solid tumor with an increasing incidence worldwide. Due to the lack of obvious early symptoms, most patients are diagnosed in the advanced stages, resulting in poor treatment outcomes and an extremely low five-year survival rate. Pancreatic cancer is devastating not only due to its high mortality rate but also its severe impact on patients' quality of life. The rapid growth and aggressive metastasis of the tumor often cause patients to experience severe pain, digestive dysfunction, and drastic weight loss.

[0003] The treatment of pancreatic cancer mainly relies on surgical resection, chemotherapy and radiotherapy, targeted therapy and immunotherapy. Surgical resection is the only possible way to cure pancreatic cancer, but it is only suitable for patients diagnosed in the early stages. Common chemotherapy drugs include gemcitabine and fluorouracil, which can prolong survival to a certain extent, but have significant side effects and are prone to drug resistance. Radiotherapy is mainly used to locally control tumor growth, but its damage to surrounding normal tissues limits its application. However, most pancreatic cancer patients are in the late stage at the time of diagnosis and cannot undergo surgery. Chemotherapy is a common treatment method, and drugs such as gemcitabine are often used in adjuvant or neoadjuvant therapy to help delay tumor progression and reduce the risk of recurrence. Radiotherapy can be used for patients with locally advanced disease, usually in combination with chemotherapy, to slow tumor growth and relieve symptoms.

[0004] Currently, CART therapy has gradually become a research hotspot. Although it has shown potential in some patients, its overall efficacy still needs further verification. Therefore, developing more effective treatments with fewer side effects is an important direction of current pancreatic cancer research.

[0005] CAR-T cells are highly effective in treating leukemia and lymphoma, but solid tumors are currently insensitive to CAR-T cell therapy. This is primarily due to the difficulty of targeted delivery of CAR-T cells to tumor tissue and their inability to effectively proliferate in the host over the long term. Traditional CAR-T cell development strategies require multiple steps, including patient blood collection, T cell extraction, viral transfection, in vitro CAR-T cell culture, and expansion, before administration to patients. These strategies can also cause serious side effects, such as cytokine release syndrome (CRS), and can damage normal tissues due to target selectivity. Furthermore, the process requires a customized, cumbersome, and expensive procedure for each patient, limiting universal access to effective treatment. This approach, coupled with its tedious, time-consuming, and expensive nature, limits the potential for widespread benefit to a limited number of patients. To address these clinical challenges, a universal, in vivo, targeted transfection of T cells to generate CAR-T cells is urgently needed for the treatment of solid tumors. Summary of the Invention

[0006] In view of this, the main purpose of the present invention is to provide a chimeric antigen receptor exosome targeting CD8 T cells, the construction of CAR-T cells and their application to solve the problems existing in the prior art.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for preparing chimeric antigen receptor exosomes targeting CD8+ T cells comprises the following steps:

[0009] S1, sequentially connect NB60, G4S-Strep tagⅡ-CD8 hinge, and N6-SP to form the gene sequence fragment N6-SP-NB60-G4S-Strep tagⅡ-CD8 hinge;

[0010] S2. Ligate the gene sequence fragment obtained in step S1 to the plasmid pcDNA 3.1N6(SP)-T20-VRC C / D box to obtain pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid;

[0011] S3. Construct two plasmids, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae;

[0012] The nucleotide sequence of pcDNA3.1-N6(SP)-Nb60-T20-C / D box is shown in SEQ ID NO: 1; the amino acid sequence of Anti-CD8-Lamp2b in BRD-PTK-CD8+-Lamp2b is shown in SEQ ID NO: 2; the nucleotide sequence of CD63-L7Ae in pcDNA3.1-CD63-L7Ae is shown in SEQ ID NO: 3;

[0013] S4. The three plasmids pcDNA3.1-N6(SP)-Nb60-T20-C / D box, BRD-PTK-CD8+-Lamp2b, and pcDNA3.1-CD63-L7Ae were co-transfected into 293F cells, and exosomes were obtained after incubation and purification.

[0014] Furthermore, in step S4,

[0015] The co-transfection specifically includes: mixing pcDNA3.1-N6(SP)-T20-Nb60-CDbox, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae with complete culture medium to obtain a plasmid mixture; at the same time, adding PEI solution to another portion of F complete culture medium to obtain a PEI mixture, standing for 5 minutes, combining the plasmid mixture and the PEI mixture, standing for 30 minutes, and then adding to 293F cells for co-transfection;

[0016] Among them, the weight ratio of pcDNA3.1-N6(SP)-T20-Nb60-CDbox, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae is 3:1:1.

[0017] Furthermore, in step S4,

[0018] The incubation specifically includes: adding OPM-CHO PFF06 medium and L-glutamine 24 hours after co-transfection, and collecting the cell supernatant 72 hours later;

[0019] The purification specifically comprises the following steps: centrifuging the obtained cell supernatant at 4°C and 300×g for 5 minutes to remove live cells, then centrifuging at 3000×g for 30 minutes to remove dead cells and debris, and finally centrifuging at 10000×g for 60 minutes to remove large extracellular vesicles to obtain a pretreated supernatant;

[0020] Add Universal Benzo nuclease and magnesium chloride to the pretreated supernatant. After overnight at 4°C, filter through a 0.22 μm filter cup. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000 × g for 90 min. Discard the supernatant, resuspend the pellet in PBS, and centrifuge again at 100,000 × g for 90 min. Finally, resuspend the pellet in PBS and store at -80°C.

[0021] The present invention also aims to provide chimeric antigen receptor exosomes targeting CD8+ T cells prepared by any of the above methods.

[0022] The present invention also aims to provide a method for constructing CAR-T cells, comprising co-incubating the exosomes described in claim 4 with PBMC cells to obtain CAR-T cells capable of targeting Trop2.

[0023] Specifically, after collecting peripheral blood samples, the plasma is removed and the resulting blood cell pellet is diluted with 0.9% sodium chloride injection at 2 times the original volume; then 30 mL of blood cell suspension is slowly layered on 15 mL of lymphocyte separation fluid (Ficoll-Paque), ensuring that the interface is clearly visible, and centrifuged at 800 g for 20 minutes at room temperature. After centrifugation, the buffy coat is collected with a pipette and transferred to a new centrifuge tube, and 0.9% sodium chloride injection is added to dilute the cell suspension at a volume ratio of ≥2:1, and centrifuged at 500 g for 7 minutes. The remaining lymphocyte separation solution was removed; the supernatant was discarded, and 10 mL of red blood cell lysis buffer was added to resuspend the cells. The cells were allowed to stand at room temperature for 10 minutes, and then 3 volumes of 0.9% sodium chloride injection were added and centrifuged at 500 g for 7 minutes. The cell pellet was resuspended in 0.9% sodium chloride injection and mixed thoroughly. 100 μL of the cell suspension was collected for cell counting and viability testing. The remaining suspension was centrifuged at 500 × g for 10 minutes to obtain PBMCs. Anti-CD3 / CD28 activation magnetic beads (Miltenyi Biotec) and 1000 IU / mL recombinant human IL-2 were added for stimulation and activation for 24 hours. CAR-T cells were then co-incubated with exosomes to obtain CAR-T cells in vitro.

[0024] Alternatively, the obtained exosomes can be directly introduced into the body to obtain CAR-T cells in vivo.

[0025] The present invention also aims to provide CAR-T cells prepared by the above method.

[0026] The present invention also aims to provide the use of the chimeric antigen receptor exosomes targeting CD8+ T cells or the CAR-T cells described above in the preparation of products for the treatment of pancreatic cancer.

[0027] The present invention also aims to provide a drug for treating pancreatic cancer, comprising the exosomes according to claim 4 or the CAR-T cells according to claim 6.

[0028] The beneficial effects of the present invention include at least:

[0029] In the present invention, the NB60 nanobody consists of only one variable region (VHH), while traditional antibodies are composed of two pairs of heavy and light chains. VHH is composed of a single amino acid sequence and has high stability and antigen-binding ability. Trop2 is used as the target of the model CAR-T to construct the CAR cassette plasmid. T cell-targeted exosomes are used as a carrier to encapsulate the CAR cassette plasmid, thereby increasing the specificity of the vector binding to T cells. T cells are then transfected in vivo to convert them into CAR-T cells while minimizing off-target effects.

[0030] This invention achieves enhanced helper function of CAR-T cells and their in vivo production by optimizing the design of Trop2-specific nanobodies and combining them with an LNP-mRNA delivery system. This provides a Trop2-targeted CAR-T cell that can be produced in vivo and specifically binds to the Trop2 antigen. In a cell killing assay of a pancreatic cancer cell line, the in vivo constructed CAR-T cell with the Trop2 target modification was able to kill malignant tumor cells, achieving an in vivo CAR-T effect.

[0031] That is, the chimeric antigen receptor exosomes targeting CD8+ T cells constructed by the present invention can deliver CAR-mRNA to T cells and convert CD8+ cells into CAR-T cells without the need to draw blood from infected people; CAR molecules targeting malignant cells in the body can be prepared in vivo; chemotherapy and radiotherapy are not required, and there are no side effects; and it is universal. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the connection between N6-SP, NB60, G4S-Strep tagⅡ-CD8 hinge fragments.

[0033] Figure 2 Schematic diagram of the pcDNA 3.1N6(SP)-T20-VRC C / D box plasmid.

[0034] Figure 3 Schematic diagram of the pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid.

[0035] Figure 4Diagram of the construction and lentiviral packaging of PLV-Luc-GFP. (A) Schematic diagram of the PLV-Luc-GFP plasmid structure; (B) A lentiviral titer rapid test card was used to assess lentiviral vector packaging efficiency; (C) 293T cells were transfected with lentivirus, and fluorescence intensity was measured using a fluorescence microscope 48 hours later.

[0036] Figure 5 Figure 1: Detection of GFP and Luc in GFP- and Luc-double-labeled cells. (A) Flow cytometry was used to detect GFP expression on the surface of BXPC-3 double-labeled cells. (B) Fluorescence microscopy was used to observe the fluorescence intensity of GFP expression in BXPC-3 double-labeled cells. (C) In vivo imaging was used to detect the fluorescence intensity of luciferase expression in BXPC-3 double-labeled cells.

[0037] Figure 6 Schematic diagram of engineered EXO mRNA targeted delivery.

[0038] Figure 7 Figure 1 shows the isolation and characterization of CAR-engineered EXOs derived from 293F cells. (A) Schematic diagram of the experimental workflow for engineered EXO isolation; (B) TEM image of engineered EXO morphology; (C) NTA analysis of engineered EXO particle size distribution and concentration; (D) analysis of engineered EXO surface markers.

[0039] Figure 8 This is the expression result of CD63, CD81 and CD9 on the surface of engineered EXO.

[0040] Figure 9 This is a diagram showing the expression of Strep2 tag and VHH nanoantibody on the surface of targeted engineered EXO.

[0041] Figure 10 This is an analysis of the positive expression of CAR-T cells in CD8+T cells at different doses and time points after incubation with engineered EXO targeting Trop2.

[0042] Figure 11 Quantitative statistical curve of the proportion of Trop2-targeted CAR+T cells under three dose conditions. (A) 10 4 Analysis of CAR-T cell positive rate under different particle / cell dosage conditions; (B) 10 5 Analysis of CAR-T cell positive rate under Particles / cell dosage conditions; (C) 10 6 Analysis of CAR-T cell positivity rate under different particle / cell dosage conditions.

[0043] Figure 12Figure 2 shows the cytotoxicity analysis of CAR-T cells generated from CD8+ T cells using engineered EXO targeting Trop2. (A) Flow cytometry analysis of the positive rate of conversion of CD8+ T cells into CAR-T cells using two engineered EXOs. (B) Cytotoxicity analysis of CAR-T cells generated from CD8+ T cells using engineered EXO targeting Trop2 against pancreatic cancer cells (n=3, *p<0.05, **p<0.01, ***p<0.001).

[0044] Figure 13 The figure shows the proportion of human immune cells in humanized mice detected by flow cytometry.

[0045] Figure 14 Figure 3. Establishment of pancreatic cancer animal models and in vivo evaluation of the antitumor activity of engineered EXOs targeting Trop2. (A) Mouse experimental timeline; (B) Fluorescence analysis of in vivo mouse imaging before and after treatment with the two engineered EXOs; (C) Comprehensive analysis of luciferase expression before and after treatment in each group of mice; (D) Body weight change curves of each group of mice; (E) Survival curves of each group of mice; (F) Growth curves of subcutaneous xenograft tumor volume in each group of mice (n > 3, *p < 0.05, **p < 0.01).

[0046] Figure 15 Figure 3: Analysis of the positive rate of CAR-T cells after incubation of in vivo-derived PBMCs with engineered EXO targeting Trop2. (A) After 24 hours of incubation of mouse PBMCs with the two engineered EXOs, flow cytometry was used to assess Strep2 expression in CD8+ T cells. (B) Statistical analysis of the proportion of CAR-T cells (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). DETAILED DESCRIPTION

[0047] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0048] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0049] The solution proposed by the present invention is described in detail below through specific embodiments:

[0050] Example 1. Construction of pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid

[0051] 1. Sequence Nb60 was synthesized by Sangon Biotechnology Co., Ltd. and cloned into the pUC57 vector. The sequence of Nb60 is shown in SEQ ID NO: 4.

[0052] 2. Using a human cDNA library as a template, primers were designed to amplify the N6-SP, NB60, and G4S-StreptagⅡ-CD8 hinge fragments. Overlap PCR was used to sequentially amplify NB60, G4S-Strep tagⅡ-CD8 hinge, and N6-SP fragments, which were then linked to form fragments with restriction sites NheⅠ and SgrAⅠ. ​​The schematic diagram of the structure is shown in Figure 2. Figure 1 shown.

[0053] Table 1 Amplification primer sequence list

[0054]

[0055]

[0056] 3. The plasmid pcDNA 3.1N6(SP)-T20-VRC C / D box was double-digested with NheⅠ and SgrAⅠ restriction enzymes. The product was subjected to 0.8% agarose gel electrophoresis and the gel was cut and recovered in an Eppendorf tube. The corresponding fragment was recovered using agarose gel recovery kit from Kangwei Century Company, and the purity and concentration of the product were determined ( Figure 2 ). The sequence of the plasmid pcDNA3.1 N6(SP)-T20-VRC C / Dbox is shown in SEQ ID NO:11.

[0057] 4. Add the recovered vector fragment and the N6-SP-NB60-G4S-Strep tag II-CD8 hinge fragment to an Eppendorf tube at a 1:2 molar ratio. Add Exnase II ligase (Vazyme) and homologous recombination enzyme 5×CE II buffer and react at 37°C for 0.5 hours. Remove 10 μL of the ligation solution and add 100 μL of DH5α competent cells. Incubate on ice for 30 minutes, then heat shock at 42°C for 90 seconds. Add 500 μL of SOC medium and incubate at 37°C at 220 rpm for 2 hours. After 2 hours, centrifuge the Eppendorf tube at 4000g for 1 minute to remove 400 μL of excess liquid. Spread the remaining liquid onto an LB plate containing kanamycin and incubate at 37°C for 12 hours. Pick a single colony from each plate and inoculate 5 mL of LB liquid medium at 37°C at 220 rpm for 12 hours. The ligation solution is composed of the following: linearized vector: 2ul, insert fragment (N6-SP-NB60-G4S-Strep tagⅡ-CD8hinge): 1ul, 5×CEMultis buffer: 4ul, C113 Exnase: 2ul, ddH2O: 11ul, total volume: 20ul.

[0058] 5. Use the Kangwei Century Small Extraction Kit to extract the plasmid and obtain the plasmid pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox. After the plasmid is sent to Shanghai Biotech Co., Ltd. for first generation sequencing verification, the DH5α strain containing the plasmid pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox is preserved. The complete map of pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox is shown below. Figure 3 shown.

[0059] Example 2: PLV-Luc-GFP lentiviral packaging

[0060] The present invention constructs a PLV-Luc-GFP lentiviral expression vector, the structure of which is as follows: Figure 4 As shown in A, the corresponding sequence is shown in SEQ ID NO: 12. The vector drives the co-expression of Luciferase and GFP (green fluorescent protein) with the EF-1α promoter, and connects the puromycin resistance gene (Puro) through the (Internal Ribosoe Entry Site, IRES) sequence to ensure the stable co-expression of fluorescent labeling and resistance screening. The results of lentiviral packaging and titer detection showed that after 48 hours of co-transfection of PLV-Luc-GFP plasmid and auxiliary packaging plasmid into 293T cells, the culture supernatant was collected and the virus particles were concentrated. The lentiviral titer rapid detection card ( Figure 4B) Verify the virus packaging efficiency. The detection band is clearly visible, indicating that the lentivirus is successfully packaged. The lentivirus infection efficiency is further verified by detecting GFP expression under a fluorescence microscope ( Figure 4 C). At the same time, bright field microscopy showed that the cells were in good condition, with no obvious apoptosis or toxic effects.

[0061] Example 3: Construction of GFP and Luc dual-labeled target cells

[0062] 1. Infection of BXPC-3 cells (human pancreatic adenocarcinoma cells in situ)

[0063] 1) Take BXPC-3 cells in good growth state and adjust the density to 1×10 6 cells / mL, and 1×10 6 cells / well were seeded into 6-well plates.

[0064] 2) Add 100 μL of concentrated PLV-Luc-GFP lentiviral solution to each well and add Polybrene to a final concentration of 8 μg / mL to enhance viral infection efficiency. Incubate the cells at 37°C, 5% CO₂ for 4–6 hours. Discard the viral culture medium and add fresh, prewarmed RPMI-1640 complete medium for continued culture.

[0065] 2. BXPC-3 dual-labeled target cell resistance screening

[0066] 1) 24-48 hours after infection, add puromycin at a final concentration of 4 μg / mL for resistance screening. During the screening process, observe cell morphology, survival rate, and green fluorescence signal expression daily to dynamically evaluate the screening process.

[0067] 2) When the cell density reaches 80-90% confluency, digest with 0.05% Trypsin-EDTA and passage at a ratio of 1:3 to 1:5 to maintain cell proliferation and long-term stability. Screening continues for approximately 2 weeks. After complete apoptosis of uninfected cells, reduce the puromycin concentration to 1 μg / mL to maintain stable screening.

[0068] 3. Phenotypic and functional identification of BXPC-3 and U251 dual-labeled target cells

[0069] 1) Fluorescence Microscopy was used to observe and record the GFP expression of cells, and the expression level of the exogenous gene and the intensity of the fluorescence signal were visually evaluated. The screened cells were collected, washed twice with PBS, and resuspended in flow cytometry buffer. The proportion of GFP-positive cells was then detected using a BD flow cytometer, and data analysis was performed using FlowJo-V10 software to quantify the proportion of GFP-positive cells and analyze the purity and transfection efficiency of the cell population. The results showed that the GFP positivity rate of BXPC-3-Luc-GFP cells was 94.2%, and the GFP positivity rate of U251-Luc-GFP cells was 99.6%, while the untransfected control cells (BXPC-3-Control) had no GFP signal. The results showed that the lentiviral vector successfully mediated the efficient transfection of the GFP gene in BXPC-3 cells, and the purity of the screened cell population was high ( Figure 5 A).

[0070] Further observation of GFP expression using a fluorescence microscope revealed that BXPC-3-Luc-GFP cells exhibited strong green fluorescence signals under the GFP channel, while no GFP signal was detected in the control group (BXPC-3-Control). This indicates that GFP expression after lentiviral transfection is stable and uniform, and that GFP expression levels are high throughout the cell population, meeting the criteria for establishing a stable cell line ( Figure 5 B).

[0071] 2) The luciferase activity of cells was assessed using the In Vivo Imaging System (IVIS).

[0072] Collect the cells to be tested and adjust the cell density to 1×10 6 cells / mL, and cells were evenly seeded in 6-well culture plates. After 24 hours, the cell supernatant was aspirated, and after washing twice with PBS, 1 mL of bioluminescent substrate solution (final concentration of 150 μg / mL) was added to each well and incubated at 37°C for 5 minutes. After the incubation, the 6-well plate was immediately placed in an IVIS (in vivo imaging system) for exposure imaging at a luciferase emission wavelength of 560 nm. Background correction and signal normalization analysis were performed using 1.7.05_amiview.sav software to quantitatively evaluate the luciferase expression of the target cells.

[0073] The results showed that BXPC-3-Luc-GFP cells all showed significant luciferase activity with high luminescence intensity, while no obvious bioluminescence signal was detected in the control group (BXPC-3-Control). This further proves that the Luciferase gene is stably expressed in the target cells and can be detected in real time by bioluminescence imaging ( Figure 5 C).

[0074] Example 4. Construction of an engineered EXO (exosome) targeted delivery system

[0075] 1. Construction of CAR mRNA expression plasmid and exosome packaging plasmid

[0076] The nucleotide sequence of the CAR structure + C / D box is shown in SEQ ID NO: 1:

[0077] 2. Construction of CD8-targeted nanoantibody expression plasmid

[0078] The amino acid sequence of Anti-CD8-Lamp2b is shown in SEQ ID NO: 2;

[0079] 3. Construction of CD63 exosome packaging plasmid

[0080] The nucleotide sequence of CD63-L7Ae is shown in SEQ ID NO: 3;

[0081] Establish an L7Ae-C / D box specific packaging system by adding C / D box to the 3'UTR region of CAR-mRNA and fusing the EXO-specific tetraspanin CD63 with L7Ae to form a fusion protein. By utilizing the specific binding of the RNA recruitment element C / D box with the RNA binding protein L7Ae, CAR-mRNA is enriched into EXO, thereby improving its loading efficiency in EXO. By fusing a nanobody specifically targeting human CD8+ with the N-terminus of Lamp2b, it is displayed on the membrane surface of the engineered EXO, exerting its receptor recognition function to mediate its recognition of specific receptor cells, allowing its content mRNA to translate and express the target protein in the target cell ( Figure 6 An engineered EXO targeted delivery system was constructed by co-transfection of three plasmids: pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox; BRD-PTK-CD8+(Flag)-Lamp2b; and pcDNA3.1-CD63-L7Ae. BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae plasmids were constructed in our laboratory.

[0082] Example 5. Preparation and characterization of exosomes

[0083] 1. 293F cell culture and exosome preparation

[0084] 293F cells were cultured in a 1:1 mixture of antibiotic-free OPM-293CD05 medium (purchased from Shanghai Aopmin Biotech Co., Ltd., catalog number: 81075-001) and SMM293-TII medium (purchased from Beijing Sino Biological Technology Co., Ltd., catalog number: RZ18OC2401-A) at 125 rpm, 37°C, and 5% CO2. Mix 60 μg of pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid, 20 μg of BRD-PTK-CD8+-Lamp2b, and 20 μg of pcDNA3.1-CD63-L7Ae with 4 mL of complete culture medium (293CD05 culture medium and SMM293-TII culture medium were mixed in a 1:1 ratio). At the same time, 300 μL of PEI (polyethyleneimine) solution (1 mg / mL) was added to another 4 mL of complete culture medium. After standing for 5 minutes, the plasmid mixture was combined with the PEI mixture, and after standing for 30 minutes, 293F cells were added for co-transfection. After 24 hours, OPM-CHO PFF06 medium (1 / 20) and L-glutamine (1 / 50) were added, and the cell supernatant was collected after 72 hours. OPM-CHO PFF06 medium was purchased from Shanghai Aopuma Biotechnology Co., Ltd., catalog number: 1265F05-001. Live cells were removed by centrifugation at 300×g for 5 minutes at 4°C, and dead cells and debris were removed by centrifugation at 3000×g for 30 minutes. Finally, large extracellular vesicles were removed by centrifugation at 10000×g for 60 minutes. Universal Benzo nuclease (2U) and magnesium chloride (2μmol / L) were added to the pretreated supernatant, and the supernatant was filtered through a 0.22μm filter cup after overnight at 4°C. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 100,000×g for 90 min using an OptimaXE-100 ultracentrifuge. The supernatant was discarded, and the pellet was resuspended in 20mL PBS and centrifuged again at 100,000×g for 90 min. Finally, the pellet was resuspended in 200μL PBS and stored at -80°C ( Figure 7 A).

[0085] 2. Exosome Characterization

[0086] (1) Engineered EXO Transmission Electron Microscopy (TEM) Characterization

[0087] TEM is used to observe the morphology, size, and integrity of engineered EXOs to confirm the nanostructure characteristics of the sample. In this experiment, the isolated and purified engineered EXOs were analyzed by transmission electron microscopy using the negative staining method. The specific method is as follows:

[0088] 1) Sample preparation: Take an appropriate amount of engineered EXO suspension (concentration of about 10 9 particles / mL), diluted appropriately with PBS, and 10 μL was added dropwise onto the copper grid and incubated at room temperature for 10 min to allow the engineered EXO to be fully adsorbed.

[0089] 2) Negative staining: Use filter paper to absorb excess liquid, then add 2% phosphotungstic acid or 2% uric acid solution for negative staining for 30-60 seconds, and gently absorb the stain again with filter paper to prevent the sample from drying out.

[0090] 3) Drying and Observation: After the copper grid was dried at room temperature, it was imaged using a TEM at 80-120 kV to observe the morphology, size, and integrity of the engineered EXOs, and the images were recorded.

[0091] The results are as follows Figure 7 As shown in Figure B, TEM observation shows that the engineered EXO has a typical "cup-shaped" vesicle structure of approximately 100 nm.

[0092] (2) Nanoparticle Tracking Analysis (NTA)

[0093] NTA technology is used to analyze the particle size distribution and concentration of engineered EXO. It is based on the principle of dynamic light scattering and calculates the particle size and determines the particle concentration by tracking the Brownian motion of particles. The experiment uses the ZetaView Nanoparticle Tracking Analyzer for measurement. The specific method is as follows:

[0094] 1) Sample preparation: Take an appropriate amount of engineered EXO suspension (about 10 9 particles / mL), and diluted appropriately with sterile PBS to the instrument detection range (usually 10 7 particles / mL), mix well and test.

[0095] 2) Instrument calibration: Before the experiment, standard nanoparticles were used to calibrate the instrument to ensure the accuracy of light source, focus, and particle concentration measurements.

[0096] 3) Detection Process: The sample is injected into the detection cell. Laser scattering and Brownian motion analysis are used to record particle size distribution data across multiple fields of view while the camera captures the motion trajectory of the engineered EXO. Detection parameters are typically set at 25°C and a detection time of 60 seconds.

[0097] The NTA results showed that the main peak size of the collected engineered EXO particles ranged from 120 to 150 nm and presented a single peak, which was consistent with the normal distribution ( Figure 7 C), which is basically consistent with the engineered EXO particle size shown in TEM images.

[0098] (3) Western Blot (WB)

[0099] 72 hours after transfection, remove 1 ml of cell suspension, centrifuge and discard the culture medium. Gently wash the cells twice with pre-chilled PBS, collect the cells, add 200 μL of RIPA lysis buffer (containing 1% PMSF protease inhibitor), and lyse on ice for 30 minutes. Subsequently, centrifuge at 10,000 × g for 10 minutes at 4°C, and collect the supernatant.

[0100] 2) Determine the protein content of the lysed cell suspension using the Beyotime BCA Protein Assay Kit. Mix the lysed cell suspension with 5× Loading bµffer, heat at 100°C for 10 minutes, and then store at -20°C until use.

[0101] 3) Perform SDS-PAGE electrophoresis with a sample load of 20 μg of protein per well at 80 V for 20 min, until bromophenol blue enters the separation gel; then adjust to 120 V for 60-90 min, until bromophenol blue migrates to the bottom of the gel.

[0102] 4) Transfer the protein to a nitrocellulose membrane using a wet transfer method at a rate of 1 kD protein / min. Then, place the nitrocellulose membrane in a protein-free rapid blocking buffer and block it at room temperature for 20 minutes.

[0103] 5) Dilute the primary antibody in TBST buffer according to the antibody instructions and incubate overnight at 4°C. Then, wash the NC membrane three times with TBST buffer for 10 minutes each to remove unbound primary antibody.

[0104] 6) Incubate with secondary antibody: Soak the NC membrane in a 1:5000 dilution of secondary antibody solution and incubate in the dark for 1 hour with shaking. Then, wash with TBST buffer three times for 10 minutes each to remove unbound secondary antibody.

[0105] 7) Finally, ECL luminescent solution A and B were mixed in a ratio of 1:1 for development, and the expression of the target protein was detected using a gel imaging analysis system.

[0106] The results are as follows Figure 7 As shown in D, WB results showed that typical EXO markers such as CD63, CD81, and CD9 were present on the surface of engineered EXO.

[0107] (4) Nano-flow Cytometry (nFCM)

[0108] Nano-flow cytometry was used to detect the expression of CD63, CD81, and CD9 on the surface of targeted engineered EXO and unmodified blank EXO derived from 293F cells. The results showed that the positive rates of CD63, CD81, and CD9 on the surface of CD8+ / Nb60+CAR+EXO were 55.6%, 54.2%, and 48.3%, respectively; while the expression rates of CD63, CD81, and CD9 in unmodified EXO derived from 293F were 20.0%, 48.0%, and 35.1%, respectively. Figure 8 The results showed that the targeted engineered EXOs were able to stably express typical EXO surface proteins, and CD63 expression was significantly upregulated in the targeted EXOs, far exceeding that of unmodified 293F-derived EXOs. These results indicate that the targeted engineered EXOs successfully enriched overexpressed CD63 during the packaging process.

[0109] (5) Verification of specific protein expression on the surface of engineered EXOs

[0110] To evaluate whether the engineered EXO surface successfully expresses the specific tag Strep2 and nanoantibody sequence in the CAR+ plasmid, the present invention directly stains unmodified EXO derived from 293F cells and targeted engineered EXO by nFCM (nano flow cytometry). FITC (fluorescein isothiocyanate)-labeled Strep2 antibody and PC5-labeled VHH antibody were used to stain the surface of the EXO samples to identify whether the engineered EXO carries the Strep tag (representing the expression of the CAR structure) and the nanoantibody structure represents the nanoantibody targeting sequence of the Lamp2b N-terminus fusion targeting CD8+T cells).

[0111] Flow cytometry results showed that the positive rate of Strep2 expression in CD8+ / Nb60+CAR+EXO engineered targeting EXO was 13.9%, significantly higher than the 0.59% expression of 293F-EXO. The results showed that the CAR structure was successfully expressed and effectively loaded into the engineered EXO. The positive rate of VHH expression in CD8+ / Trop2+CAR+EXO was 37.2%, while that of 293F-EXO was 0.16%, indicating that the scFv / VHH, Strep2 structure and Lamp2b N-terminal fused targeting CD8+T cell nanoantibodies in the CAR+ plasmid can be stably expressed and localized on the surface of the engineered EXO ( Figure 9 ).

[0112] The above results show that 293F cells can be transfected to construct a targeting CD8+T cell nanoantibody carrying a CAR structure fused to the N-terminus of Lamp2b, and effectively secrete engineered EXOs expressing dual-specific functions.

[0113] Example 6. Preparation of CAR+T cells by incubation of exosomes and detection of CD8+T cell transduction efficiency

[0114] Peripheral blood samples were collected, plasma removed, and the resulting blood cell pellet diluted 2-fold to its original volume with 0.9% sodium chloride injection. Next, 30 mL of the blood cell suspension was slowly layered onto 15 mL of lymphocyte separation medium (Ficoll-Paque), ensuring a clear interface. The sample was centrifuged at 800 g for 20 min at room temperature (the centrifuge was set to 1:1 ratio). After centrifugation, the buffy coat was carefully collected using a pipette and transferred to a new centrifuge tube. The cell suspension was diluted with 0.9% sodium chloride injection to a volume ratio of ≥2:1, and the remaining lymphocyte separation medium was removed by centrifugation at 500 g for 7 min. The supernatant was discarded, and the cells were resuspended in 10 mL of red blood cell lysis buffer and allowed to stand at room temperature for 10 min. Three volumes of 0.9% sodium chloride injection were added and centrifuged at 500 g for 7 min. The cell pellet was resuspended in 0.9% sodium chloride injection and mixed thoroughly. 100 μL of the cell suspension was collected for cell count and viability testing. The remaining suspension was centrifuged at 500×g for 10 min to obtain PBMCs (peripheral blood mononuclear cells). Anti-CD3 / CD28 activation magnetic beads (Miltenyi Biotec) and 1000 IU / mL recombinant human IL-2 were added for stimulation and activation for 24 h, and then a dose gradient of 10 4 , 10 5 and 10 6 The particles / cells were co-incubated with human PBMC cells, and the cells were detected by flow cytometry at 12h, 24h, 48h, 72h, Day 5, Day 9 and Day 12. The strep-II positive cells were CAR-T cells.

[0115] The results show that ( Figure 10 ), in the CD8+ / Trop2+CAR+EXO group, with the increase of engineered EXO dose, the positive expression ratio of CAR-T cells in CD8+T cells increased significantly. Especially in the high-dose group (10 6The positive rate of particles / cell) CAR+T cells reached 97.6% at 12h, peaked at 98.2% at 24h, and still had high-level expression of 98.9% and 99.3% of CAR+T cells at 48h and 72h, respectively. The positive rate of CAR+T cells was 93.1% on the 5th day, and then gradually decreased to 10.9% on the 9th day. On the 12th day, 2.81% of CAR+T cells were still detected, indicating that the expression of CARmRNA in cells had a significant time decay trend. In contrast, at 10 5 Under the particle / cell condition, the positive expression rate of CAR+T cells can reach 65.9% in 24 hours, but it is lower than 10 6 The decay rate is faster under the particle / cell condition; while 10 4 The CAR+T cell positivity rate in the particles / cell group was below 2% at all time points, indicating that low-dose engineered EXOs were inefficient in converting CD8+T cells into CAR-T cells. In contrast, the CAR+T cell positivity rate in the control group, Flag / Trop2+CAR+EXO, remained extremely low at the same dose and time conditions, with the highest rate not exceeding 1.41%. At most time points, the results were close to background expression (<1%), further demonstrating that engineered EXOs that do not target CD8+T cells lack the ability to effectively target CD8+T cells and convert them into CAR-T cells.

[0116] 10 4 , 10 5 , 10 6 Quantitative analysis of the particles / cell group showed that the CD8+ / Trop2+CAR+EXO group had the highest efficiency in converting CD8+ T cells into CAR-T cells under high-dose conditions, with the longest expression duration and a clear "dose-dependent" and "time-dependent" variation pattern. The CAR+T cell positive rate maintained a peak value for 24-72 hours and then gradually declined, which was consistent with the kinetic characteristics of intracellular expression and degradation after mRNA delivery ( Figure 11 AC).

[0117] In summary, engineered EXO targeting Trop2 can efficiently deliver CAR mRNA to CD8+ T cells in a dose-dependent manner and induce them to transform into functional CAR-T cells in a short period of time. The expression of CAR-mRNA is time-dependent, and the expression level begins to decline after 5 days, significantly attenuates after 9 days, and is basically degraded after 12 days ( Figure 11 ). This indicates that this type of engineered EXO can achieve an efficient, controllable, and short-cycle CAR-T cell generation process.

[0118] Example 7: CAR-T cells targeting Trop2 kill pancreatic cancer in vitro

[0119] To verify the ability of Trop2-targeted engineered EXO to induce CD8+ T cells to generate CAR-T cells in vitro and its tumor killing activity, the present invention co-incubated two engineered EXOs, CD8+ / Trop2+CAR+EXO and Flag / Trop2+CAR+EXO, with activated human PBMCs for 24 hours. Flow cytometry results showed that ( Figure 12 A), the proportion of Strep2-positive cells in CD8+ T cells in the CD8+ / Trop2+CAR+EXO group was as high as 94.6%, while that in the Flag group was only 1.83%, indicating that Trop2-targeted engineered EXO can efficiently deliver CAR structures to CD8+ T cells and induce their expression, thereby achieving effective generation of CAR-T cells in vitro.

[0120] To further evaluate the anti-tumor function of the CAR-T cells induced in vitro, a pancreatic cancer cell line BXPC-3-Luci-GFP stably expressing GFP and Luciferase dual-labeled was constructed and used as a target cell. It was co-incubated with CAR-T cells at different effector cell to target cell ratios (E:T = 1:1, 5:1, 10:1) for 4 hours, and the changes in the luminescence intensity of Luciferase activity were detected using a multifunctional microplate reader. The results showed that under E:T=1:1 conditions, the anti-tumor activity of CAR-T cells in the CD8+ / Trop2+CAR+EXO group was 31.2%, while that in the Flag / Trop2+CAR+EXO group was 18.5%; when E:T=1:5 conditions, the anti-tumor activity of CAR-T cells in the CD8+ / Trop2+CAR+EXO group increased to 65.8%, significantly higher than the 42.3% in the Flag / Trop2+CAR+EXO group; under E:T=1:10 conditions, the anti-tumor activity of CAR-T cells in the CD8+ / Trop2+CAR+EXO group reached 91.7%, while that in the Flag / Trop2+CAR+EXO group was 64.5% ( Figure 12 B). CAR-T cells generated by CD8+ / Trop2+CAR+EXO have potent and specific cytotoxicity against BXPC-3 tumor cells.

[0121] In summary, Trop2-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-pancreatic cancer activity.

[0122] Implementation 8: Establishment of a Humanized Mouse Model

[0123] In order to evaluate the reconstruction of the human immune system in mice, the present invention uses flow cytometry to analyze the chimerism level of human immune cells and the composition of T cell subsets in the peripheral blood of humanized mice. After joint staining with antibodies such as BV421-CD45, APC-Cy7-CD3, FITC-CD8 and APC-CD4, the expression ratio of human immune cells was detected. The test results showed that the proportion of human CD45+ cells in the peripheral blood of mice accounted for 51.4% of the total lymphocytes, of which CD3+T cells accounted for 97.4% of the CD45+ population, indicating that human T lymphocytes were effectively reconstructed in mice. Further analysis of the CD3+T cell subsets showed that CD8+T cells accounted for 66.1%, CD4+T cells accounted for 20.9%, and CD4+CD8+ double positive cells accounted for 3.84% ( Figure 13 In summary, the experiment successfully established a humanized mouse model with a high level of human T cell reconstitution, providing a reliable model basis for subsequent in vivo functional evaluation experiments of CAR-T cells.

[0124] Implementation 9: Establishment of a pancreatic cancer animal model and the in vivo anti-tumor effect of exosomes

[0125] The mice with successful humanization modeling were subcutaneously injected with BXPC-3-GFP-Luc cells, with a uniform injection dose of 1×10 per mouse. 7 The inoculation site was located in the right subcutaneous tissue. BXPC-3-GFP-Luc cells stably expressed GFP and luciferase dual reporter genes, which could monitor tumor formation and development in real time through in vitro and in vivo fluorescence signals. One week after the injection of BXPC-3-GFP-Luc cells, mice were imaged in vivo. When the fluorescence signal of tumor cells in mice reached 10 6 -10 8 photons / s / cm 2 The tumor animal model was successfully established. The mice with successful modeling were randomly divided into groups on day 0 and entered the subsequent engineered EXO treatment experiment ( Figure 14 A).

[0126] To further evaluate the anti-tumor activity of Trop2-targeted engineered EXO against BXPC-3 cells in vivo, this study, based on the successful establishment of a humanized pancreatic cancer animal model, randomly divided mice into three groups and injected via the tail vein with CD8+Trop2+CAR+EXO, Flag / Trop2+CAR+EXO, and No-CAR-EXO. All mice underwent the first small animal live imaging before treatment (day 0) as a baseline signal, and were immediately given the first injection of engineered EXO, with a uniform injection dose of 10 per mouse. 12Imaging and repeated injections of engineered EXOs were performed 4, 7, 11, 14, and 18 days after treatment to dynamically monitor changes in tumor burden and quantitatively analyze changes in tumor burden, physiological status, and survival of mice.

[0127] IVIS results showed that from the 4th to the 7th day after injection, the tumor bioluminescence signal of the No-CAR-T group and the Flag / Trop2+CAR+EXO group gradually increased, while the tumor growth of the CD8+ / Trop2+CAR+EXO group was significantly inhibited, and the tumor signal was weak. On the 11th day, the tumor burden of the No-CAR-T group and the Flag / Trop2+CAR+EXO group further increased, while the tumor growth of the CD8+ / Trop2+CAR+EXO group continued to be inhibited, and the signal intensity was significantly lower than that of the other groups. By the 14th day, some mice in the No-CAR-T group died due to excessive tumor burden, while the CD8+ / Trop2+CAR+EXO group was still able to maintain a strong anti-tumor effect. By the 18th day, the tumor bioluminescence signal of mice in the No-CAR-T group and the Flag / Trop2+CAR+EXO group was significantly enhanced, while mice in the CD8+ / Trop2+CAR+EXO group still showed significant tumor growth inhibition ( Figure 14 B).

[0128] ROI quantitative results show ( Figure 14 C), on day 18, the tumor signal in the CD8+Trop2+CAR+EXO group was only 7.9 times the initial value, significantly lower than that in the Flag / Trop2+CAR+EXO group (12.7 times) and the No-CAR-EXO group (17.2 times). Analysis of mouse survival rates showed that no deaths occurred in the CD8+Trop2+CAR+EXO group during the entire experimental period, while one mouse died in the Flag / Trop2+CAR+EXO group on day 18. One mouse in the No-CAR-EXO group died on day 11, and only one mouse survived on day 18. ( Figure 14 E). The results of the weight changes of mice showed that there was no significant difference in weight among the three groups, indicating that the overall safety of the engineered EXO treatment was good ( Figure 14 D). Further tumor size measurement and tumor volume calculation showed that the growth trend of tumor volume in the CD8+ / Trop2+CAR+EXO group was significantly slower than that in the Flag / Trop2+CAR+EXO group and the No-CAR group, indicating that tumor growth was significantly inhibited ( Figure 14 F).

[0129] The above results indicate that engineered EXO targeting Trop2 has certain anti-tumor activity in vivo, can significantly inhibit tumor growth and has good overall safety in treatment.

[0130] Implementation 10: Targeting Trop2 with engineered EXO to convert CD8+ T cells into CAR-T cells in vivo

[0131] To further verify that the engineered EXO targeting Trop2 can exert tumor killing function by generating CAR-T cells in vivo, the present invention selected mice with successfully established humanized tumor models, collected peripheral blood by mandibular blood sampling, and labeled the mouse blood PBMC cells with Anti Human-CD8 flow cytometry antibodies and then co-incubated with two engineered EXOs, CD8+Trop2+CAR+EXO and Flag / Trop2+CAR+EXO, under in vitro conditions. After incubation for 24 hours, the expression of Strep2 tags in CD8+T cells was detected by flow cytometry. The results showed that the positive rate of CAR-T cells in CD8+T cells in the CD8+Trop2+CAR+EXO group was as high as 86.3%, which was significantly higher than the positive rate of CAR-T cells in CD8+T cells in the Flag control group (1.48%). Figure 15 ), indicating that the engineered EXO targeting Trop2 has good CD8+ T cell recognition and CAR mRNA delivery capabilities, and can efficiently induce the transformation of CD8+ T cells into CAR-T cells in vivo.

[0132] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0133] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0134] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing chimeric antigen receptor exosomes targeting CD8+ T cells, characterized in that: The following steps are involved: S1, sequentially connect NB60, G4S-Strep tagⅡ-CD8 hinge, and N6-SP to form the gene sequence fragment N6-SP-NB60-G4S-Strep tagⅡ-CD8 hinge; S2. Ligate the gene sequence fragment obtained in step S1 to the plasmid pcDNA 3.1N6(SP)-T20-VRC C / D box to obtain pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid; S3. Construct two plasmids, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae; The nucleotide sequence of pcDNA3.1-N6(SP)-Nb60-T20-C / D box is shown in SEQ ID NO: 1; the amino acid sequence of Anti-CD8-Lamp2b in BRD-PTK-CD8+-Lamp2b is shown in SEQ ID NO: 2; the nucleotide sequence of CD63-L7Ae in pcDNA3.1-CD63-L7Ae is shown in SEQ ID NO: 3; S4. The three plasmids pcDNA3.1-N6(SP)-Nb60-T20-C / D box, BRD-PTK-CD8+-Lamp2b, and pcDNA3.1-CD63-L7Ae were co-transfected into 293F cells, and exosomes were obtained after incubation and purification.

2. The method for preparing chimeric antigen receptor exosomes targeting CD8+ T cells according to claim 1, characterized in that: In step S4, The co-transfection specifically includes: mixing pcDNA3.1-N6(SP)-Nb60-T20-CDbox, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae with complete culture medium to obtain a plasmid mixture; simultaneously, adding PEI solution to another portion of F complete culture medium to obtain a PEI mixture, standing for 5 minutes, combining the plasmid mixture and the PEI mixture, standing for 30 minutes, and then adding to 293F cells for co-transfection; Among them, the weight ratio of pcDNA3.1-N6(SP)-Nb60-T20-CDbox, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae is 3:1:

1.

3. The method for preparing chimeric antigen receptor exosomes targeting CD8+ T cells according to claim 2, characterized in that: In step S4, The incubation specifically includes: adding OPM-CHO PFF06 medium and L-glutamine 24 hours after co-transfection, and collecting the cell supernatant 72 hours later; The purification specifically comprises the following steps: centrifuging the obtained cell supernatant at 4°C and 300×g for 5 minutes to remove live cells, then centrifuging at 3000×g for 30 minutes to remove dead cells and debris, and finally centrifuging at 10000×g for 60 minutes to remove large extracellular vesicles to obtain a pretreated supernatant; Add Universal Benzo nuclease and magnesium chloride to the pretreated supernatant. After overnight at 4°C, filter through a 0.22 μm filter cup. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000 × g for 90 min. Discard the supernatant, resuspend the pellet in PBS, and centrifuge again at 100,000 × g for 90 min. Finally, resuspend the pellet in PBS and store at -80°C.

4. A chimeric antigen receptor exosome targeting CD8+ T cells prepared according to any one of claims 1 to 3.

5. A method for constructing CAR-T cells, characterized in that: The method comprises co-incubating the exosomes according to claim 4 with PBMC cells to obtain CAR-T cells capable of targeting Trop2.

6. A CAR-T cell constructed using the construction method of claim 7.

7. Use of the chimeric antigen receptor exosomes targeting CD8+ T cells according to claim 4 or the CAR-T cells according to claim 6 in the preparation of a product for treating pancreatic cancer.

8. A drug for treating pancreatic cancer, characterized in that: Including the exosomes according to claim 4 or the CAR-T cells according to claim 6.

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