A chimeric antigen receptor exosome targeting CD8 T cells, CAR-T cells, their construction methods and applications

Abstract

The application discloses a kind of chimeric antigen receptor exosome targeted to CD8 T cell, CAR-T cell and its construction method and application, it is related to biomedical technical field, including the three plasmids of BRD-PTK-CD8+ -Lamp2b, pcDNA3.1-CD63-L7Ae, pcDNA3.1-N6 (SP) -Nb60-T20-C / D box are co-transfected into 293F cell, after incubation, the exosome capable of targeting CD8 T cell is obtained after purification.The exosome can deliver CAR-mRNA to T cell and convert CD8+ cell into CAR-T cell, without extracting the blood of infected person;CAR molecule for malignant cell in vivo can be prepared in vivo;Without chemotherapy radiotherapy, no side effect;It has universality.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a chimeric antigen receptor exosome targeting CD8 T cells, CAR-T cells, their construction methods and applications. Background Technology

[0002] Pancreatic cancer is a highly malignant solid tumor with an increasing incidence rate worldwide. Because early symptoms are often subtle, most patients are diagnosed at an advanced stage, leading to poor treatment outcomes and an extremely low five-year survival rate. The danger of pancreatic cancer lies not only in its high mortality rate but also in its severe impact on patients' quality of life. Rapid tumor growth and aggressive metastasis often result in severe pain, digestive disorders, and rapid weight loss.

[0003] Treatment for pancreatic cancer primarily relies on surgical resection, chemotherapy and radiotherapy, targeted therapy, and immunotherapy. Surgical resection is the only potentially curative treatment for pancreatic cancer, but it is only suitable for patients diagnosed at an early stage. Commonly used chemotherapy drugs include gemcitabine and fluorouracil, which can prolong survival to some extent, but have significant side effects and are prone to developing drug resistance. Radiotherapy is mainly used to locally control tumor growth, but its application is limited by its damage to surrounding normal tissues. However, most pancreatic cancer patients are diagnosed at an advanced stage and are not eligible for surgery. Chemotherapy is a common treatment, with drugs such as gemcitabine often used as adjuvant or neoadjuvant therapy to help slow tumor progression and reduce the risk of recurrence. Radiotherapy can be used for locally advanced patients, usually in combination with chemotherapy, to slow tumor growth and relieve symptoms.

[0004] Currently, CAR-T therapy is becoming a research hotspot. Although it has shown potential in some patients, its overall efficacy still needs further validation. Therefore, developing more effective treatments with fewer side effects is an important direction for current pancreatic cancer research.

[0005] CAR-T cell therapy has shown significant efficacy in treating leukemia and lymphoma, but solid tumors are currently not very sensitive to it. This is mainly because CAR-T cells are difficult to target and deliver to tumor tissues, and cannot proliferate effectively in the host body for a long period. Traditional CAR-T cell construction strategies require multiple steps, including patient blood collection, T cell extraction, viral transfection, in vitro CAR-T cell culture, and expansion, before they can be administered to patients. This can also cause serious side effects, such as cytokine release syndrome (CRS), and due to target selectivity issues, may damage normal tissues. Furthermore, each patient must undergo the same customized process, making it difficult to achieve universality. The entire process is cumbersome, lengthy, and expensive, allowing only a small number of patients to receive effective treatment, thus hindering the benefit of many cancer patients. To address these clinical problems, it is essential to develop a universal, in vivo, targeted transfection method for constructing CAR-T cells using T cells for the treatment of solid tumors. Summary of the Invention

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

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: A method for preparing chimeric antigen receptor exosomes targeting CD8+ T cells includes the following steps: S1, connect N6(SP), NB60, and G4S-Strep tagⅡ-CD8 hinge in sequence, and merge them into the gene sequence fragment N6(SP)-NB60-G4S-Strep tagⅡ-CD8 hinge; S2. The gene sequence fragment obtained in step S1 is ligated into the plasmid pcDNA 3.1 N6(SP)-T20-VRC C / Dbox to obtain the pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid. S3. Construct two plasmids, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae, respectively. The nucleotide sequence of the N6(SP)-Nb60-T20-C / D box in the pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid 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; and the nucleotide sequence of CD63-L7Ae in pcDNA3.1-CD63-L7Ae is shown in SEQ ID NO:3. S4. 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.

[0008] Furthermore, in step S4, The co-transfection specifically includes: mixing pcDNA3.1-N6(SP)-T20-Nb60-CDbox, BRD-PTK-CD8+-Lamp2b, and pcDNA3.1-CD63-L7Ae with F 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; after standing for 5 minutes, combining the plasmid mixture with the PEI mixture; after standing for 30 minutes, adding the mixture to 293F cells for co-transfection; The weight ratio of pcDNA3.1-N6(SP)-T20-Nb60-CDbox, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae is 3:1:1.

[0009] Furthermore, in step S4, The incubation process was as follows: OPM-CHO PFF06 medium and L-glutamine were added 24 hours after co-transfection, and the cell supernatant was collected 72 hours later. The purification process specifically involves 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, resulting in a pretreated supernatant. Add Universal Benzo nuclease and magnesium chloride to the pretreated supernatant, incubate overnight at 4°C, then filter through a 0.22µm filter. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000×g for 90 min. Discard the supernatant, resuspend the precipitate in PBS, and centrifuge again at 100,000×g for 90 min. Finally, resuspend the precipitate in PBS and store at -80°C.

[0010] The present invention also aims to provide chimeric antigen receptor exosomes targeting CD8+ T cells prepared by any of the above methods.

[0011] The present invention also aims to provide a method for constructing CAR-T cells, comprising co-incubating the above-described exosomes with PBMC cells to obtain CAR-T cells capable of targeting Trop2.

[0012] Specifically, after collecting peripheral blood samples, the plasma is removed, and the resulting blood cell pellet is diluted twice its original volume with 0.9% sodium chloride injection. Then, 30 mL of the blood cell suspension is slowly spread onto 15 mL of Ficoll-Paque lymphocyte separation medium, ensuring a clear interface. The mixture is centrifuged at 800 g at room temperature for 20 min. After centrifugation, the white membrane layer is collected using a pipette and transferred to a new centrifuge tube. 0.9% sodium chloride injection is added to dilute the cell suspension at a volume ratio of ≥2:1. The mixture is centrifuged at 500 g for 7 min to remove residual lymphocyte separation medium. The supernatant is discarded, and the cells are 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 is added, and the mixture is centrifuged at 500 g for 7 min. The cell pellet is resuspended again in 0.9% sodium chloride injection and thoroughly mixed. 100 μL of the cell suspension is used for cell counting and viability testing. The remaining suspension is centrifuged at 500 × g for 10 minutes. After min, PBMCs were finally obtained; anti-CD3 / CD28 activating magnetic beads (Miltenyi Biotec) and 1000 IU / mL recombinant human IL-2 were added to stimulate and activate them for 24 h; then they were co-incubated with exosomes to obtain CAR-T cells in vitro.

[0013] Alternatively, the obtained exosomes can be directly introduced into the body to obtain CAR-T cells.

[0014] Another objective of this invention is to provide CAR-T cells prepared by the above method.

[0015] The present invention also aims to provide the application of the chimeric antigen receptor exosomes targeting CD8+ T cells or the CAR-T cells described above in the preparation of products for treating pancreatic cancer.

[0016] Another objective of this invention is to provide a drug for treating pancreatic cancer, comprising the exosomes described above or the CAR-T cells described above.

[0017] The beneficial effects of this invention include at least the following: In this invention, the NB60 nanobody consists of only one variable region (VHH), while traditional antibodies consist of two pairs of heavy and light chains. The VHH is composed of a single amino acid sequence, exhibiting high stability and antigen-binding capacity. Trop2 is used as the target of the CAR-T model to construct the CAR frame plasmid. T cell-targeted modified exosomes are used as vectors to encapsulate the CAR frame plasmid, thereby improving the specificity of the vector's binding to T cells. In vivo transfection of T cells converts them into CAR-T cells while reducing off-target effects.

[0018] This invention achieves enhanced helper function of CAR-T cells and their in vivo preparation by optimizing the design of Trop2-specific nanobodies and combining them with an LNP-mRNA delivery system. It provides a Trop2-targeted CAR-T cell that can be prepared in vivo and specifically binds to the Trop2 antigen. In cell killing assays of pancreatic cancer cell lines, the in vivo construction of CAR-T cells modified with the Trop2 target can kill malignant tumor cells, achieving an effect similar to the in vivo formation of CAR-T cells.

[0019] In other words, the chimeric antigen receptor exosomes targeting CD8+ T cells constructed in this invention can deliver CAR-mRNA to T cells and convert CD8+ cells into CAR-T cells without drawing blood from infected individuals; CAR molecules targeting malignant cells in vivo can be prepared in vivo; chemotherapy and radiotherapy are not required, and there are no side effects; it is versatile. Attached Figure Description

[0020] Figure 1 This is a schematic diagram showing the connection of the hinge segments of N6-SP, NB60, and G4S-Strep tagⅡ-CD8.

[0021] Figure 2 This is a schematic diagram of the pcDNA 3.1 N6(SP)-T20-VRC C / D box plasmid.

[0022] Figure 3 This is a schematic diagram of the pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid.

[0023] Figure 4 The diagram shows the construction and lentiviral packaging of PLV-Luc-GFP. (A) Schematic diagram of PLV-Luc-GFP plasmid structure; (B) lentiviral titer rapid detection card to detect lentiviral vector packaging efficiency; (C) lentiviral transfection of 293T cells, fluorescence intensity detected by fluorescence microscopy 48 h later.

[0024] Figure 5The diagram shows the detection of GFP and Luc in cells double-labeled with GFP and Luc. (A) Flow cytometry was used to detect the expression of GFP on the surface of BXPC-3 double-labeled cells; (B) Fluorescence microscopy was used to observe the fluorescence intensity of the expressed green fluorescent protein in BXPC-3 double-labeled cells; (C) In vivo imaging was used to detect the fluorescence intensity of the luciferase gene expressed in BXPC-3 double-labeled cells.

[0025] Figure 6 This is a schematic diagram of engineered EXO mRNA targeted delivery.

[0026] Figure 7 The images show the isolation and characterization of CAR-engineered EXO derived from 293F cells. (A) Schematic diagram of the workflow for engineered EXO isolation experiment; (B) TEM image of engineered EXO morphology; (C) NTA analysis of engineered EXO particle size distribution; (D) Analysis of surface-specific markers of engineered EXO.

[0027] Figure 8 The image shows the representation results of CD63, CD81, and CD9 on the engineered EXO surface.

[0028] Figure 9 This figure shows the expression of the Strep2 tag and VHH nanobody on the surface of the targeted engineered EXO.

[0029] Figure 10 This is a graph showing the positive expression of CAR-T cells in CD8+ T cells at different doses and time points after incubation with Trop2-engineered EXO.

[0030] Figure 11 Quantitative statistical curves of the proportion of Trop2-targeted CAR+ T cells under three dosage conditions. (A) 10 4 Analysis of CAR-T cell positivity rate under Particles / cell dosage conditions; (B) 10 5 Particles / cell dose-dependent CAR-T cell positivity analysis; (C) 10 6 Analysis of CAR-T cell positivity rate under Particles / cell dosage conditions.

[0031] Figure 12 Cytotoxicity analysis of CD8+ T cells converted into CAR-T cells using Trop2-engineered EXO. (A) Flow cytometry detection of the positive rate of CD8+ T cells converted into CAR-T cells by two engineered EXOs; (B) Cytotoxicity analysis of CAR-T cells generated from CD8+ T cells using Trop2-engineered EXO against pancreatic cancer cells (n=3, *p<0.05, **p<0.01, ***p<0.001).

[0032] Figure 13 A graph showing the proportion of human immune cells in humanized mice as detected by flow cytometry.

[0033] Figure 14 Figure 1 shows the establishment of an animal model of pancreatic cancer and the in vivo antitumor activity evaluation of Trop2-targeted engineered EXO. (A) Mouse experimental timeline; (B) Fluorescence analysis of in vivo imaging of mice before and after treatment with the two engineered EXOs; (C) Comprehensive analysis of luciferase expression changes before and after treatment in each group of mice; (D) Body weight changes in each group of mice; (E) Survival curves in each group of mice; (F) Growth curves of subcutaneous xenograft volume in each group of mice (n>3, *p<0.05, **p<0.01).

[0034] Figure 15 The graph shows the positive rate of CAR-T cells after co-incubation of PBMC cells derived in vivo with Trop2-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-T cells (n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0037] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Construction of pcDNA3.1-N6(SP)-Nb60-T20-C / D box plasmid 1. The sequence Nb60 was synthesized by Sangon Biotech Co., Ltd., and the synthesized sequence was cloned into the pUC57 vector. The sequence of Nb60 is shown in SEQ ID NO:4.

[0038] 2. Using a human cDNA library as a template, primers were designed for PCR amplification of fragments N6-SP, NB60, and G4S-StreptagⅡ-CD8 hinge, respectively. Overlap PCR was used to sequentially amplify and ligate NB60, G4S-StreptagⅡ-CD8 hinge, and N6-SP into fragments containing the restriction enzyme sites NheⅠ and SgrAⅠ; the structural diagram is shown below. Figure 1 As shown.

[0039] Table 1. Amplification Primer Sequence List

[0040] 3. The plasmid pcDNA 3.1 N6(SP)-T20-VRC C / D box was double-digested with NheⅠ and SgrAⅠ restriction endonucleases. The products were subjected to 0.8% agarose gel electrophoresis, and the gel was excised and collected in Eppendorf tubes. The corresponding fragments were recovered using agarose gel recovery kit from Kangwei Century Company, and the purity and concentration of the products were determined. Figure 2 The sequence of plasmid pcDNA 3.1 N6(SP)-T20-VRC C / D box is shown in SEQ ID NO:11.

[0041] 4. The recovered fragment of the above vector and the N6-SP-NB60-G4S-Strep tagⅡ-CD8 hinge fragment were added to an Eppendorf tube at a 1:2 molar ratio. Exnase II ligase (Vazyme) and homologous recombinase 5×CEⅡ buffer were added, and the reaction was carried out at 37°C for 0.5 hours. 10 μL of the ligation solution was added to 100 μL of DH5α competent cells and incubated on ice for 30 min, followed by heat shock at 42°C for 90 s. After that, 500 μL of SOC medium was added, and the cells were cultured at 37°C and 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 plated on LB agar plates containing kanamycin and cultured at 37°C for 12 hours. Single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium and cultured at 37°C and 220 rpm for 12 hours. The binding fluid consists of the following components: linearized carrier: 2ul, insert fragment (N6-SP-NB60-G4S-Strep tagⅡ-CD8 hinge): 1ul, 5×CE Multis buffer: 4ul, C113 Exnase: 2ul, ddH2O: 11ul, and total volume: 20ul.

[0042] 5. Using the Kangwei Century Small Prep Kit, plasmid pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for first-generation sequencing verification. After confirmation, DH5α strain containing plasmid pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox was preserved. A complete schematic diagram of pcDNA3.1-N6(SP)-Nb60-T20-C / Dbox is shown below. Figure 3 As shown.

[0043] Example 2: Packaging of PLV-Luc-GFP Lentiviral Virus This invention provides an embodiment of a PLV-Luc-GFP lentiviral expression vector, the structure of which is as follows: Figure 4 As shown in Figure A, the corresponding sequence is shown in SEQ ID NO:12. This vector uses the EF-1α promoter to drive the co-expression of Luciferase and GFP (green fluorescent protein), and links the puromycin resistance gene through the (Internal Ribosoe Entry Site, IRES) sequence to ensure stable co-expression of fluorescent labeling and resistance selection. Lentiviral packaging and titer detection results showed that after co-transfecting 293T cells with the PLV-Luc-GFP plasmid and helper packaging plasmid for 48 hours, the culture supernatant was collected, and the viral particles were concentrated. A rapid lentivirus titer detection card was used... Figure 4 B) Verifying viral packaging efficiency: Clearly visible bands indicate successful lentivirus packaging. Lentiviral infection efficiency was further verified by detecting GFP expression using fluorescence microscopy. Figure 4 C). Meanwhile, under a bright-field microscope, the cells appeared to be in good condition, with no obvious apoptosis or toxic effects.

[0044] Example 3: Construction of GFP and Luc dual-labeled target cells 1. Infects BXPC-3 cells (human in situ pancreatic adenocarcinoma cells) 1) Take BXPC-3 cells in good growth condition and adjust the density to 1×10⁻⁶. 6 cells / mL, and at 1×10 6 Cells / wells were seeded into 6-well plates.

[0045] 2) Add 100 μL of concentrated PLV-Luc-GFP lentivirus solution to each well, and add Polybrene to a final concentration of 8 μg / mL to enhance viral infection efficiency. After incubating the cells at 37℃ and 5% CO2 for 4-6 h, discard the viral culture medium and add preheated RPMI-1640 fresh complete culture medium for further culture.

[0046] 2. BXPC-3 dual-label target cell resistance screening 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.

[0047] 2) When the cell density reaches 80-90%, 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 about 2 weeks. After the uninfected cells have completely undergone apoptosis, reduce the puromycin concentration to 1 μg / mL to maintain stable screening.

[0048] 3. Phenotypic and functional identification of BXPC-3 and U251 dual-labeled target cells 1) Fluorescence microscopy was used to observe and record GFP expression in cells, providing a direct assessment of the expression level and fluorescence signal intensity of the exogenous gene. Selected 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 percentage of GFP-positive cells, analyze the purity of the cell population, and assess transfection efficiency. Results showed that the GFP positivity rate of BXPC-3-Luc-GFP cells was 94.2%, and that of U251-Luc-GFP cells was 99.6%, while untransfected control cells (BXPC-3-Control) showed no GFP signal. These results indicate that the lentiviral vector successfully mediated efficient transfection of the GFP gene in BXPC-3 cells, and the selected cell population exhibited high purity. Figure 5 A).

[0049] Further observation of GFP expression using fluorescence microscopy 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 the GFP expression level is high throughout the entire cell population, meeting the criteria for constructing a stable cell line. Figure 5 B).

[0050] 2) The In Vivo Imaging System (IVIS) was used to assess the luciferase activity of cells.

[0051] Collect the cells to be tested and adjust the cell density to 1×10⁻⁶. 6Cells were evenly seeded in 6-well plates at a concentration of 150 μg / mL. After 24 h, the cell supernatant was aspirated, and the cells were washed twice with PBS. Then, 1 mL of bioluminescent substrate solution (final concentration 150 μg / mL) was added to each well, and the plates were incubated at 37 °C for 5 min. Immediately after incubation, the 6-well plates were placed in an IVIS (in vivo imaging system), and Luciferase emission at 560 nm was selected for exposure imaging. Background correction and signal normalization analysis were performed using 1.7.05_amiview.sav software to quantitatively assess the luciferase expression of the target cells.

[0052] The results showed that BXPC-3-Luc-GFP cells all exhibited significant luciferase activity and high luminescence intensity, while no obvious bioluminescent signal was detected in the control group (BXPC-3-Control). This further demonstrates the stable expression of the Luciferase gene in the target cells and its real-time detection via bioluminescence imaging. Figure 5 C).

[0053] Example 4: Construction of an engineered EXO (exosome) targeted delivery system 1. Construction of CAR mRNA expression plasmid and exosome packaging plasmid The nucleotide sequence of the CAR structure + C / D box is shown in SEQ ID NO:1: 2. Construction of CD8-targeting nanobody expression plasmid The amino acid sequence of Anti-CD8-Lamp2b is shown in SEQ ID NO:2; 3. Construction of CD63 exosome packaging plasmid The nucleotide sequence of CD63-L7Ae is shown in SEQ ID NO:3; A specific packaging system using the L7Ae-C / D box was established. This system involved adding a C / D box to the 3'UTR region of CAR-mRNA and fusing the EXO-specific tetraspanic membrane protein CD63 with L7Ae to form a fusion protein. By utilizing the specific binding of the RNA recruitment element C / D box to the RNA-binding protein L7Ae, CAR-mRNA was enriched within the EXO, improving its loading efficiency. Furthermore, a nanobody specifically targeting human CD8+ was fused to the N-terminus of Lamp2b, displaying it on the membrane surface of an engineered EXO. This nanobody then mediated the recognition of specific receptor cells, enabling the mRNA contents to be translated and expressed as the target protein in the target cells. Figure 6An engineered EXO targeted delivery system was constructed by co-transfection with three plasmids: pcDNA3.1-N6(SP)-Nb60-T20-C / D box, BRD-PTK-CD8+(Flag)-Lamp2b, and pcDNA3.1-CD63-L7Ae. The BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae plasmids were constructed in our laboratory.

[0054] Example 5: Preparation and characterization of exosomes 1. 293F cell culture and preparation of exosomes 293F cells were cultured in a 1:1 mixture of antibiotic-free OPM-293 CD05 medium (purchased from Shanghai OPM Biotechnology Co., Ltd., catalog number: 81075-001) and SMM293-TII medium (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: RZ18OC2401-A) under the following conditions: 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, 20 μg of pcDNA3.1-CD63-L7Ae, and 4 mL of F complete medium (293 CD05 medium and SMM293-TII medium mixed in a 1:1 ratio). At the same time, add 300 μL of PEI (polyethyleneimine) solution (1 mg / mL) to another 4 mL of F complete medium. After standing for 5 minutes, combine the plasmid mixture with the PEI mixture, and after standing for 30 minutes, add it to 293F cells for co-transfection. 24 hours later, OPM-CHO PFF06 medium (1 / 20) and L-glutamine (1 / 50) were added. After 72 hours, the cell supernatant was collected. OPM-CHO PFF06 medium was purchased from Shanghai Aopumai Biotechnology Co., Ltd., catalog number: 1265F05-001. Live cells were removed by centrifugation at 300×g for 5 min at 4℃, followed by centrifugation at 3000×g for 30 min to remove dead cells and debris. Finally, large extracellular vesicles were removed by centrifugation at 10000×g for 60 min. After pretreatment, Universal Benzo nuclease (2U) and magnesium chloride (2μmol / L) were added to the supernatant. The mixture was incubated overnight at 4°C, then filtered through a 0.22µm filter. 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 precipitate was resuspended in 20mL PBS. The precipitate was centrifuged again at 100,000×g for 90 min. Finally, the precipitate was resuspended in 200μL PBS and stored at -80°C. Figure 7 A).

[0055] 2. Exosome Characterization (1) Engineering EXO transmission electron microscopy (TEM) characterization TEM is used to observe the morphology, size, and integrity of engineered EXO to confirm the nanostructural characteristics of the samples. In this experiment, negative staining was used to analyze the isolated and purified engineered EXO using transmission electron microscopy. The specific method is as follows: 1) Sample preparation: Take an appropriate amount of engineered EXO suspension (concentration approximately 10). 9 The particle count (in mL) was appropriately diluted with PBS and 10 µL was added to a copper grid. The mixture was then incubated at room temperature for 10 min to allow the engineered EXO to be fully adsorbed.

[0056] 2) Negative staining treatment: 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 staining solution again with filter paper to avoid the sample drying and cracking.

[0057] 3) Drying and observation: After the copper mesh is dried at room temperature, it is imaged using TEM at a voltage of 80-120kV to observe the morphology, size and integrity of the engineered EXO and record the images.

[0058] The results are as follows Figure 7 As shown in Figure B, TEM observation reveals that the engineered EXO exhibits a typical "cup-shaped" vesicle structure of approximately 100 nm.

[0059] (2) Nanoparticle Tracking Analysis (NTA) NTA (Nano-Taking Analysis) technology was used to analyze the particle size distribution and concentration of engineered EXO. Based on the principle of dynamic light scattering, it calculates particle size and determines particle concentration by tracking the Brownian motion of particles. The experiment was conducted using a Zeta View nanoparticle tracking analyzer, and the specific method is as follows: 1) Sample preparation: Take an appropriate amount of engineered EXO suspension (approximately 10... 9 particles / mL), diluted appropriately with sterile PBS to the instrument's detection range (usually 10). 7 (particles / mL), mix well and test.

[0060] 2) Instrument calibration: Before the experiment, the instrument is calibrated using standard nanoparticles to ensure the accuracy of the light source, focusing, and particle concentration measurement.

[0061] 3) Detection process: The sample is injected into the detection cell, and laser scattering and Brownian motion analysis are used. While the camera captures the motion trajectory of the engineered EXO, particle size distribution data from multiple fields of view are recorded. The detection parameters are typically set as follows: temperature 25℃, detection time 60s.

[0062] NTA results showed that the main peak particle size of the collected engineered EXO particles ranged from 120 to 150 nm, and exhibited a single peak, conforming to a normal distribution. Figure 7 C), which is basically consistent with the particle size of engineered EXO shown in the TEM image.

[0063] (3) Western Blot (WB) 1) 72 h after transfection, take 1 ml of cell suspension, centrifuge to discard the culture medium, gently wash twice with pre-cooled PBS, collect the cells, add 200 µL of RIPA lysis buffer (containing 1% PMSF protease inhibitor), and lyse on ice for 30 min. Subsequently, centrifuge at 10000×g for 10 min at 4 °C and collect the supernatant; 2) The protein content in the lysed cell suspension was determined using the Beyotime BCA protein quantification kit. The lysed cell suspension was mixed with 5× Loading bµffer, heated at 100℃ for 10 min, and then stored at -20℃ for later use. 3) Perform SDS-PAGE electrophoresis with a sample loading of 20µg protein per well. Set the electrophoresis conditions to 80V constant voltage for 20min until bromophenol blue enters the separating gel; then adjust to 120V constant voltage for 60-90min until bromophenol blue migrates to the bottom of the gel. 4) The protein was transferred to the NC membrane (nitrocellulose membrane) at a rate of 1 kD protein / min using wet transfer. The NC membrane was then placed in a protein-free rapid blocking solution and blocked at room temperature for 20 min. 5) Dilute the primary antibody with TBST buffer according to the corresponding ratio in the antibody instructions and incubate overnight at 4°C. Then, wash the NC membrane three times with TBST buffer for 10 minutes each time to remove unbound primary antibody. 6) Perform secondary antibody incubation: Immerse the NC membrane in a 1:5000 diluted secondary antibody solution and incubate with shaking in the dark for 1 h. Then, wash three times with TBST buffer for 10 min each time to remove unbound secondary antibody. 7) Finally, ECL luminescent solutions A and B were mixed in a 1:1 ratio for development, and the expression of the target protein was detected using a gel imaging analysis system.

[0064] The results are as follows Figure 7 As shown in Figure D, the WB results indicate that the engineered EXO surface contains typical EXO markers such as CD63, CD81, and CD9.

[0065] (4) Nano-flow cytometry (nFCM) The expression of CD63, CD81, and CD9 on the surface of targeted engineered EXO and unmodified blank EXO derived from 293F cells was detected using nanoflow cytometry. 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 EXO surface could stably express typical EXO surface proteins, and the expression of CD63 in the targeted delivery EXO was significantly upregulated, much higher than that in the unmodified EXO derived from 293F. These results indicate that the targeted engineered EXO successfully enriched the overexpressed CD63 during the packaging process.

[0066] (5) Validation of the expression of specific proteins on the surface of engineered EXO To assess whether the engineered EXO successfully expressed the specific tag Strep2 and nanobody sequence from the CAR+ plasmid, this invention used nFCM (nanoflow cytometry) to directly stain unmodified EXO and targeted engineered EXO derived from 293F cells. EXO samples were stained with FITC-labeled Strep2 antibody and PC5-labeled VHH antibody, respectively, to identify whether the engineered EXO carried the Strep tag (representing CAR structure expression) and whether the nanobody structure represented the Lamp2b N-terminal fusion-targeting nanobody sequence for CD8+ T cells.

[0067] Flow cytometry results showed that the positive rate of Strep2 expression in the CD8+ / Nb60+CAR+EXO engineered EXO targeting EXO was 13.9%, significantly higher than the 0.59% expression rate of 293F-EXO, indicating 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 in 293F-EXO was 0.16%, indicating that the scFv / VHH, Strep2 structure, and Lamp2b N-terminus-fused Targeting CD8+ T cell nanobody in the CAR+ plasmid can be stably expressed and localized on the surface of the engineered EXO. Figure 9 ).

[0068] The above results collectively demonstrate that 293F cells can effectively secrete engineered EXO with dual specific functions by transfecting and constructing a Targeting CD8+ T cell nanobody carrying a CAR structure fused to the N-terminus of Lamp2b.

[0069] Example 6: Preparation of CAR+ T cells by exosome incubation and detection of CD8+ T cell transduction efficiency Peripheral blood samples were collected, plasma was removed, and the resulting blood cell pellet was diluted twice 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 (peripheral blood mononuclear cells). Anti-CD3 / CD28 activating magnetic beads (Miltenyi Biotec) and 1000 IU / mL recombinant human IL-2 were added for stimulation and activation for 24 h. 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 12h, 24h, 48h, 72h, Day 5, Day 9, and Day 12. Strep-II positive cells were identified as CAR-T cells.

[0070] The results show that ( Figure 10 In the CD8+ / Trop2+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...). 6The positivity rate of CAR+T cells (particles / cells) reached 97.6% at 12h, peaked at 98.2% at 24h, and remained high at 98.9% and 99.3% at 48h and 72h, respectively. The positivity rate of CAR+T cells was 93.1% on day 5, subsequently gradually decreasing to 10.9% on day 9. A positivity rate of 2.81% was still detectable on day 12, indicating a significant time-decay trend in CAR mRNA expression within cells. In contrast, at 10... 5 Under particle / cell conditions, the CAR+ T cell expression positivity rate can reach 65.9% at 24h, but it is lower than that under 10 particle / cell conditions. 6 The decay rate is faster under particle / cell conditions; while 10 4 The CAR+T cell positivity rate in the particles / cell group was below 2% at all time points, indicating that the low-dose engineered EXO had a low efficiency 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 under the same dose and time conditions, with a maximum of no more than 1.41%, and the results at most time points were close to background expression (<1%), further demonstrating that the engineered EXO that does not target CD8+T cells lacks the ability to effectively target CD8+T cells and convert them into CAR-T cells.

[0071] For 10 respectively 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 exhibited obvious dose-dependent and time-dependent changes. The CAR+T cell positivity rate remained at its peak for 24-72 hours and then gradually declined, consistent with the kinetic characteristics of mRNA expression and degradation in cells after delivery. Figure 11 AC).

[0072] 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. Furthermore, CAR-mRNA expression is time-dependent, with expression levels beginning to decline after 5 days, significantly decreasing after 9 days, and essentially degrading after 12 days. Figure 11 This indicates that this type of engineered EXO can achieve a highly efficient, controllable, and short-cycle CAR-T cell generation process.

[0073] Example 7: In vitro killing of pancreatic cancer by CAR-T cells targeting Trop2. 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, this 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 ( 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 the CAR structure to CD8+ T cells and induce their expression, thus achieving effective generation of CAR-T cells in vitro.

[0074] 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 labeling, was constructed. This line was used as the target cell and 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. The changes in luminescence intensity of Luciferase activity were detected using a multifunctional microplate reader. The results showed that under the E:T=1:1 condition, the antitumor 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 the E:T=1:5 condition, the antitumor 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 the E:T=1:10 condition, the antitumor 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). This indicates that CAR-T cells generated by CD8+ / Trop2+CAR+EXO have potent and specific cytotoxicity against BXPC-3 tumor cells.

[0075] In summary, Trop2-targeted engineered EXO can not only efficiently transform CD8+ T cells into CAR-T cells in vitro, but the generated CAR-T cells also have strong specific anti-pancreatic cancer activity.

[0076] 8. Establishment of a humanized mouse model To assess the reconstitution of the human immune system in mice, this invention employed flow cytometry to analyze the chimerism level and T cell subset composition of human immune cells in the peripheral blood of humanized mice. After combined 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 results showed that human CD45+ cells accounted for 51.4% of total lymphocytes in mouse peripheral blood, with CD3+ T cells accounting for 97.4% of the CD45+ population, indicating effective reconstitution of human T lymphocytes in mice. Further analysis of CD3+ T cell subsets revealed that CD8+ T cells accounted for 66.1%, CD4+ T cells for 20.9%, and CD4+CD8+ double-positive cells for 3.84%. Figure 13 In summary, the experiment successfully constructed a humanized mouse model with a high level of human T cell regeneration, providing a reliable model basis for subsequent in vivo functional evaluation experiments of CAR-T cells.

[0077] Implementation 9: Establishment of an animal model of pancreatic cancer and the in vivo antitumor effect of exosomes. Mice that successfully underwent humanization modeling were subcutaneously injected with BXPC-3-GFP-Luc cells at a uniform dose of 1×10⁻⁶ per mouse. 7 One cell line was injected into the right subcutaneous tissue. BXPC-3-GFP-Luc cells stably express GFP and luciferase dual reporter genes, allowing for real-time monitoring of tumor formation and development via in vitro and in vivo fluorescence signals. In vivo imaging of mice was performed one week after BXPC-3-GFP-Luc cell injection. When the fluorescence signal related to tumor cells in the mice reached 10... 6 -10 8 photons / s / cm 2 The successful establishment of the tumor animal model was demonstrated. The mice that successfully developed the model were randomly assigned to groups on day 0 for subsequent engineered EXO treatment experiments. Figure 14 A).

[0078] To further evaluate the antitumor 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, which were injected via tail vein with CD8+Trop2+CAR+EXO, Flag / Trop2+CAR+EXO, and No-CAR-EXO, respectively. All mice underwent their first small animal in vivo imaging before treatment (day 0) as baseline signal, and were immediately given their first engineered EXO injection. The injection dose was uniformly 10 mg / m³ per mouse. 12Exosomes were collected. Imaging and engineered EXO were repeated at 4, 7, 11, 14, and 18 days after treatment to dynamically monitor changes in tumor burden. Quantitative analyses were performed on changes in tumor burden, physiological status, and survival in mice.

[0079] IVIS results showed that from day 4 to day 7 post-injection, tumor bioluminescence signals gradually increased in the No-CAR-T group and the Flag / Trop2+CAR+EXO group, while tumor growth was significantly inhibited and the tumor signal was weak in the CD8+ / Trop2+CAR+EXO group. On day 11, tumor burden further increased in the No-CAR-T group and the Flag / Trop2+CAR+EXO group, while tumor growth in the CD8+ / Trop2+CAR+EXO group remained inhibited, and the signal intensity was significantly lower than other groups. By day 14, some mice in the No-CAR-T group died due to excessive tumor burden, while the CD8+ / Trop2+CAR+EXO group maintained a strong anti-tumor effect. By day 18, tumor bioluminescence signals were significantly enhanced in the No-CAR-T group and the Flag / Trop2+CAR+EXO group, while the CD8+ / Trop2+CAR+EXO group still showed significant tumor growth inhibition. Figure 14 B).

[0080] 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). Survival analysis of mice showed that no mice died in the CD8+Trop2+CAR+EXO group throughout the experimental period, while one mouse died in the Flag / Trop2+CAR+EXO group on day 18. In the No-CAR-EXO group, one mouse died on day 11, and only one mouse survived on day 18. Figure 14 E). The results of mouse body weight changes showed no significant difference in body weight among the three groups, indicating that the overall safety of this engineered EXO treatment was good. Figure 14 D). Further measurements of tumor size and calculation of tumor volume showed that the tumor volume growth over time 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).

[0081] The above results indicate that engineered EXO targeting Trop2 possesses certain anti-tumor activity in vivo, can significantly inhibit tumor growth, and has good overall safety in treatment.

[0082] Implementation 10: Targeting Trop2-engineered EXO to convert CD8+ T cells into CAR-T cells in vivo. To further verify that the Trop2-targeted engineered EXO can exert tumor-killing function by generating CAR-T cells in vivo, this invention selected mice with successfully established humanized tumor models. Peripheral blood was collected through jaw sampling. Mouse blood PBMCs were first labeled with an Anti Human-CD8 antibody by flow cytometry and then co-incubated with two engineered EXOs, CD8+Trop2+CAR+EXO and Flag / Trop2+CAR+EXO, under in vitro conditions. After 24 hours of incubation, the expression of the Strep2 tag 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%, significantly higher than the 1.48% positive rate of CAR-T cells in CD8+ T cells in the Flag control group. Figure 15 This indicates 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.

[0083] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0084] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of chimeric antigen receptor exosomes targeting CD8+ T cells in the preparation of products for treating pancreatic cancer, characterized in that, The preparation method of chimeric antigen receptor exosomes targeting CD8+ T cells includes the following steps: S1. Connect N6-SP, NB60, and G4S-Strep tagⅡ-CD8 hinge sequentially to merge them into the gene sequence fragment N6-SP-NB60-G4S-Strep tagⅡ-CD8 hinge. S2. The gene sequence fragment obtained in step S1 is ligated into the plasmid pcDNA3.1N6-SP-T20-VRC C / Dbox to obtain the pcDNA3.1-N6-SP-Nb60-T20-C / D box plasmid. S3. Construct two plasmids, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae, respectively. The nucleotide sequence of the N6-SP-Nb60-T20-C / D box in the pcDNA3.1-N6-SP-Nb60-T20-C / D box plasmid 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; and the nucleotide sequence of the pcDNA3.1N6-SP-T20-VRC C / D box is shown in SEQ ID NO:

11. S4. 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 application 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 F complete medium to obtain a plasmid mixture; simultaneously, adding PEI solution to another portion of F complete medium to obtain a PEI mixture; after standing for 5 minutes, combining the plasmid mixture with the PEI mixture; after standing for 30 minutes, adding the mixture to 293F cells for co-transfection; The weight ratio of pcDNA3.1-N6-SP-Nb60-T20-CDbox, BRD-PTK-CD8+-Lamp2b and pcDNA3.1-CD63-L7Ae is 3:1:

1. The F complete culture medium is a mixture of OPM-293CD05 culture medium and SMM293-TII culture medium in a 1:1 ratio.

3. The application according to claim 2, characterized in that, In step S4, The incubation process was as follows: OPM-CHO PFF06 medium and L-glutamine were added 24 hours after co-transfection, and the cell supernatant was collected 72 hours later. The purification process specifically involves 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, resulting in a pretreated supernatant. Add Universal Benzo nuclease and magnesium chloride to the pretreated supernatant, incubate overnight at 4°C, then filter through a 0.22µm filter. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000×g for 90 min. Discard the supernatant, resuspend the precipitate in PBS, and centrifuge again at 100,000×g for 90 min. Finally, resuspend the precipitate in PBS and store at -80°C.

4. Chimeric antigen receptor exosomes targeting CD8+ T cells prepared by any one of claims 1 to 3.

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

6. A CAR-T cell constructed using the method of claim 5.

7. The use of the CAR-T cells according to claim 6 in the preparation of products for treating pancreatic cancer.

8. A drug for treating pancreatic cancer, characterized in that, Includes the exosomes of claim 4 or the CAR-T cells of claim 6.

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