Preparation method and application of tumor exosome loaded gamma delta T cell
Through the preparation method of tumor exosome-loaded γδT cells, the problem of insufficient number of γδT cells and dysfunction in the tumor microenvironment was solved, and the efficient anti-tumor effect and tumor-specific response of γδT cells were achieved, which significantly improved the treatment effect of lung cancer patients.
Patent Information
- Application Number
- CN202411736172.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-08-29
AI Technical Summary
It is difficult to obtain a sufficient number of γδ T cells and improve their tumor immunotherapy effects in the prior art, and γδ T cells are dysfunctional in the tumor microenvironment and lack tumor-specific responses, which affect their anti-tumor ability.
The preparation method of tumor exosome-loaded γδT cells was adopted, and the treated tumor cell exosomes were co-cultured, and the anti-tumor activity of γδT cells was activated and enhanced by using the antigen loading capacity of the exosomes. The dual-load technology was used to ensure that γδT cells fully loaded exosomes.
The anti-tumor effect of γδT cells was significantly improved and tumor-specific response was enhanced. The CD3+ and CD4+ positive cells were significantly increased, the CD8+ positive cells were significantly reduced, the CD4+/CD8+ ratio was upregulated, the number of NK cells increased, the tumor cells were effectively attacked, and the lesions in lung cancer patients were reduced.
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Figure CN120555348A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of anti-tumor drugs, and in particular to a method for preparing tumor exosomes loaded with γδT cells and applications thereof. Background Art
[0002] T cells can be divided into αβT cells and γδT cells based on the type of surface antigen receptor (AAR). In cancer immunotherapy, αβT cells primarily participate in adaptive immunity. However, their dependence on the major histocompatibility complex (MHC), their ability to recognize only mutant peptides, and their low tropism for tumor sites, particularly in solid tumors, have hindered their clinical application. γδT cells serve as a bridge between the innate and adaptive immune systems in cancer immunotherapy and serve as a first line of defense for tumors, possessing antitumor effects absent in normal cells and potentially useful for assessing patient prognosis. Furthermore, their genetic makeup allows for easy therapeutic intervention, allowing them to recognize a variety of antigens, including lipids, phosphorylated antigens, and peptides, in both MHC-dependent and -independent manners. This suggests that γδT cells can also exert antitumor effects against tumors with low mutational burden and MHC downregulation.
[0003] Studies have reported that using non-peptide phosphoantigens or phosphoantigen-induced aminobisphosphonates can activate γδT cells in vitro. These cells have been shown to inhibit the growth of various tumors in vitro and in vivo, and Phase I clinical studies have been completed in advanced lung cancer, renal cancer, and malignant melanoma. Currently, the anti-lung cancer effect of γδT cells has been confirmed in numerous studies using cell-based and animal models. For example, Liu et al. reported that continuous low-dose γδT cell intervention had an anti-lung cancer cell killing effect both in vitro and in vivo. Xie Ling et al. established a technique for culturing γδT cells in vitro. At an effector-target ratio (E / T ratio) of 50 / 1, the anti-SK-MES-1, Ho8910, A549, and K562 cells cultured in vitro achieved an anti-tumor efficiency exceeding 65%. In vivo experiments demonstrated that tumor volume in γδT-treated mice was significantly lower than that in control mice.
[0004] These results suggest that, although all current results remain in the experimental stage, adoptive cell transfer of γδT cells has demonstrated remarkable efficacy in the treatment of lung cancer and warrants clinical promotion. However, the lack of robust, consistent, and GMP-compliant expansion protocols has made it difficult to obtain sufficient numbers of γδT cells to overcome the immunosuppressive tumor microenvironment in cancer patients, a major factor contributing to the slow clinical progress of γδT cells. Furthermore, tumors harbor multiple inhibitory factors, such as suppressive cytokines secreted by tumor cells and suppressive cells in the tumor microenvironment, which inhibit effector γδT cells through various mechanisms, leading to γδT cell dysfunction and promoting tumor cell immune escape, hindering the further application of γδT cells. The lack of T cells capable of recognizing tumor antigens expressed in many individuals and the lack of sufficient tumor-specific responses are also important factors that affect the anti-tumor ability of γδT cells. Therefore, obtaining sufficient numbers of γδT cells, improving the efficacy of γδT cell-based tumor immunotherapy, and enhancing tumor-specific responses have become current research focuses.
[0005] Exosomes are extracellular vesicles secreted by most eukaryotic cells. They carry proteins, lipids, sugar structures, nucleic acids (mRNA, miRNAs, and DNA), and metabolites that can alter the fate of recipient cells through autocrine and paracrine signaling. The mechanisms by which exosomes regulate cancer progression primarily include three aspects: ① Exosomal proteins can alter the fate of the exosome-releasing cells through autocrine pathways; ② Exosomal DNA alters cell survival; and ③ Exosome cargo can act as external stimuli in recipient cells, thereby altering signaling pathways in recipient cells, mediating intercellular interactions and regulating the microenvironment through paracrine pathways. Previous studies have shown that exosomes are more abundant in the circulating fluids of cancer patients compared to healthy controls, and can promote tumor cell proliferation, metastasis, drug resistance, epithelial-mesenchymal transition, induction of angiogenesis, establishment of a pre-metastatic microenvironment, and immune escape. In addition, because exosomes are part of the disease process and are very stable in biological fluids (such as plasma and urine), they can be isolated for clinical evaluation even in the early stages of the disease. Therefore, exosome-based biomarkers have been rapidly adopted in the clinical field. The first prostate cancer test based on exosomal RNA has helped more than 50,000 patients make decisions and is now included in the National Comprehensive Cancer Network's early prostate cancer detection guidelines. Therefore, exosomes may be biomarkers and new therapeutic targets for cancer diagnosis and prognosis.
[0006] Furthermore, studies have shown that exosomes are promising drug delivery agents for therapeutic delivery due to their natural intercellular communication, excellent biocompatibility, low immunogenicity, low toxicity, long-distance transport, biodegradability, and ability to cross various biological barriers. The key to using exosomes as drug carriers is the efficient loading of exogenous drugs into exosomes. Currently, methods such as sonication, electroporation, transfection, incubation, extrusion, saponin-assisted loading, genetic modification, freeze-thaw cycling, heat shock, pH gradient methods, and hypotonic dialysis are common methods for loading these drugs into exosomes. Studies have shown that exosomes can deliver various molecules to adjacent cells or tissues located in different anatomical sites, making exosomes unique candidates for vaccine development.
[0007] Based on the fact that various immune cells have been widely used to induce anti-tumor immunity in clinical studies, a promising immunotherapy to improve the anti-tumor specificity of immune cells is to load autologous immune cells (such as dendritic cells, γδT cells, NK cells, etc.) with autologous tumor antigens from self-renewing autologous cancer cells in vitro (tumor antigen loading), activate them in vitro, and then inject them into the patient to induce an immune response specific for the tumor antigen. As the most effective antigen-presenting cells, DCs (moDCs) derived from the patient's peripheral blood mononuclear cells (PBMCs) can be loaded with different forms of tumor antigens. Therefore, DCs are currently the most widely studied antigen-loaded cells. Currently, antigen-loaded immunotherapies, such as chimeric antigen receptor (CAR)-engineered T cell (CAR-T) therapy and multi-antigen-stimulated cell therapy (MASCT), have shown clinical benefits and manageable safety in solid cancers such as hepatocellular carcinoma, gastric cancer, and bone and soft tissue sarcomas.
[0008] Studies have reported that tumor-derived exosomes (Texos), released from tumor cells into the extracellular environment, are rich in tumor-associated antigens, major MHC molecules, tetranectin (CD9 and CD63), and costimulatory molecules. These exosomes can induce anti-tumor immune responses by cytotoxic T lymphocytes (CTLs), acting as potent stimulators of the immune response. In recent years, exosomes have become a research hotspot in cancer immunity. DC vaccines pulsed with exosomes have been shown to elicit a range of tumor antigens and to elicit stronger immune responses than cell lysates in vitro and in vivo. Studies have reported that HeLa-exo loading of DCs in vitro by co-culturing HeLa exosomes with DCs has been shown to promote T cell proliferation and induce CTL responses, thereby inhibiting the growth of cervical cancer cells in vitro. Therefore, loading tumor-derived exosomes may also be an effective method to enhance the anti-tumor activity and specificity of immune cells. However, studies have yet to report the tumor-suppressive effects of tumor exosome-loaded γδ T cells. Summary of the Invention
[0009] In response to the deficiencies of the prior art, the present invention provides a method for preparing tumor exosomes loaded with γδT cells and its application, thereby improving the anti-tumor activity and specificity of immune cells.
[0010] To solve the above technical problems, the technical solution of the present invention is as follows: a method for preparing tumor exosomes loaded with γδT cells, the preparation method is performed as follows:
[0011] The isolated tumor cell exosomes were inactivated under sterile conditions by keeping them at 55-65°C for 20-40 minutes, repeated freezing and thawing 3-5 times, and centrifuged at 2500-3500 rpm for 8-15 minutes. The supernatant was sterilized by filtration using a membrane filter.
[0012] On the 1st to 7th day of γδT cell culture, the treated tumor cell exosomes were added to the culture medium for co-culture with γδT cells. The amount of exosomes added to the culture medium was 10 8 -10 10 particles / mL to obtain exosome-loaded γδT cells; the culture medium can be replaced every day or every other day.
[0013] Preferably, the tumor cell exosomes are lung cancer tumor cell exosomes.
[0014] Preferably, the tumor cell exosome-loaded γδT cells are loaded with exosomes for a second time before use, and the amount of exosomes added to the culture medium is 10 8 -10 10 The second loading method is used because during the culture process, some γδT cells are not loaded with exosomes due to medium replacement and cell proliferation. A second loading method can fully load the cells with exosomes. Therefore, the dual loading method of the present invention ensures that γδT cells are fully loaded with tumor exosomes before injection into the patient.
[0015] As a further description of the above scheme: the isolation process of the exosomes is as follows:
[0016] The tumor tissue was cut into small pieces, and the cells were separated from the tissue using mechanical dispersion and enzymatic digestion. The cells were cultured in serum-free DMEM medium, and the cell viability and apoptosis levels were detected by CCK-8 and flow cytometry. After the cells reached 70-80% confluence and continued to be cultured for 36-60 hours, the digested lung cancer tumor cell suspension was collected for the isolation of exosomes.
[0017] Add lung cancer tumor cell suspension to an ultrafiltration centrifuge tube, centrifuge at 3500-4500g for 20-40min, add PBS, and gently pipette to obtain an ultrafiltration concentrate containing exosomes; the ultrafiltration concentrate is subjected to gradient centrifugation according to the steps of centrifugation at 200-400g for 8-15min, centrifugation at 1500-2500g for 8-15min, centrifugation at 9000g-12000g for 20-40min, and centrifugation at 80000-120000g for 60-80min. The final centrifugation is performed 1-3 times. The resulting precipitate attached to the bottom wall of the test tube is the exosomes of lung cancer tumor cells; use PBS to gently and thoroughly pipette to mix, fully resuspend the exosomes, and store in a -80°C refrigerator for use.
[0018] As a further description of the above scheme: the isolation and purification process of the γδT cells is as follows:
[0019] Cell isolation: Obtain peripheral blood mononuclear cells, adjust the cell density using serum-free medium containing 7-10% human platelet lysate, add zoledronic acid at a final concentration of 50-100 μg / mL and IL-2 at a final concentration of 50-100 μg / mL, seed the cells into cell flasks, and culture in a CO2 incubator for 1-4 days;
[0020] Cell activation and expansion: Replace the culture medium with fresh complete medium containing IL-2 at a final concentration of 20-50 μg / mL every other day to maintain the cell density at 1×10 6 / mL to expand and culture the cells;
[0021] For the sorting of γδT cells, anti-γδTCR antibodies combined with magnetic beads are used to label γδT cells in the single-cell suspension, and magnetic field adsorption and washing steps are performed in a magnetic separation column to finally collect the purified γδT cells.
[0022] The present invention also provides tumor exosomes loaded with γδT cells obtained by the preparation method.
[0023] The present invention also provides the use of the tumor exosomes loaded with γδT cells in the preparation of anti-lung cancer drugs.
[0024] Compared with the existing technology, the present invention has the following beneficial effects: The present invention studies the use of tumor exosomes loaded in γδT cells for the treatment of lung cancer. After γδT cells and tumor cell exosomes are co-cultured with cancer cells, a large number of tumor cell exosomes γδT cells surround the tumor cells and attack the tumor cells, while the γδT cells do not attack the tumor cells around them. Tumor cell exosomes loaded γδT cells were intravenously injected into multiple lung cancer patients. Compared with before treatment, the proportion of CD3+ and CD4+ positive cells increased significantly after 4 cycles and 6 months after treatment, the proportion of CD8+ positive cells decreased significantly, the CD4+ / CD8+ ratio increased significantly after 6 months, and the number of NK cells increased significantly after 4 cycles of treatment. Therefore, tumor exosomes loaded γδT cells have a good anti-tumor effect, providing a valuable scientific basis and experience for industrial production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 For the isolation and identification of exosomes;
[0026] Figure 2 γδT cell induction detection; A: γδT cell morphology under a 400x ordinary microscope; B: CD3+γδT expression detected by flow cytometry;
[0027] Figure 3 The effect of tumor cell exosome loading on γδT cells;
[0028] Figure 4 The effect of tumor cell exosomes γδT cells on tumor cells;
[0029] Figure 5 Imaging changes in lung tissue after treatment with tumor cell exosomes loaded with γδT cells in lung cancer patients. DETAILED DESCRIPTION
[0030] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.
[0031] Example 1 Preparation of tumor cell exosomes loaded with γδT cells
[0032] (1) Exosome isolation and preparation methods
[0033] Tumor cell extraction, exosome isolation and quality control
[0034] Non-small cell lung cancer (NSCLC) tumor tissue was cut into small pieces, and cells were isolated from the tissue using mechanical dispersion and enzymatic digestion. The cells were then cultured in DMEM medium containing 10% FBS. Cell viability and apoptosis were assessed using CCK-8 and flow cytometry, and non-small cell lung cancer cell markers CEA, TTF-1, and Napsin A were measured by flow cytometry. Tumor cells were cultured in serum-free DMEM medium until they reached 70-80% confluence. The digested lung cancer cell suspension was collected and 1200 mL of the cell suspension was added to a 100 kD ultrafiltration centrifuge tube. The tube was centrifuged at 4000 g for 30 minutes, and then PBS (pH 7.4) was added and gently pipetted to obtain approximately 120 mL of an ultrafiltration concentrate containing exosomes.
[0035] The suspension was subjected to gradient centrifugation at 300 g for 10 min, 2000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 70 min × 2 times. The resulting precipitate attached to the bottom wall of the test tube was the exosomes of lung cancer cells.
[0036] Gently pipette and mix thoroughly with 1 mL of PBS to fully resuspend the exosomes and temporarily store in a -80°C refrigerator;
[0037] The exosome solutions prepared each time are combined, and after passing the inspection, they are placed on a freeze dryer for freeze drying to obtain lung cancer tumor cell exosomes for loading γδ T cells.
[0038] Exosome characteristic quality detection
[0039] Figure 1 Results showed that transmission electron microscopy revealed a clear, saucer-shaped structure of exosomes from lung cancer cells. NTA analysis revealed a particle size of 152 nm. The exosome size range was 30-200 nm, which is consistent with the exosome size range, demonstrating successful extraction of exosomes from lung cancer cells. Protein quantification of the exosomes is shown in Table 1.
[0040] Table 1 Quantification of exosome proteins
[0041]
[0042]
[0043] As can be seen from Table 1, the total protein concentration of exosomes is 27.5 g / L, the albumin (miscellaneous protein) concentration is <3 g / L, the apolipoprotein concentration is negligible, and the protein purity of this sample is >90%.
[0044] In addition, the exosome concentration at each particle size was summed to obtain the number of particles per milliliter of the sample exosomes of 8.77×10 8The sample was 20ul and diluted to 1mL for determination. Therefore, after the reduction multiple was calculated, the exosome concentration of the original sample was 4.38×10 10 particles / mL. The standard concentration of exosome products is >1.0×10 9 particles / mL, so the sample meets the standard.
[0045] γδT cell isolation and preparation method
[0046] γδT cells are mainly derived from autologous or immediate family PBMCs (peripheral blood mononuclear cells). Preparation mainly includes the following steps:
[0047] Cell isolation: Obtain peripheral blood mononuclear cells, adjust the cell density using serum-free medium containing 7-10% human platelet lysate, add zoledronic acid at a final concentration of 50-100 μg / mL and IL-2 at a final concentration of 50-100 μg / mL, seed the cells into cell flasks, and culture in a CO2 incubator.
[0048] Cell activation and expansion: On days 3, 5, 7, 9, and 11, replace the culture medium with fresh complete medium containing IL-2 at a final concentration of 20 to 50 μg / mL to maintain the cell density at 1 × 10 6 / mL to expand and culture the cells.
[0049] For the sorting of γδT cells, anti-γδTCR antibodies combined with magnetic beads were used to label γδT cells in the single-cell suspension, and magnetic field adsorption and washing steps were performed in a magnetic separation column to finally collect the purified γδT cells; subsequently, the purity and characteristics of the γδT cells were further analyzed and verified by flow cytometry using anti-γδTCR and anti-CD3 antibody labeling.
[0050] After 7 days of induction culture, the formation of cell aggregates was observed under a microscope ( Figure 2 A). After 14 days of culture, flow cytometry detection of γδT revealed that 90% of the cells were positive for CD3+γδT ( Figure 2 B), The results indicate that γδT induction was successful.
[0051] (3) Lung cancer cell exosomes load (induce) γδT cells
[0052] Exosome loading: The exosomes of lung cancer cells obtained above were inactivated under sterile conditions at 56°C for 30 minutes, repeatedly frozen and thawed 3-5 times, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and sterilized by filtration with a 0.22 mm filter membrane and stored at -80°C until use. γδT cells were seeded in 24-well plates at a density of 1.5×10 per well. 6On the 3rd to 5th day of γδT cell culture, lung cancer tumor exosomes were added to RPMI-1640 culture medium for co-culture with γδT cells. The amount of exosomes added to the culture medium was 10 9 Co-culture was performed with 500 μg / mL of γδT cells. The aggregation and morphological changes of γδT cells after co-culture were observed under a microscope. The activity and proliferation of γδT cells were determined using MTT. The secretion levels of cytokines (IFN-γ, TNF-α, granzyme B, and perforin) in the culture supernatant after co-culture were measured by ELISA.
[0053] After γδT cells and exosomes were co-cultured, the culture medium was replaced and culture was continued. Tumor cell exosomes stimulated the proliferation of γδT cells, and the number of cell clusters increased (e.g. Figure 3 This suggests that tumor exosome loading may promote the proliferation of γδT cells.
[0054] Considering that the amount of exosomes obtained in actual application is not enough, the γδT cells loaded with tumor cell exosomes can be loaded with exosomes for a second time before being put into use. The amount of exosomes added to the culture medium is 10 8 -10 10 The cells were loaded with exosomes at a concentration of 1000 μg / mL and cultured for 6-24 hours. After the first exosome loading, as the γδT cells proliferate and the exosomes are released from the cells, some γδT cells are not loaded with exosomes. Therefore, the dual loading method of the present invention ensures that the γδT cells are fully loaded with tumor exosomes before injection into the patient.
[0055] Example 2 Verification of the effect of tumor cell exosomes loaded with γδT cells
[0056] Tumor cell exosomes loaded with γδT cells can specifically kill tumor cells
[0057] like Figure 4 The results showed that after γδT cells and tumor cell exosome-loaded γδT cells were co-cultured with lung cancer tumor cells respectively, a large number of tumor cell exosome-loaded γδT cells surrounded the tumor cells and attacked the tumor cells, while the γδT cells attacked the tumor cells around them weakly.
[0058] Preliminary clinical observation of tumor cell exosomes loaded with γδT cells
[0059] (1) Effects of tumor cell exosomes loaded with γδT cells on T lymphocyte subsets
[0060] 32 lung cancer patients were intravenously injected with tumor cell exosomes loaded with γδT cells. The changes in T lymphocyte subsets before treatment, after 2 cycles of treatment, 4 cycles of treatment, and 6 months are shown in Table 2. Compared with before treatment, the proportion of CD3+ and CD4+ positive cells increased significantly after 4 cycles and 6 months of treatment, while the proportion of CD8+ positive cells decreased significantly. The CD4+ / CD8+ ratio was significantly increased after 6 months, and the number of NK cells increased significantly after 4 cycles of treatment.
[0061] Table 2 Comparison of changes in T lymphocyte subsets before and after treatment n = 32, x ± s
[0062] project Before treatment 2 weeks after treatment 4 weeks after treatment 6 months later CD3+ 54.7±13.8 58.4±7.2 65.6±9.2* 68.6±8.4* CD4+ 27.2±5.4 29.8±3.4 32.2±2.8* 33.6±3.2* CD8+ 23.3±8.8 24.5±6.2 17.6±6.1* 18.2±7.2* CD4+ / CD8+ 1.2±0.2 1.3±0.3 1.4±0.4* 1.5±0.5* NK 24.3±9.6 23.2±8.2 29.6±8.6* 28.5±8.2
[0063] Note: *P<0.05 compared with before treatment
[0064] (2) Blood test and chemotherapy side effects of a lung cancer patient receiving tumor cell exosome-loaded γδT cell therapy
[0065] During treatment, one lung cancer patient receiving tumor cell exosome-loaded γδT cells underwent blood testing and observation for side effects after chemotherapy. The patient's carcinoembryonic antigen (CEA) level decreased significantly after treatment, while his white blood cell count increased significantly. Prior to treatment with tumor cell exosome-loaded γδT cells, the patient had a significant chemotherapy reaction, with side effects including fatigue, nausea, vomiting, dizziness, loss of appetite, and weakness. However, these side effects resolved after treatment, and the patient was able to complete chemotherapy.
[0066] (3) Imaging changes of lung tissue in patients with lung cancer and lung metastasis after treatment with tumor cell exosome-loaded γδT cells
[0067] like Figure 5 CT imaging results showed that the lung tissue lesions of lung cancer patients were significantly reduced after six months of treatment with tumor cell exosome-loaded γδT cells.
[0068] The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above-mentioned embodiments. Any other changes made without departing from the spirit and principles of the present invention shall be considered equivalent replacements and shall be included in the scope of protection of the present invention.
Claims
1. A method for preparing tumor exosomes loaded with γδT cells, characterized in that: The preparation method is as follows: The isolated tumor cell exosomes were inactivated under sterile conditions by keeping them at 55-65°C for 20-40 minutes, repeated freezing and thawing 3-5 times, and centrifuged at 2500-3500 rpm for 8-15 minutes. The supernatant was sterilized by filtration using a membrane filter. On the 1st to 7th day of γδT cell culture, the treated tumor cell exosomes were added to the culture medium for co-culture with γδT cells. The amount of exosomes added to the culture medium was 10 8 -10 10 particles / mL to obtain exosome-loaded γδT cells.
2. The method for preparing tumor exosomes loaded with γδT cells according to claim 1, characterized in that: The tumor cell exosomes are lung cancer tumor cell exosomes.
3. The method for preparing tumor exosomes loaded with γδT cells according to claim 1, characterized in that: The tumor cell exosome-loaded γδT cells were loaded with exosomes for a second time before use, and the amount of exosomes added to the culture medium was 10 8 -10 10 particles / mL.
4. The method for preparing tumor exosome-loaded γδT cells according to claim 2 or 3, characterized in that: The exosome isolation process is as follows: The tumor tissue was cut into small pieces, and the cells were separated from the tissue using mechanical dispersion and enzymatic digestion. The cells were cultured in serum-free DMEM medium, and the cell viability and apoptosis levels were detected by CCK-8 and flow cytometry. After the cells reached 70-80% confluence and continued to be cultured for 36-60 hours, the digested lung cancer tumor cell suspension was collected for the isolation of exosomes. Add lung cancer tumor cell suspension to an ultrafiltration centrifuge tube, centrifuge at 3500-4500g for 20-40min, add PBS, and gently pipette to obtain an ultrafiltration concentrate containing exosomes; the ultrafiltration concentrate is subjected to gradient centrifugation according to the steps of centrifugation at 200-400g for 8-15min, centrifugation at 1500-2500g for 8-15min, centrifugation at 9000g-12000g for 20-40min, and centrifugation at 80000-120000g for 60-80min. The final centrifugation is performed 1-3 times. The resulting precipitate attached to the bottom wall of the test tube is the exosomes of lung cancer tumor cells; use PBS to gently and thoroughly pipette to mix, fully resuspend the exosomes, and store in a -80°C refrigerator for use.
5. The method for preparing tumor exosomes loaded with γδT cells according to claim 4, characterized in that: The separation and purification process of the γδT cells is as follows: Cell isolation: Obtain peripheral blood mononuclear cells, adjust the cell density using serum-free medium containing 7-10% human platelet lysate, add zoledronic acid at a final concentration of 50-100 μg / mL and IL-2 at a final concentration of 50-100 μg / mL, seed the cells into cell flasks, and culture in a CO2 incubator for 1-4 days; Cell activation and expansion: Replace the culture medium with fresh complete medium containing IL-2 at a final concentration of 20-50 μg / mL every other day to maintain the cell density at 1×10 6 / mL to expand and culture the cells; For the sorting of γδT cells, anti-γδTCR antibodies combined with magnetic beads are used to label γδT cells in the single-cell suspension, and magnetic field adsorption and washing steps are performed in a magnetic separation column to finally collect the purified γδT cells.
6. Tumor exosomes loaded with γδT cells obtained by the preparation method according to claim 5.
7. Use of the tumor exosomes loaded with γδT cells according to claim 6 in the preparation of anti-lung cancer drugs.