Method for transforming CD8 + T cells through LIFUS and application

Abstract

The invention relates to a method for transforming CD8 + T cells through LIFUS and application, and belongs to the technical field of biological medicine. The method comprises the following steps: culturing a certain number of T cells in vitro, extracting GVs expressed by an acoustic response reporter gene, co-culturing the T cells and the GVs, carrying out acoustic cavitation treatment by using LIFUS, and finally, carrying out flow separation to obtain the treated T cells. The T cells comprise CD8 < + > T cells. The invention provides the application of the method in preparation of the cell therapy medicine, and provides a new idea for preparing the cell therapy medicine.

Description

Technical Field

[0001] The present invention relates to a method for modifying CD8 + T cells by LIFUS and its applications, belonging to the field of biomedical technology. Background Art

[0002] Tumors, as diseases that pose a serious threat to human health, have always been the focus of medical research in terms of treatment options. Especially for solid tumors, the dual barriers formed by their tumor microenvironment (TME): the physical barrier (dense stromal tissue hinders drug penetration) and the immune barrier (the inhibitory microenvironment leads to T cell dysfunction) are the main challenges in treatment. Research shows that cancer-associated fibroblasts (CAFs), as the main component of the TME stroma, inhibit the activity of T cells by proliferating and secreting various immunosuppressive factors. Although innovative progress has been made in tumor treatment with immune checkpoint inhibitors (ICI), insufficient T cell infiltration and functional exhaustion in solid tumors remain the main bottlenecks in tumor treatment. Therefore, how to break through the physical barrier of the TME and reshape the tumor immune microenvironment is the key to improving the treatment effect.

[0003] In recent years, engineering bacteria-mediated tumor treatment has been rapidly becoming an effective treatment modality. Research shows that the hypoxic and immunosuppressive microenvironment inside tumors provides favorable conditions for bacterial colonization. Bacteria can break through the tumor vascular barrier and high interstitial pressure, proliferate in the hypoxic and necrotic core regions of tumors, activate the body's innate immune response and adaptive immune response. At the same time, through genetic engineering modification of bacteria, their toxicity can be reduced and effective factors can be loaded or released to enhance the anti-tumor effect. VNP20009 is an engineered attenuated Salmonella typhimurium obtained by knocking out purI and mutating the msbB gene. In addition to having an obvious preference for anaerobic environments, it also exhibits a unique ability to penetrate through dense matrices. However, the phase I clinical trials using these bacteria as independent tumor treatments have not resulted in substantial tumor regression, which reveals the limitations of these bacteria for single treatment: insufficient toxicity and targeting. More and more evidence shows that bacterial treatment can be synergistically combined with other treatment modalities to achieve a more effective anti-tumor effect; the above-mentioned prominent permeability and biosafety make VNP2009 a candidate for gene delivery in the field of solid tumor treatment.

[0004] Although breakthroughs have been made in engineering bacteria as a carrier, real-time monitoring of bacterial colonization and gene expression still mainly relies on fluorescence or bioluminescence techniques, which have the disadvantages of low sensitivity and poor penetrability. The team of Shapiro et al. at the University of California, USA, proposed to use acoustic reporter genes (ARG) to express gas vesicles (GVs), which can generate an acoustic response under the action of focused ultrasound (FUS) to achieve deep tissue imaging, providing a new strategy for real-time monitoring of gene expression and therapeutic effects. At the same time, they found that the introduction of the FUS-GVs technology can not only accurately monitor the distribution of bacteria in the body, but also enhance the targeting and penetrability of engineering bacteria therapy through the non-thermal effect generated by FUS, improving the therapeutic effect of tumors. These studies have confirmed the potential of GVs as acoustic cavitation nuclei. However, the ultrasound parameters selected in these studies and the ultrasonic responses studied mainly focus on the thermal effects generated by higher-parameter FUS. There are relatively few studies on the treatment of tumors by low-intensity ultrasound (LIFUS) in combination with engineering bacteria (ARG-GVs). LIFUS is a mode of FUS that uses the focusing characteristics of ultrasound to concentrate ultrasonic energy on a specific target area in the body, and the energy density is 0.1-1W / cm 2 (focused area). LIFUS has the advantages of non-invasiveness and little damage to surrounding normal tissues. In the local area, it mainly generates mechanical effects by acoustic cavitation, and the damage to surrounding tissues caused by the thermal effect is relatively small. Some studies have confirmed that the mechanical force generated by the acoustic response of LIFUS can have a certain impact on the interaction of signal pathways in the tumor microenvironment, and can enhance the opening of the blood-brain barrier in neuromodulation, stimulate the release of a large number of immune-stimulating factors, and even increase the infiltration of CD8 + T cells. However, the effect of LIFUS in combination with ARG-GVs in treating solid tumors, especially the mechanism of cell interaction between the mechanical force generated by LIFUS-GVs and the tumor microenvironment, remains to be further studied.

[0005] In summary, in order to overcome the challenges in the treatment of solid tumors, the present invention proposes a treatment method that combines the use of VNP2009 as an acoustic response gene vector encoding GVs with LIFUS. The ARG cluster expressing GVs is introduced into VNP2009 to break through the physical barrier of the tumor while activating the immune response of the tumor, achieving visual collaborative anti-tumor immunotherapy, and further exploring the mechanism of cell interaction between the mechanical force generated by LIFUS-GVs and the tumor immune microenvironment, providing a new target for the mechanical immunotherapy of tumors. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to obtain a technical solution that can overcome the bottleneck in the current treatment of solid tumors and provide a new technical solution for tumor treatment.

[0007] To achieve the purpose of solving the above problems, the technical solution adopted by the present invention is to provide a method for enhancing the killing ability of T cells. The method includes culturing a certain number of T cells in vitro, extracting GVs expressed by an acoustic response reporter gene, co-culturing the T cells with GVs, then performing acoustic cavitation treatment using LIFUS, and finally obtaining the treated T cells by flow separation; the T cells include CD8 + T cells.

[0008] The present invention provides the application of the above method in the preparation of cell therapy drugs, and the application includes the application in the preparation of anti-tumor drugs.

[0009] Preferably, the application includes the application in the preparation of anti-tumor metastasis drugs.

[0010] The present invention provides the application of the above method in the preparation of cell therapy drugs that increase the release of serine protease GZMA and IFN-γ by activating T cells.

[0011] The present invention provides the application of the above method in the preparation of cell therapy drugs that increase the adhesion to tumor cells by activating T cells.

[0012] The present invention provides the application of the above method in the preparation of cell therapy drugs for inhibiting the interaction between tumor-associated fibroblasts and CD8 + T cells.

[0013] The present invention provides the application of the above method in the preparation of cell therapy drugs for inhibiting the NOTCH signaling pathway of tumor-associated fibroblasts.

[0014] The present invention provides the application of the above method in the preparation of cell therapy drugs for regulating the NOTCH signaling pathway on the CD8 + T cell membrane.

[0015] The present invention provides a method and application for modifying CD8 + T cells by LIFUS.

[0016] In the first aspect of the present invention, there is provided the application of low-intensity ultrasound in the preparation of cell therapy drugs, including the CD8 activated by the mechanical force generated by the action of the low-intensity ultrasound on gas vesicles + T cells.

[0017] Preferably, the application in the preparation of cell therapy drugs includes the preparation of drugs that activate CD8 +Application of T cells in increasing the release of serine protease GZMA and IFN-γ in cell therapy drugs.

[0018] Preferably, the application in preparing cell therapy drugs includes the application in preparing cell therapy drugs that increase the adhesion of CD8 + T cells to tumor cells.

[0019] In the second aspect of the present invention, there is provided an application of low-intensity ultrasound in preparing cell therapy drugs for inhibiting the interaction between tumor-associated fibroblasts and CD8 + T cells in tumors.

[0020] Preferably, the application in preparing cell therapy drugs includes the application of the mechanical force generated by the action of the low-intensity ultrasound on gas vesicles in preparing cell therapy drugs for inhibiting the expression of Notch1 receptor and Jagged-1 ligand in the NOTCH signaling pathway on the membrane of tumor-associated fibroblasts.

[0021] Preferably, the application in preparing cell therapy drugs includes the application of the mechanical force generated by the action of the low-intensity ultrasound on gas vesicles in preparing cell therapy drugs for increasing the expression of Notch1 receptor and inhibiting the expression of Jagged-1 ligand in the NOTCH signaling pathway on the membrane of CD8 + T cells.

[0022] In the third aspect of the present invention, there is provided an application of low-intensity ultrasound in preparing cell therapy drugs for inhibiting the tumor microenvironment, including the application of the mechanical force generated by the action of the low-intensity ultrasound on gas vesicles in preparing cell therapy drugs for inhibiting the expression of fibroblasts in tumors.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The mechanical force generated by the action of low-intensity ultrasound on gas vesicles inhibits the NOTCH axis on the membrane of tumor-associated fibroblasts and activates CD8 + T cells, promotes the release of anti-tumor factors, increases the adhesion to tumor cells, reduces the interaction between CAFs and CD8 + T cells in tumors, and provides a new path for preparing cell therapy drugs. Description of the Drawings

[0025] Figure 1 Experimental result diagram of the change of the NOTCH signaling pathway of tumor-associated fibroblasts (CAFs)-CD8 + T cells under the action of dual mechanical forces.

[0026] Figure 2 For CD8+ Schematic diagram of experimental results showing that the immunotherapeutic effect of T cells is improved through the mechanical force-NOTCH pathway. Detailed implementation manners

[0027] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows:

[0028] Example 1

[0029] Extract CAFs and CD8 + Co-culture CAFs and CD8 T lymphocytes in vitro. After adding the pre-extracted GVs, perform acoustic response treatment with LIFUS, and separate the two types of cells by flow cytometry. Figure 1 The multi-color immunofluorescence in Figure B shows the expression of representative proteins on the NOTCH pathway in CAFs. It can be seen that the fluorescence intensities of representative Notch1 and Jagged-1 in the group of CAFs without LIFUS acoustic response treatment (-US group) are significantly higher than those in the group of CAFs with LIFUS acoustic response (+US group). Figure 1 The WB analysis in Figure C shows the expression of these two proteins on the NOTCH pathway in CAFs. The results are consistent with the fluorescence staining. The expression of these two proteins in the +US group of CAFs is significantly lower than that in the -US group, indicating that after sonoporation of CAFs, the expressions of Notch1 receptor and Jagged-1 ligand on the cell membrane have both decreased significantly.

[0030] Similarly, the same verification was also performed on the NOTCH pathway proteins on CD8 T cells (+US group) that had been co-cultured and subjected to LIFUS acoustic response treatment and on those in the group without LIFUS acoustic response treatment (-US group) (Figure D, Figure E). The results showed that, compared with the -US group, the expression of Jagged-1 ligand on CD8 T cells in the +US group was the same as that in CAFs, and the expression in the +US group decreased compared with the -US control group. However, the expression of Notch1 receptor protein on CD8 T cells in the +US group was basically the same as the expression of Notch1 protein in the control group, without decrease or increase. The changes in the expression of NOTCH pathway proteins after co-culture of the above two types of cells in vitro can further explain the reason for the reduced interaction between CAFs and CD8 T cells in tumors, and it can be speculated that it is mainly due to the decreased expression of CAFs, because the expression of Notch1 receptor protein on CD8 T cells did not decrease, but instead showed an upward trend. The increase in Nocth1 receptor protein and the decrease in Jagged-1 ligand protein may contribute to CD8 + T cells, and the expression of Jagged-1 ligand on CD8 T cells in the +US group decreased compared with the -US control group. However, the expression of Notch1 receptor protein on CD8 T cells in the +US group was basically the same as the expression of Notch1 protein in the control group, without decrease or increase. The changes in the expression of NOTCH pathway proteins after co-culture of the above two types of cells in vitro can further explain the reason for the reduced interaction between CAFs and CD8 T cells in tumors, and it can be speculated that it is mainly due to the decreased expression of CAFs, because the expression of Notch1 receptor protein on CD8 T cells did not decrease, but instead showed an upward trend. The increase in Nocth1 receptor protein and the decrease in Jagged-1 ligand protein may contribute to CD8 + T cells, and the expression of Jagged-1 ligand on CD8 T cells in the +US group decreased compared with the -US control group. However, the expression of Notch1 receptor protein on CD8 T cells in the +US group was basically the same as the expression of Notch1 protein in the control group, without decrease or increase. The changes in the expression of NOTCH pathway proteins after co-culture of the above two types of cells in vitro can further explain the reason for the reduced interaction between CAFs and CD8 T cells in tumors, and it can be speculated that it is mainly due to the decreased expression of CAFs, because the expression of Notch1 receptor protein on CD8 T cells did not decrease, but instead showed an upward trend. The increase in Nocth1 receptor protein and the decrease in Jagged-1 ligand protein may contribute to CD8 + T cells, and the expression of Jagged-1 ligand on CD8 T cells in the +US group decreased compared with the -US control group. However, the expression of Notch1 receptor protein on CD8 T cells in the +US group was basically the same as the expression of Notch1 protein in the control group, without decrease or increase. The changes in the expression of NOTCH pathway proteins after co-culture of the above two types of cells in vitro can further explain the reason for the reduced interaction between CAFs and CD8 T cells in tumors, and it can be speculated that it is mainly due to the decreased expression of CAFs, because the expression of Notch1 receptor protein on CD8 T cells did not decrease, but instead showed an upward trend. The increase in Nocth1 receptor protein and the decrease in Jagged-1 ligand protein may contribute to CD8 + T cells, and the expression of Jagged-1 ligand on CD8 T cells in the +US group decreased compared with the -US control group. However, the expression of Notch1 receptor protein on CD8 T cells in the +US group was basically the same as the expression of Notch1 protein in the control group, without decrease or increase. The changes in the expression of NOTCH pathway proteins after co-culture of the above two types of cells in vitro can further explain the reason for the reduced interaction between CAFs and CD8 T cells in tumors, and it can be speculated that it is mainly due to the decreased expression of CAFs, because the expression of Notch1 receptor protein on CD8 T cells did not decrease, but instead showed an upward trend. The increase in Nocth1 receptor protein and the decrease in Jagged-1 ligand protein may contribute to CD8 + T cells, and the expression of Jagged-1 ligand on CD8 T cells in the +US group decreased compared with the -US control group. However, the expression of Notch1 receptor protein on CD8 T cells in the +US group was basically the same as the expression of Notch1 protein in the control group, without decrease or increase. The changes in the expression of NOTCH pathway proteins after co-culture of the above two types of cells in vitro can further explain the reason for the reduced interaction between CAFs and CD8 T cells in tumors, and it can be speculated that it is mainly due to the decreased expression of CAFs, because the expression of Notch1 receptor protein on CD8 T cells did not decrease, but instead showed an upward trend. The increase in Nocth1 receptor protein and the decrease in Jagged-1 ligand protein may contribute to CD8 +The activation of T cells binds to tumor cells, thereby inhibiting the growth of tumor cells.

[0031] Example 2

[0032] To verify the speculation in Example 1, after co-culture and sonoporation, CD8 + T GVs+US cells and CD8 + T GVs-US cells in the group without sonoporation during co-culture were co-cultured with tumor cells. As Figure 1 shown in the immunofluorescence results of Figures F, G, and H, it can be seen that after co-culture of tumor cells with CD8 + T cells treated with sonoporation, in the group of CD8 + T GVs+US cells treated with sonoporation, the fluorescence intensity of CD8 + T cells around the tumor cells was significantly higher than that of the control group without co-culture and sonoporation and the group of CD8 + T GVs-US cells (51.89±10.469 VS. 3.25±1.194 and 15.72±7.526), indicating that the adhesion ability of CD8 + T GVs+US cells to tumor cells was significantly improved, about 17 times higher than that of the control group. The increase in the adhesion of CD8 + T cells contributed to the improvement of the killing ability against tumors. After further culturing for 12 h and 36 h respectively, the apoptosis of tumor cells was observed. Figure 1 As shown in Figure I, there were no significant differences among the groups at 12 h, but after 36 h, the tumor cells in the group of CD8 + T GVs+US cells had basically undergone apoptosis, significantly inhibiting the growth of tumor cells. The 4T1-Luc tumor cells labeled with luciferase showed almost no fluorescence ( Figure 1 Figure J). Further, the supernatant of the co-culture was extracted for detection. Figure 1 As shown in Figures K-L, it can be seen that the release amounts of the related cytotoxic protease serine protease GZMA and the related pro-inflammatory cytokine IFN-γ were significantly higher than those of the control group and the group of CD8 + T GVs-US cells, playing an effective role in inhibiting tumor growth. Therefore, CD8 + T GVs+US cells can inhibit tumor growth more effectively than CD8 + T GVs-US cells. This effect may be due to the dual action of the exogenous tissue of GVs and the mechanical force generated by the acoustic response of LIFUS, which effectively activates the activity of CD8 + T cells, and the change in the Notch pathway reduces CD8+ T cell exhaustion. This anti-tumor immunotherapeutic effect of LIPUS-VNP / ARG-GVs provides a new idea for the subsequent modification of CD8 + T cells using the mechanical force-NOTCH axis for tumor treatment.

[0033] As Figure 1 shown, Figure 1 is the experimental result graph of the NOTCH signaling pathway changes in tumor-associated fibroblasts (CAFs)-CD8 + T cells under dual mechanical forces.

[0034] Among them, Figure A is a schematic diagram of the co-culture system constructed by tumor-associated fibroblasts (CAFs) and CD8 + T cells and ultrasound-responsive gas vesicles (GVs);

[0035] Figure B is the confocal imaging result graph of the expression of NOTCH1 receptor and Jagged1 ligand in tumor-associated fibroblasts (CAFs) in the GVs-US group and GVs+US group;

[0036] Figure C is the WB experimental result graph of the expression of NOTCH1 receptor and Jagged1 ligand in tumor-associated fibroblasts (CAFs) in the GVs-US group and GVs+US group;

[0037] Figure D is the confocal imaging result graph of the expression of Jagged1 ligand and Notch1 receptor in the GVs+US group and GVs-US group of CD8 + T cells.

[0038] Figure E is the WB experimental result graph of the expression of Jagged1 ligand and Notch1 receptor in the GVs+US group and GVs-US group of CD8 + T cells;

[0039] Figure F is a schematic diagram of the co-culture of CD8 + T cells (+US) group after co-culture and sonoporation and CD8 + T cells (-US) group without sonoporation treatment and tumor cells (4T1 cells);

[0040] Figure G is the immunofluorescence experimental graph of the adhesion of CD8 + T cells in the co-culture+US group, co-culture-US group, and control group to tumor cells;

[0041] Figure H is the statistical graph of the adhesion number of CD8 + T cells to tumor cells in the co-culture+US group, co-culture-US group, and control group;

[0042] Figure I is a statistical chart of the fluorescence intensity of tumor apoptosis after co-culture + US group, co-culture - US group, and control group were cultured for 12 h and 36 h respectively;

[0043] Figure J is a graph showing the results of the tumor apoptosis fluorescence experiment after the co-culture + US group, co-culture - US group, and control group were cultured for 12 h and 36 h respectively;

[0044] Figure K is a statistical chart of the secretion amount of cytotoxic protease serine protease GZMA detected by extracting the supernatant of co-culture after 36 h for each group;

[0045] Figure L is a statistical chart of the secretion amount of interferon - γ detected by extracting the supernatant of co-culture after 36 h for each group.

[0046] Example 3

[0047] Directly use mechanically - NOTCH axis - treated CD8 + T cells to verify the immunosuppressive effect on tumors.

[0048] Two different tumor models were used in the study to verify the inhibitory effects of unmodified and modified CD8 + T cells on tumors. Figure 2 In Figure A, it shows that a B16 - OVA mouse model was established by injecting B16 - OVA cells, and then 0T - 1 cells (i.e., 0T - 1 GVs-US and 0T - 1 GVs+US ) treated with GVs - US and GVs + US were injected. After 15 days, different changes in the tumor volume could be seen. The tumors of mice in the blank control group grew the fastest, while the 0T - 1 GVs-US group also showed certain killing ability after being stimulated by GVs and had a certain inhibitory effect on tumors, but its inhibitory effect was significantly inferior to that of the 0T - 1 GVs+US group, which had an obvious inhibitory effect on tumor growth ([Figure Figure 2 B - C), and the change in the body weight of mice was also the smallest ([Figure Figure 2 D). Figure 2 In Figure E, flow cytometry was used to detect the combined amount of Tim3 and PD - l in tumors. With the enhancement of anti - tumor activity, the combined amount of Tim3 and PD - l was the least expressed (2.55 ± 1.273) in the 0T - 1 GVs+US group, while the most expressed (43 ± 4.601) in the control group, a decrease of about 94%. At the same time, the combined expression amount of anti - tumor factors TNF and IFN - γ was also the highest in the 0T - 1 GVs+US group, representing the highest anti - tumor activity. And Ki67, representing the apoptosis index, showed different degrees of apoptosis in tumors of each group, among which the 0T - 1 GVs+USThe apoptosis rate of the group was higher than that of the other two groups (63.20±8.433 VS. WT, 10.57±2.247 and 0T-1 GVs -US , 33.32±3.962), showing significant statistical significance.

[0049] 4T1-Luc tumor cells were intravenously injected into mice to establish a 4T1 lung metastasis model ( Figure 2 Figure F in), and the model was treated with three different CAR-T cells. Compared with the CAR-T cell control group without any treatment, the CAR-T GVs+US cell group effectively inhibited the metastasis of 4T1 in the lung. Figure 2 It can be seen from Figure G in that the number of lung metastases in the three groups was significantly different. There was almost no lung metastasis in the CAR-T GVs +US cell group, and there was metastasis in the CAR-T GVs-US cell group, but the amount of metastasis was also significantly less than that in the control group. Figure 2 Small animal imaging in Figure H in showed obvious lung metastasis imaging in the control group, and the mice began to die on the 15th day after injecting 4T1 tumor cells, and metastases also appeared in other organs of the mice. On the 25th day, the mice in the control group were basically dead, while the CAR-T GVs-US cell group began to die on the 20th day. The CAR-T GVs+US cell group did not show death during the observation period, and the metastases in the group returned to normal after appearing on the 10th day, proving the inhibitory effect of CAR-T GVs+US cells on metastatic tumors. In summary, CD8 + T cells treated with GVs+US achieved the effect of effectively inhibiting tumor growth.

[0050] As Figure 2 shown, Figure 2 it is the experimental result diagram that the immunotherapy effect of CD8 + T cells was improved through the mechanical force-NOTCH pathway.

[0051] Among them, Figure A is a schematic diagram of establishing a mouse orthotopic B16-OVA model.

[0052] Figure B is the tumor volume change diagram of 0T-1 cells (0T-1 GVs-US group, 0T-1 GVs+US group) and the control group treated with GVs-US and GVs+US from 10 days before treatment to 15 days after treatment;

[0053] Figure C is the 0T-1 cells (0T-1 GVs-US group, 0T-1GVs+US Statistical chart of tumor weights of the treatment group and the control group;

[0054] Panel D shows the statistical chart of the weights of mice in the 0T-1 cell (0T-1 GVs-US group, 0T-1 GVs+US group) and the control group within 0 - 15 days after different treatments of GVs-US and GVs+US;

[0055] Panel E shows the flow cytometry results of tumor-infiltrating lymphocytes in the 0T-1 cell (0T-1 GVs-US group, 0T-1 GVs+US group) and the control group after different treatments of GVs-US and GVs+US; Compared with the control group, the co-expression of Tim-3 / PD-L1 in the 0T-1 GVs+US group decreased by 94.1%.

[0056] Panel F shows a schematic diagram of the establishment of a mouse 4T1-luciferase metastasis model;

[0057] Panel G shows the statistical chart of the fluorescence intensity of metastatic tumors treated with chimeric antigen receptor T cell (CAR-T) monotherapy, chimeric antigen receptor T cell combined with targeted gas vesicles (CAR-T GVs-US ), chimeric antigen receptor T cell combined with targeted gas vesicles + ultrasound (CAR-T GVs+US ); Compared with CAR-T monotherapy, CAR-T GVs+US treatment reduced lung metastasis by 92.7% (p<0.001).

[0058] Panel H shows the small animal imaging results of tumors treated with chimeric antigen receptor T cell (CAR-T) monotherapy, chimeric antigen receptor T cell combined with targeted gas vesicles (CAR-T GVs-US ), chimeric antigen receptor T cell combined with targeted gas vesicles + ultrasound (CAR-T GVs+US );

[0059] Panel I shows the Kaplan-Meier survival curves of tumors treated with chimeric antigen receptor T cell (CAR-T) monotherapy (control group), chimeric antigen receptor T cell combined with targeted gas vesicles (CAR-T GVs-US ), chimeric antigen receptor T cell combined with targeted gas vesicles + ultrasound (CAR-T GVs+US ); Compared with the CAR-T GVs-US group and the control group, the survival period of the CAR-T GVs+US group (median survival period > 30 days) was prolonged.

[0060] As described above, it is only the preferred embodiment of the present invention and does not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as within the protection scope of the present invention. For those skilled in the art, without departing from the spirit and scope of the present invention, any equivalent changes such as slight modifications, decorations, and evolutions made by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the substantial technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for enhancing the killing ability of T cells, characterized in that, The method includes culturing a certain number of T cells in vitro, extracting GVs expressed by an acoustic response reporter gene, co-culturing the T cells with the GVs, then performing acoustic cavitation treatment using LIFUS, and finally obtaining the treated T cells by flow separation; the T cells include CD8 + T cells.

2. Use of the method according to claim 1 in the preparation of a cell therapy drug, characterized in that, The application includes the application in the preparation of anti-tumor drugs.

3. The application according to claim 2, wherein The application includes the application in the preparation of anti-tumor metastasis drugs.

4. The application of the method according to claim 1 in the preparation of a cell therapy drug that increases the release of serine protease GZMA and IFN-γ by activating T cells.

5. The application of the method according to claim 1 in the preparation of a cell therapy drug that increases the adhesion to tumor cells by activating T cells.

6. Use of the method according to claim 1 in the preparation of a cell therapy drug for inhibiting the interaction between tumor-associated fibroblasts and CD8 + T cells.

7. The application of the method according to claim 1 in the preparation of a cell therapy drug that inhibits the NOTCH signaling pathway of tumor-associated fibroblasts.

8. Use of the method according to claim 1 in the preparation of a cell therapy drug for regulating the NOTCH signaling pathway on CD8 + T cell membranes.

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