Engineered exosome for breaking through pyroptosis immune starting and maintaining barrier as well as preparation method and application of engineered exosome

Abstract

The invention discloses an engineered exosome for breaking through a pyroptosis immune starting and maintaining barrier as well as a preparation method and application of the engineered exosome. The exosome is constructed by loading an immune checkpoint inhibitor into a pre-stimulated exosome, and the exosome is derived from immune cells, high-expression interferon gamma and granzyme A. The expression of the tumor cell GSDMB is up-regulated by delivering the interferon gamma through the exosome, and the exogenous granzyme A cuts the GSDMB to trigger pyroptosis; meanwhile, an immune checkpoint inhibitor blocks a PD-1 / PD-L1 pathway in situ, T cell depletion is reversed, and release of endogenous granzyme A is promoted; the pyroptosis cells release antigens to activate the T cells, the activated T cells continuously secrete granzyme A / interferon gamma to form a cascade amplification effect, and the problem of double barriers existing in the pyroptosis immune cycle of granzyme A-GSDMB is solved. The engineered exosome can improve the infiltration degree of CD8 + T cells, and is suitable for treating tumors such as liver cancer and non-small cell lung cancer.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly discloses an engineered exosome for breaking through a pyroptosis immune starting and maintaining barrier and a preparation method and application thereof. BACKGROUND

[0002] In recent years, cell pyroptosis has become an important strategy in the field of tumor treatment due to its significant advantages of immune activation and overcoming tumor cell apoptosis resistance. The occurrence of cell pyroptosis depends on the cleavage of gasdermin (GSDM) family proteins. When these proteins are cleaved, the NT domains of the proteins are combined with cell membrane phospholipids and form pores, leading to cell swelling and eventually rupture. It is worth mentioning that the NT domain (GSDMB-NT) protein of Gasdermin B (GSDMB) in the GSDM family can cause more obvious cell pyroptosis effect and has become a research hotspot.

[0003] At present, important progress has been made in the molecular mechanism of GSDMB-N induced cell pyroptosis. GSDMB is specifically cleaved at the Lys244 site by Granzyme A (GZMA) secreted by activated cytotoxic T cells and natural killer (NK) cells, and GSDMB-NT with membrane penetration activity is released. This process leads to the loss of tumor cell membrane integrity, and further induces cell pyroptosis. Pyroptotic cells release inflammatory cytokines and damage associated molecular patterns (DAMPs), activate anti-tumor immune response, promote cytotoxic T cells to infiltrate tumor tissues and release more GZMA. Starting the GZMA-GSDMB pyroptosis immune cycle cascade may provide an efficient treatment strategy for cancer treatment.

[0004] Wu et al. developed a PD-1 / Her2 bispecific antibody (IBI315) based on this mechanism. This antibody bridges tumor cells expressing her2 and T cells expressing PD-1, on the one hand, blocks the PD-1 / PD-L1 inhibitory signal, and on the other hand, promotes the recruitment and activation of T cells at the tumor site. In a Her2-positive gastric cancer model, the positive feedback loop of “pyroptosis-immune activation” was successfully triggered by releasing GZMA to lyse GSDMB, showing good therapeutic effect. However, the core defect of this strategy is that its effect is highly dependent on the pre-existence and activation of T cells in the tumor microenvironment. In the case of insufficient infiltration of immune cells (especially T cells), this carefully designed “pyroptosis-immune cycle” cascade cannot start by itself due to the lack of initial source of GZMA release. In specific tumor cells such as breast cancer, lung cancer, and liver cancer, the lack of sufficient GSMDB further exacerbates the difficulty of starting the pyroptosis-immune cycle cascade. In order to solve the problem of insufficient expression of GSDMB in tumor tissues and insufficient infiltration of immune cells, Li et al. proposed an innovative strategy, i.e. using electrostatic interaction to encapsulate GSDMB-NT mRNA in liposomes to alleviate the problem of insufficient expression of GSDMB. Exogenous GSDMB-NT mRNA is used to directly initiate cell pyroptosis and activate anti-tumor immune response. The liposome combined with anti-PD-1 treatment can significantly inhibit the growth of multiple tumors. However, since this liposome lacks tumor targeting mechanism, its range of action is not accurate and can cause systemic inflammation and damage to normal cells. Therefore, specifically increasing the expression of GSDMB in tumor cells to obtain sufficient amount of GZMA to precisely initiate tumor cell pyroptosis is still a key problem.

[0005] Interferon-γ (IFN-γ) has been shown to effectively upregulate GSDMB expression in tumor cells, offering a potential solution to overcome its underexpression in malignant tumors. Given that IFN-γ and GZMA are primarily secreted by activated cytotoxic T cells and natural killer (NK) cells, adoptive transfer of exogenous NK cells theoretically represents a strategy to simultaneously increase the levels of these molecules within the local tumor, thereby establishing a GZMA-GSDMB-mediated pyroptosis-immune cycle. However, a large number of exogenous NK cells pose a risk of inducing cytokine release syndrome in vivo, significantly limiting their clinical translation. To address these challenges, NK cell-derived exosomes (NK-exos) have emerged as a promising therapeutic option due to their unique advantages. NK-exos exhibit low toxicity, high biocompatibility, and good safety profiles. Their expressed NK cell-specific receptors confer inherent tumor-targeting capabilities. More importantly, NK-exos are enriched with effector molecules such as IFN-γ and GZMA. This payload enables a dual-activation strategy of targeted delivery to tumor cells. This method achieves precise delivery of effector molecules while effectively avoiding the safety risks associated with adoptive NK cell therapy. However, although NK-exos-initiated GZMA-GSDMB pyroptosis releases damage-related molecular patterns and cytokines to recruit T cells into the tumor microenvironment (TME), it encounters a key "sustainability barrier." This barrier mainly stems from the pervasive immunosuppressive signals within the TME, particularly the PD-L1 / PD-1 pathway. Specifically, the binding of PD-L1 on tumor cells to PD-1 on infiltrating T cells significantly inhibits the cytotoxic activity and effector function of T cells. This inhibition drastically reduces the ability of T cells to release endogenous IFN-γ and GZMA, making it difficult to maintain the initiated GZMA-GSDMB pyroptosis immune cascade.

[0006] Therefore, establishing an efficient and durable anti-tumor immune response is a core challenge currently facing the field. Summary of the Invention

[0007] The purpose of this invention is to address the problem that existing therapies cannot establish a highly efficient GSDMB-mediated pyroptosis immune cycle due to two obstacles: initiation barrier (low GSDMB expression in tumor cells and insufficient GZMA in the microenvironment) and maintenance barrier (PD-1 / PD-L1 pathway-mediated T cell exhaustion). Based on this, this invention proposes an engineered exosome for overcoming the barriers to pyroptosis initiation and maintenance, along with its preparation method and applications. This enhances GSDMB expression in tumor cells and delivers GZMA, cleaving GSDMB to induce pyroptosis. Pyroptotic tumor cells release DMAPs to activate T cells. Furthermore, by blocking PD-1 / PD-L1 with an immune checkpoint inhibitor, T cells are activated to continuously secrete IFN-γ and GZMA, establishing a pyroptosis immune cycle. This invention systematically solves the barriers to pyroptosis initiation (low GSDMB and GZMA) and maintenance (immunosuppression), providing an innovative solution for constructing a highly efficient and durable anti-tumor immune response and overcoming the bottleneck in tumor treatment.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides engineered exosomes for overcoming the pyroptotic immune initiation and maintenance barrier. The engineered exosomes are constructed by loading immune checkpoint inhibitors into exosomes derived from immune cells, and the exosomes from immune cells express IFN-γ and GZMA. In tumor cells, the engineered exosomes overcome the pyroptotic immune initiation and maintenance barrier through the following pathways: (1) Upregulates the expression of Gasdermin B in tumor cells and activates pyroptosis; (2) Blocking the PD-1 / PD-L1 pathway reverses T cell functional exhaustion; (3) Establish a positive pyroptosis immune cycle of granzyme A-interferon γ-Gasdermin B.

[0009] Furthermore, the immune checkpoint inhibitors include BMS202, JQ-1, CA-170, INCB086550, Atezolizumab, Durvalumab, and Avelumab.

[0010] Furthermore, the immune cells include natural killer cells and T cells.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned engineered exosomes, comprising the following steps: (1) Pretreatment of immune cells: Immune cells were co-incubated with drugs that stimulate the expression of interferon-γ for 24 h; (2) Extraction of cell exosomes: Remove the original culture medium, and continue to culture the immune cells obtained in step (1) in serum-free culture medium for 48 h. Collect the cell culture supernatant and extract cell exosomes by ultracentrifugation. (3) Preparation of immune checkpoint inhibitor solution: The immune checkpoint inhibitor is dissolved in DMSO to prepare an immune checkpoint inhibitor solution with a concentration of 10-100 mg / mL and a volume of 1-50 µL. (4) Preparation of engineered exosomes: The immune checkpoint inhibitor solution and the cell exosomes obtained in step (2) were mixed evenly in a PBS buffer system, sonicated under ice bath and then placed in a shaker for 1 h to obtain crude engineered exosomes; (5) Purification of engineered exosomes: Add the crude engineered exosomes obtained in step (4) into a 50 K ultrafiltration tube, 3000 × g, 10 min, repeat 3 times to obtain engineered exosomes; Furthermore, the drug that stimulates interferon-γ expression in step (1) includes one or more of interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18).

[0012] Furthermore, the ultrasound conditions in step (4) are: 20% amplitude, on for 30 seconds, off for 1 minute, repeated 5 times.

[0013] Furthermore, in step (4), the mass ratio of cell exosomes to immune checkpoint inhibitors is 1:1 to 100:1.

[0014] A third aspect of the present invention provides the application of the above-described engineered exosomes in the preparation of antitumor drugs, wherein the tumor is a PD-L1 positive, GSDMB negative tumor, including liver cancer, breast cancer, and non-small cell lung cancer.

[0015] This invention overcomes the barriers to pyroptosis immune initiation and maintenance through the following pathways: (1) Overcoming the initiation barrier: Exogenous endogenous IFN-γ upregulates GSDMB expression in tumor cells, and exogenous GZMA cleaves activated GSDMB, triggering pyroptosis in tumor cells; (2) Breakthrough of maintenance barrier: The co-loaded immune checkpoint inhibitor blocks the PD-L1 / PD-1 pathway in situ, reverses T cell exhaustion and promotes the release of endogenous GZMA; (3) Establishing a positive immune cycle: Pyroptosis cells release antigens to activate T cells, and the activated T cells continuously secrete GZMA / IFN-γ, forming an anti-tumor immune cascade amplification effect.

[0016] Compared with the prior art, the advantages of this invention are as follows: (1) Overcoming the initiation and maintenance barriers: This invention directly triggers pyroptosis by delivering IFN-γ via engineered exosomes to upregulate tumor GSDMB expression and by directly cleaving GSDMB with GZMA. It also targets and delivers immune checkpoint inhibitors via engineered exosomes, blocking the PD-1 / PD-L1 pathway in situ and reversing T cell exhaustion without triggering a systemic inflammatory storm. A self-circulating pyroptosis cascade is constructed, where pyroptotic cells release DAMPs to activate T cells. The activated T cells secrete endogenous IFN-γ / GZMA, amplifying the pyroptosis signal and achieving a high tumor suppression rate in a liver cancer model. Furthermore, CD8+... + T-cell infiltration continued to increase.

[0017] (2) Safety breakthrough: Based on the natural low immunogenicity of NK exosomes and with precise local action, no liver / nephrotoxicity was observed in the PBMC-CDX mouse model.

[0018] (3) The engineered exosomes pioneered in this invention have broken through the triple technical bottleneck of "pyroptosis triggering - immune maintenance - cycle amplification" in tumor immunotherapy, and achieved synergistic breakthroughs in three dimensions: anti-tumor efficacy, immune response persistence and system safety, providing a transformative solution for the treatment of solid tumors. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the engineered exosome mechanism.

[0020] Figure 2 Characterization diagrams of engineered exosomes; (a) Morphological characteristics of exosome NKExo pre-stimulated NK cells and (b) engineered exosome SupNKExo-BMS; (c) Verification of BMS202 encapsulation efficiency in engineered exosomes; (d)~(e) Content of GZMA and IFN-γ in exosomes; (f) Verification diagram of the cleavage ability of engineered exosomes for recombinant GSDMB protein.

[0021] Figure 3 Figure 1 shows the results of in vitro performance tests of engineered exosomes; (a) a typical morphological image of tumor cell pyroptosis induced by exosomes; (b) the amount of lactate dehydrogenase released.

[0022] Figure 4 Effect of engineered exosomes activating in vivo immunity for anti-tumor therapy; (a) CD8 infiltrating tumor tissue. + (a) T cell content detection graph; (b) graph of exosome tumor treatment efficacy.

[0023] Figure 5 The image shows the H&E staining results for in vivo safety testing of engineered exosomes. Detailed Implementation

[0024] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0025] Unless otherwise specified, the methods used in the following embodiments are all conventional techniques.

[0026] Example 1: Engineered exosomes and their preparation method (1) Immunocyte pretreatment: Recombinant proteins interleukin-18 (IL-18) and interleukin-15 (IL-15) were dissolved in PBS buffer (pH=7.4) to prepare IL-18 and IL-15 stock solutions with a concentration of 50 ng / µL, respectively. 1 µL of IL-18 stock solution and 1 µL of IL-15 stock solution were added to each mL of NK cell culture medium to obtain NK cell culture medium containing IL-18 and IL-15. The composition of the NK cell culture medium was MEMα + 0.2 mM Inositol + 0.1 mM β-mercaptoethanol + 0.02 mM Folic Acid + 12.5% ​​horse serum + 12.5% ​​fetal bovine serum. NK cells were seeded in the NK cell culture medium containing IL-18 and IL-15 and cultured at 37 ℃ in a 5% CO2 cell culture incubator for 24 h.

[0027] (2) Extraction of exosomes: The original culture medium was removed, and the above NK cells were cultured in serum-free culture medium (MEMα + 0.2 mM Minositol + 0.1 mM β-mercaptoethanol + 0.02 mM Folic Acid) at 37 ℃ in a 5% CO2 cell culture incubator for 48 h. The supernatant of the culture medium was collected and centrifuged for 70 min (12000 × g, 4℃). After centrifugation, the precipitate was collected and resuspended in PBS buffer solution, which is the pre-stimulated NK cell exosome (SupNKExo). The concentration was further quantified with BCA and adjusted to 5 mg / mL in a volume of 200 µL.

[0028] (3) Preparation of engineered exosomes: BMS202 was dissolved in DMSO to prepare a concentration of 100 mg / mL. 200 µL of pre-stimulated NK cell exosomes with a concentration of 5 mg / mL and 1 µL of BMS202 with a concentration of 100 mg / mL were mixed evenly in PBS buffer solution (pH=7.4), and the volume was adjusted to 250 µL by adding PBS buffer solution. The mixture was placed in a cell sonicator and sonicated under the following conditions: 20% amplitude, 30 s on, 1 min off, repeated 5 times, and ice bath. After sonication, the mixture was placed in a shaker at 37℃ and reacted for 1 h to obtain crude engineered exosomes. The crude engineered exosomes were added to a 50 K ultrafiltration tube, heated at 3000 × g for 10 min, and repeated 3 times to obtain purified engineered exosomes (SupNKExo-BMS).

[0029] Morphological characterization results of SupNKExo exosomes and SupNKExo-BMS exosomes are as follows: Figure 2 As shown in a and 2b, the ultrasound process did not disrupt the morphology of SupNKExo-BMS exosomes, such as Figure 2 As shown in Figure c, the content of BMS202 in engineered exosomes was detected by high performance liquid chromatography, proving that engineered exosomes encapsulate BMS202 within the cavity and retain the morphological characteristics of the exosomes.

[0030] To investigate the effect of prestimulation on NK cells, four groups were set up: NKExo, NKExo-BMS, SupNKExo, and SupNKExo-BMS. The NKExo group used NKExo exosomes, the NKExo-BMS group used NKExo-BMS exosomes, the SupNKExo group used prestimulated NK cell exosomes prepared in step (2), and the SupNKExo-BMS group used purified engineered exosomes SupNKExo-BMS prepared in step (3).

[0031] The extraction steps for exosomes from the NKExo group cells are as follows: NK cells were cultured in serum-free medium (MEMα + 0.2 mM Inositol + 0.1 mM β-mercaptoethanol + 0.02 mM Folic Acid) at 37 ℃ in a 5% CO2 cell culture incubator for 48 h. The supernatant was collected and centrifuged for 70 min (12000 × g, 4 ℃). After centrifugation, the pellet was collected and resuspended in PBS buffer to obtain NK cell exosomes (NKExo). The concentration was quantified using BCA and adjusted to 5 mg / mL in a volume of 200 µL.

[0032] The preparation steps of the above NKExo-BMS are as follows: BMS202 was dissolved in DMSO to prepare a concentration of 100 mg / mL. 200 µL of NKExo (5 mg / mL) and 1 µL of BMS202 (100 mg / mL) were mixed thoroughly in PBS buffer (pH=7.4), and the volume was adjusted to 250 µL with PBS buffer. The mixture was placed in a cell sonicator under the following conditions: 20% amplitude, 30 s on, 1 min off, repeated 5 times, and kept on ice. After sonication, the mixture was placed in a shaker at 37°C and reacted for 1 h to obtain crude engineered exosomes. The crude engineered exosomes were added to a 50 K ultrafiltration tube and incubated at 3000 × g for 10 min, repeated 3 times to obtain purified engineered exosomes (NKExo-BMS).

[0033] The levels of GZMA and IFN-γ in the four groups of exosomes were detected by ELISA, and the results are as follows: Figure 2 As shown in d and 2e, the results indicate that prestimulation significantly increased the levels of GZMA and IFN-γ in exosomes. A 0.5 µg / µL GSDMB recombinant protein solution and a 5 mg / mL SupNKExo-BMS solution were prepared using PBS buffer. 1 µL of the GSDMB recombinant protein solution and 20 µL of the SupNKExo-BMS solution were mixed thoroughly and reacted at 37°C for 1 h. The reaction products were then subjected to SDS-PAGE electrophoresis. The electrophoresis image showed a GSDMB-NT band, demonstrating the cleavage effect of engineered exosomes on the recombinant GSDMB protein. The results are shown in [Figure 1]. Figure 2 f. Engineered exosomes exhibit cleavage of recombinant GSDMB protein.

[0034] Example 2: Application of engineered exosomes in tumor cells Using Huh-7 as the verification model, the Control group, BMS202 group, NKExo group, NKExo-BMS group, SupNKExo group, and SupNKExo-BMS group were set up. The control group was the untreated group. The BMS202 group received the immune checkpoint inhibitor BMS202 alone; specifically, BMS202 was dissolved in DMEM medium to a final concentration of 1.5 µg / mL, and Huh-7 cells were then seeded and co-cultured in this medium. The NKExo group used NKExo; specifically, NKExo was dissolved in PBS buffer to a concentration of 5 mg / mL, and 40 µL was added to DMEM medium to a final exosome concentration of 200 µg / mL, and Huh-7 cells were then seeded and co-cultured in this medium. The NKExo-BMS group used NKExo-BMS; specifically, NKExo-BMS was dissolved in PBS buffer to a concentration of 5 mg / mL, and 40 µL was added to DMEM medium to a final exosome concentration of 200 µg / mL. The Huh-7 cell group was co-cultured with the above-mentioned medium after being seeded with 5 mg / mL SupNKExo. The SupNKExo group used SupNKExo, specifically prepared by dissolving SupNKExo in PBS buffer to a concentration of 5 mg / mL, adding 40 µL to DMEM medium to achieve a final exosome concentration of 200 µg / mL, and then seeding Huh-7 cells in the same medium for co-culture. The SupNKExo-BMS group used SupNKExo-BMS, specifically prepared by dissolving SupNKExo-BMS in PBS buffer to a concentration of 5 mg / mL, adding 40 µL to DMEM medium to achieve a final exosome concentration of 200 µg / mL, and then seeding Huh-7 cells in the same medium for co-culture. All groups were co-cultured with Huh-7 cells at 37 ℃ in a 5% CO2 cell culture incubator for 12 h. Typical cell morphology was observed using confocal microscopy. Results are as follows: Figure 3 As shown in figure a, it is demonstrated that SupNKExo-BMS induces pyroptosis in tumor cells.

[0035] Lactate dehydrogenase (LDH) was detected using a lactate dehydrogenase release assay kit. The culture wells were divided into the following groups: cell-free culture medium wells (background blank control wells), a control group, a control group for subsequent lysis (sample maximum enzyme activity control wells), and BMS202, NKExo, NKExo-BMS, SupNKExo, and SupNKExo-BMS groups. Exosomes were prepared to a concentration of 200 µg / mL using serum-free DMEM, and BMS202 was prepared to a concentration of 1.5 µg / mL using serum-free DMEM (preparation process as above). The cells were co-cultured with Huh-7 cells at 37 °C, 5% CO2 for 24 h. One hour before the scheduled detection time, the cell culture plates were removed from the cell culture incubator, and the LDH release reagent provided in the kit was added to the "sample maximum enzyme activity control wells" at a volume of 10% of the original culture medium. After adding the lactate dehydrogenase release reagent, mix thoroughly by repeatedly pipetting several times, and then continue incubation in a cell culture incubator. After 1 h, centrifuge the cell culture plate at 400g for 5 min using a multi-well centrifuge. Take 120 μl of the supernatant from each well and add it to the corresponding well of a new 96-well plate, then add 60 μl of lactate dehydrogenase detection working solution to the sample. Mix well and incubate at room temperature in the dark for 30 min. Then measure the absorbance at 490 nm. The high lactate dehydrogenase release in the SupNKExo group and the SupNKExo-BMS group further demonstrates that engineered exosomes induce pyroptosis in tumor cells (…). Figure 3 b).

[0036] Example 3: Application of engineered exosomes in liver cancer A PBMC-CDX model was constructed using Huh-7 and PBMCs. The specific steps involved setting a concentration of 2.5 × 10⁻⁶ ppm. 7 PBMCs at a concentration of 100 µL (cells / mL) were injected into NGS mice via the tail vein. Seven days later, a concentration of 1×10⁻⁶ cells / mL was added to the PBMCs. 7100 µL of Huh-7 cells / mL were subcutaneously seeded into the back of the NGS mice. Drug administration via tail vein began 14 days later. The mice were divided into four groups: Control, BMS202, NKExo, NKExo-BMS, SupNKExo, and SupNKExo-BMS. The Control group was untreated; the BMS202 group received the immune checkpoint inhibitor BMS202 alone; the NKExo group received NKExo; the NKExo-BMS group received NKExo-BMS; the SupNKExo group received SupNKExo; and the SupNKExo-BMS group received SupNKExo. Dosage was determined based on mouse body weight. The Control group received 100 µL of PBS buffer, and exosomes from each group were dissolved in PBS buffer to a concentration of 100 µg / g body weight (based on mouse body weight), in 100 µL volumes. The BMS202 group was administered at a concentration of 0.75 µg / g, in a volume of 100 µL. It was injected via tail vein once every other day for a total of three injections. Tumor tissue was collected after 32 days. Figure 4 Flow cytometry was used to detect immune cells in tumor tissue. The results showed that engineered exosomes could promote T cell infiltration into the tumor microenvironment and had a good therapeutic effect on tumors.

[0037] After the experiment, major tissues from the model mice were collected and stained with H&E, such as... Figure 5 As shown, no significant damage was observed in the major tissues of mice in each group, demonstrating the safety of engineered exosomes.

[0038] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An engineered exosome for overcoming the pyroptosis immune initiation and maintenance barrier, characterized in that: The engineered exosomes are constructed by loading immune checkpoint inhibitors into exosomes derived from immune cells, and the exosomes from immune cells express interferon-γ and granzyme A; in tumor cells, the engineered exosomes overcome the pyroptosis immune initiation and maintenance barriers through the following pathways: (1) Upregulates the expression of Gasdermin B in tumor cells and activates pyroptosis; (2) Blocking the PD-1 / PD-L1 pathway reverses T cell functional exhaustion; (3) Establish a positive pyroptosis immune cycle of granzyme A-interferon γ-Gasdermin B.

2. The engineered exosomes according to claim 1, characterized in that: The immune checkpoint inhibitors include BMS202, JQ-1, CA-170, INCB086550, Atezolizumab, Durvalumab, and Avelumab.

3. The engineered exosomes according to claim 1, characterized in that: The immune cells include natural killer cells and T cells.

4. The method for preparing engineered exosomes according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Pretreatment of immune cells: Immune cells were co-incubated with drugs that stimulate the expression of interferon-γ for 24 h; (2) Extraction of cell exosomes: Remove the original culture medium, continue to culture the immune cells obtained in step (1) in serum-free culture medium for 48 h, collect the cell culture supernatant, and extract cell exosomes by ultracentrifugation. (3) Preparation of immune checkpoint inhibitor solution: The immune checkpoint inhibitor is dissolved in DMSO to prepare an immune checkpoint inhibitor solution with a concentration of 10-100 mg / mL and a volume of 1-50 µL. (4) Preparation of engineered exosomes: The immune checkpoint inhibitor solution and the cell exosomes obtained in step (2) were mixed evenly in a PBS buffer system, sonicated under ice bath and then placed in a shaker for 1 h to obtain crude engineered exosomes; (5) Purification of engineered exosomes: Add the crude engineered exosomes obtained in step (4) into a 50 K ultrafiltration tube, 3000 × g, 10 min, repeat 3 times to obtain engineered exosomes.

5. The preparation method according to claim 4, characterized in that: The drugs that stimulate interferon-γ expression in step (1) include one or more of interleukin-12, interleukin-15, and interleukin-18; According to the preparation method of claim 4, the ultrasonic conditions in step (4) are: 20% amplitude, on for 30 seconds, off for 1 minute, repeated 5 times.

6. The preparation method according to claim 4, characterized in that: In step (4), the mass ratio of exosomes to immune checkpoint inhibitors is 1:1 to 100:

1.

7. The use of engineered exosomes as described in any one of claims 1 to 3 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that: The tumors are PD-L1 positive and GSDMB negative, including liver cancer, breast cancer, and non-small cell lung cancer.

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