Induced pluripotent stem cell-derived exosomes with anti-tumor activity and their application in enhancing car-t cell therapy

Abstract

This invention belongs to the field of biomedical technology and discloses exosomes derived from induced pluripotent stem cells (iPSCs) with anti-tumor activity and their application in enhancing CAR-T cell therapy. This invention is the first to discover that iPSC-derived exosomes possess inherent anti-tumor activity by directly inhibiting tumor cell proliferation, inducing apoptosis, and inhibiting migration, with a yield hundreds of times higher than that of traditional cell-derived exosomes. Through genetic engineering, the surface of the exosomes is made to display a bispecific single-chain variable fragment against PD-1 / MSLN and loaded with indole-3-propionic acid, constructing a multifunctional therapeutic platform. This platform, administered via nebulized inhalation, achieved a 66.7% complete remission rate, a 100% 80-day survival rate, and an 83.3% resistance to tumor re-challenge in an orthotopic lung cancer model when combined with CAR-T therapy, significantly outperforming existing treatment strategies. This invention provides an innovative enhancement strategy for CAR-T cell therapy in solid tumors and has significant clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to exosomes derived from induced pluripotent stem cells with anti-tumor activity and their application in enhancing CAR-T cell therapy. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has achieved revolutionary success in the treatment of hematological malignancies, with complete remission rates of 80-90% in B-cell acute lymphoblastic leukemia and 40-70% in refractory multiple myeloma. However, the efficacy of CAR-T therapy in solid tumors is extremely limited. Pooled data from multiple clinical trials showed an overall response rate of only 9% in 262 patients. This disparity is particularly pronounced in lung cancer, where, despite 69% of adenocarcinomas expressing targetable antigens such as mesothelin (MSLN), the efficacy of CAR-T therapy is negligible.

[0003] Biological barriers limiting the efficacy of CAR-T therapy in solid tumors include: (1) T cell depletion; (2) less than 5% of CAR-T cells reaching the tumor site; and (3) an immunosuppressive tumor microenvironment. Current combination therapy strategies, such as those using immune checkpoint inhibitors or bispecific T-cell connectives (BiTEs), have shown promise but still face many limitations.

[0004] Exosomes, as natural nanoscale extracellular vesicles, possess excellent biocompatibility and targeted delivery capabilities. However, traditional exosome sources (such as T cells, mesenchymal stem cells, and tumor cells) suffer from low yields. While exosomes derived from embryonic stem cells (ESCs) exhibit high yields and anti-tumor effects, they face ethical and sourcing limitations.

[0005] Induced pluripotent stem cells (iPSCs) are functionally similar to ESCs and can be mass-produced and genetically engineered. However, there are currently no reports on the inherent antitumor activity of iPSC-derived exosomes, and their potential application value in cancer treatment has not yet been recognized and developed. Summary of the Invention

[0006] This invention reports on the inherent antitumor activity of iPSC-derived exosomes and their potential application value in tumor treatment.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention claims protection for the use of exosomes derived from induced pluripotent stem cells in the preparation of agents for the prevention and / or treatment of tumors.

[0009] Furthermore, the induced pluripotent stem cell-derived exosomes exhibit antitumor activity by directly inhibiting tumor cell proliferation, inducing apoptosis, and inhibiting migration.

[0010] Furthermore, the induced pluripotent stem cell exosomes are obtained by the following method: culturing induced pluripotent stem cells in cell culture medium, and extracting the induced pluripotent stem cell exosomes from the supernatant of the induced pluripotent stem cell culture medium; the cell culture medium is a serum-free cell culture medium.

[0011] Furthermore, the induced pluripotent stem cells are derived from mouse or human sources.

[0012] This invention also claims protection for the use of engineered induced pluripotent stem cell-derived exosomes in the preparation of antitumor and / or therapeutic agents for tumors.

[0013] The present invention also provides an engineered induced pluripotent stem cell-derived exosome, wherein the surface of the exosome displays a bispecific single-chain variable fragment against PD-1 / MSLN, and the exosome is internally loaded with indole-3-propionic acid.

[0014] Preferably, the anti-PD-1 / MSLN bispecific single-stranded variable fragment has a nucleotide sequence as shown in SEQ ID NO: 3.

[0015] Preferably, the anti-PD-1 single-stranded variable fragment displayed on the exosome surface has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] A nucleic acid molecule encoding a single-stranded variable fragment for anti-PD-1 / MSLN bispecificity, the nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 3.

[0017] A nucleic acid molecule encoding a single-stranded variable fragment against PD-1, said nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0018] This invention further protects the expression vector of the aforementioned nucleic acid molecule, the host cell transformed from the expression vector, and the application of the aforementioned nucleic acid molecule, expression vector, or host cell in the preparation of engineered induced pluripotent stem cell-derived exosomes.

[0019] This invention also provides a method for preparing the engineered induced pluripotent stem cell-derived exosomes, comprising the following steps:

[0020] 1) Construct a lentiviral vector expressing PD-1 / MSLN bispecific scFv;

[0021] 2) Lentiviral transduction of iPSCs, screening for stable expression strains;

[0022] 3) Cultivate engineered iPSCs and collect the culture supernatant;

[0023] 4) Centrifugation to separate and purify exosomes;

[0024] 5) Electroporation loading of indole-3-propionic acid.

[0025] Furthermore, the present invention also claims the use of the engineered induced pluripotent stem cell-derived exosomes in the preparation of antitumor drugs.

[0026] Furthermore, the tumor is a solid tumor. More specifically, the solid tumor is lung cancer.

[0027] Furthermore, the antitumor drug is administered via nebulized inhalation.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] The first discovery that iPSC-derived exosomes possess inherent antitumor activity: This is the most important innovation of this invention. iPSC exosomes can directly inhibit tumor cell proliferation (dose-dependent), induce apoptosis, inhibit migration, and downregulate the expression of proliferation and metastasis-related genes.

[0030] Significant yield advantage: iPSC exosome yield is hundreds of times higher than that of traditional cell sources, solving the yield bottleneck for clinical application of exosomes.

[0031] Multimodal synergistic anti-tumor activity: combines multiple mechanisms such as the inherent anti-tumor activity of exosomes, bispecific T cell bridging, checkpoint blockade, and metabolic reprogramming.

[0032] Significantly improves CAR-T efficacy: In an orthotopic lung cancer model, the combination of BIEXO@IPA and CAR-T achieved a complete remission rate of 66.7%, an 80-day survival rate of 100%, and 83.3% resistance to tumor re-challenge.

[0033] Good biocompatibility: no obvious toxic side effects and no tumorigenic risk. Attached Figure Description

[0034] Figure 1 This is a diagram showing the preparation process and physicochemical characterization of engineered exosomes BIEXO@IPA in this embodiment of the invention. Figure 1 a: A schematic diagram of the gene construction of the anti-PD-1 / MSLN bispecific single-chain antibody (scFv) on the iPSC surface, showing the linking sequence of the signal peptide, PD-1 scFv, linker peptide, MSLN scFv, PDGFR transmembrane domain and His tag. Figure 1b: Morphological image of BIEXO exosomes observed under a transmission electron microscope (TEM), showing that they have a typical saucer-like membrane vesicle structure, with a scale bar of 50 nm. Figure 1 c: Exosome particle size distribution determined by nanoparticle tracking analysis (NTA), showing that the particle size is mainly distributed in the range of 30-150 nm. Figure 1 d: Western blot analysis, showing the expression of exosome characteristic marker proteins (CD9, CD63, TSG101) by engineered exosome BIEXO. Figure 1 e: A bar chart of zeta potentials for various exosomes (iPSC-Exo, MiPSC-Exo, PiPSC-Exo, BiPSC-Exo), with values ​​between -20mV and -30mV, which is conducive to mucus penetration. Figure 1 f: Western blotting analysis of His-tagged protein expression in exosome lysate confirmed successful bispecific scFv expression. Figure 1 g: Immunofluorescence images of His tags (green fluorescence) on the surface of exosomes were observed using confocal microscopy, confirming that scFv was successfully displayed on the surface of exosomes. Figure 1 h: Particle size stability changes of BIEXO after incubation in 10% serum buffer for different times (0-7 days). Figure 1 i: Flow cytometry was used to detect the retention rate of scFv (His tag) on ​​the surface of BIEXO after incubation in serum for 7 days, showing that it has good stability. Figure 1 j: Standard curve of indole-3-propionic acid (IPA) by ultraviolet spectrophotometry. Figure 1 k: Line graphs of encapsulation efficiency and loading capacity at different IPA concentrations, determining 100 μg / mL as the optimal dosing concentration. Figure 1 l: The cumulative drug release curve of BIEXO@IPA in an in vitro simulated physiological environment (PBS, pH 7.4, 37°C) shows that it has sustained-release properties.

[0035] Figure 2 Characterization and antitumor properties of iPSC-derived exosomes (IEXO). Figure 2 a: A bar chart comparing the exosomal protein production of iPSCs with Lewis lung cancer (LLC) cells, T cells, and mesenchymal stem cells (MSCs), showing that iPSCs have a significant production advantage. Figure 2 b: Evaluation of growth inhibition rate after co-incubation of IEXO (100 μg / mL) with various tumor cell lines for 24 hours. Figure 2c: The dose-dependent inhibition curve of IEXO on cell proliferation after co-incubation with LLC cells for 24 hours. Figure 2 de: Analysis diagram of cell apoptosis detected by Annexin V / PI staining method, where (d) is a representative flow cytometry scatter plot and (e) is a quantitative statistical graph of apoptosis rate. Figure 2 fg: Scratch healing assay to detect the inhibition of LLC cell migration ability by IEXO, where (f) is a representative microscopic image and (g) is a quantitative analysis of the healing rate of the migration area. Figure 2 h: Immunofluorescence analysis of changes in the expression of vimentin and cleaved caspase-3 in LLC cells after IEXO treatment. Figure 2 i: A statistical graph showing the expression levels of proliferation and migration-related genes in LLC cells after IEXO treatment, analyzed by qRT-PCR using fibroblast exosomes (FEXO) as a control. Figure 2 jl: In vivo therapeutic efficacy evaluation of IEXO in a subcutaneous LLC tumor-bearing mouse model, including tumor volume growth curve over time (j, k) and final weight statistics of tumors removed on day 15 (l). Figure 2 m,n: In vivo therapeutic efficacy evaluation of human iPSC exosomes in a subcutaneous A549 tumor-bearing nude mouse model, including tumor volume growth curve over time (m) and final weight statistics of tumors removed on day 15 (n).

[0036] Figure 3 This diagram illustrates the in vitro targeting binding ability, cell bridging function, and antitumor activity of BIEXO in this embodiment of the invention. Figure 3 a: The binding affinity curve of BIEXO to recombinant MSLN protein was detected by ELISA and showed a concentration-dependent relationship. Figure 3 b: ELISA assay curve of the binding affinity of BIEXO to recombinant PD-1 protein. Figure 3 c: Flow cytometry was used to detect the binding of BIEXO to 293T cells that highly express PD-1. The right side shows the statistical graph of mean fluorescence intensity (MFI). Figure 3 d: Flow cytometry was used to detect the binding of BIEXO to 293T cells that highly express MSLN. The right side shows the MFI statistics. Figure 3 e: BIEXO (green) targeting and binding to PD-1 as captured by confocal microscopy + Image of T cells (red). Figure 3 f: Image of BIEXO (green) targeting and binding LLC-MSLN tumor cells (red) by confocal microscopy. Figure 3g: Confocal microscopy observation of BIEXO-mediated PD-1 + Images of intercellular physical bridging between T cells and LLC-MSLN tumor cells, and quantitative analysis of the corresponding contact distance. Figure 3 h: A bar graph of cell bridging efficiency detected by FRET technology shows that energy transfer efficiency increases with increasing BIEXO concentration, confirming that the distance between cells is shortened. Figure 3 ij: A bar chart of T cell killing activity detected by LDH release assay shows that BIEXO can reverse PD-L1-induced T cell exhaustion and restore the killing effect on tumor cells. Figure 3 k: Schematic diagram of the experimental procedure for in vitro T cell induction and differentiation and drug treatment. Figure 3 l: Flow cytometry detection of PD-1 + CD8 + Histogram of T cell stem cell marker TCF-1 expression levels shows increased expression in the IPA-treated group. Figure 3 m: Line graph showing the inhibition rate of LLC-MSLN cell proliferation in vitro by different treatment groups (PBS, IEXO, BIEXO, etc.) as detected by CCK-8 assay. Figure 3 n: Simulated tumor microenvironment (TILs) + The LDH release cytotoxicity assay in the co-culture system of tumor cells showed that the BIEXO@IPA group had the strongest killing effect.

[0037] Figure 4 This is a diagram showing the in vivo biodistribution and cellular uptake of BIEXO@IPA after nebulized inhalation administration in an embodiment of the present invention. Figure 4 ad: In vivo IVIS images (a, c) and quantitative fluorescence intensity analysis of major organs (heart, liver, spleen, lung, kidney) at different time points (2-48 h) after intravenous injection (iv) and nebulized inhalation (inh.) of DiR-labeled exosomes in mice confirmed that nebulized inhalation significantly increased lung accumulation. Figure 4 ef: Quantitative graph of lung fluorescence intensity after nebulized inhalation of different doses of BIEXO@IPA, showing dose-dependent accumulation. Figure 4 g: Comparison of uptake efficiency of different formulations (Lipo@IPA, IEXO@IPA, BIEXO@IPA) by total lung cells as detected by flow cytometry. Figure 4 hj: Pie chart showing the distribution ratio of different formulations in various cell subpopulations (tumor cells, macrophages, epithelial cells, and DC cells) in the lungs, indicating that BIEXO@IPA has the highest enrichment in tumor cells. Figure 4kn: Flow cytometry analysis of the bar chart showing the uptake positivity rate of each cell subset (LLC-MSLN, macrophages, epithelial cells, and DC cells) confirmed that BIEXO@IPA has excellent tumor cell-specific targeting ability (>79%).

[0038] Figure 5 This is a diagram illustrating the therapeutic effect and immune microenvironment remodeling analysis of BIEXO@IPA in an in situ lung cancer model in this embodiment of the invention. Figure 5 a: Schematic diagram of the timeline for the establishment of the orthotopic lung cancer mouse model and BIEXO@IPA nebulization therapy. Figure 5 b: IVIS imaging of Luciferase bioluminescence intensity in mouse lung tumors at different time points during treatment, visually showing tumor growth. Figure 5 c: Corresponding Figure 5 The quantitative curve of tumor bioluminescence intensity (b) shows that tumor growth in the BIEXO@IPA group was significantly inhibited. Figure 5 d: Statistical graph of lung tumor volume / weight in mice in each group at the experimental endpoint. Figure 5 e: Kaplan-Meier survival curves of mice in each treatment group, showing that the survival time of the BIEXO@IPA group was significantly prolonged. Figure 5 fg: Flow cytometry detection of CD8 in tumor tissue + Scatter plot and bar chart of T cell infiltration frequency. Figure 5 Hi: CD8 in tumor tissue sections + Immunofluorescence staining and quantitative analysis of T cells (green) confirmed increased T cell infiltration. Figure 5 j: Flow cytometry detection of PD-1 + CD8 + The statistical graph of TCF-1 expression level in T cells suggests enhanced T cell stemness. Figure 5 km: Flow cytometry detection of regulatory T cells (Treg, CD4) in tumor tissue + FoxP3 + Scatter plots and statistical graphs of the proportions show that Treg in the BIEXO@IPA group is significantly reduced. Figure 5 nr: Flow cytometry plot of the expression of other immune cell subsets (NK cells, macrophages, DC cells, etc.) and their functional molecules (IFN-γ, TNF-α) in the tumor microenvironment.

[0039] Figure 6 This is a biosafety evaluation diagram of BIEXO@IPA in an embodiment of the present invention. Figure 6 a: Curves showing the changes in body weight of mice in each group during treatment. Figure 6b: Bar chart of cytotoxicity of BIEXO@IPA against normal human hepatocytes (L02), lung epithelial cells (BEAS-2B), and kidney cells (HK2) as determined by the CCK-8 assay. Figure 6 cf: At the end of treatment, the results of serum biochemical indicators (alkaline phosphatase ALP, alanine aminotransferase ALT, blood urea nitrogen BUN, creatinine Cr) in mice were all within the normal range. Figure 6 gi: Results of detection of routine blood parameters (white blood cells WBC, red blood cells RBC, platelets PLT) in mice. Figure 6 j: H&E pathological sections of major organs (heart, liver, spleen, lung, and kidney) of mice, with no obvious tissue damage observed. Figure 6 ko: Graph showing the levels of tumor markers (AFP, CEA, MUC1, CA125, CA19-9) in mouse serum, confirming that iPSC-derived exosomes are non-tumorigenic.

[0040] Figure 7 This is a diagram illustrating the efficacy of BIEXO@IPA combined with CAR-T cell therapy for orthotopic lung cancer and metastatic tumors in an embodiment of the present invention. Figure 7 a: Images of T cells transduced with anti-MSLN CAR and mCherry reporter gene observed under a fluorescence microscope. Figure 7 b: Flow cytometry was used to detect the positive transduction rate of CAR molecules on the surface of T cells. Figure 7 c: Schematic diagram of the experimental protocol and timeline of BIEXO@IPA combined with CAR-T therapy. Figure 7 de: IVIS imaging and quantitative curve of bioluminescence of lung tumors in mice in the combined treatment group, showing that the combined group achieved the best tumor inhibition effect (CR rate 66.7%). Figure 7 f: Kaplan-Meier survival curves of mice in the combined treatment group, showing a 100% long-term survival rate. Figure 7 gi: Immunological analysis of the tumor microenvironment after combined therapy, showing CD8 + Increased T cells, decreased Treg cells, and elevated TCF-1 expression in CAR-T cells. Figure 7 j: Immunofluorescence staining was used to observe the deep infiltration of CAR-T cells (Myc tag, yellow) in tumor tissue. Figure 7 k: Curves showing the change in the proportion of CAR-T cells persisting in peripheral blood at different time points after treatment. Figure 7 lm: A statistical graph showing the ability of tumor-infiltrating CAR-T cells to secrete IFN-γ and TNF-α by flow cytometry. Figure 7 No: IVIS imaging and quantitative map of the Rechallenge experiment confirmed the establishment of immune memory. Figure 7pq: Flow cytometry analysis of the proportion of memory T cells (central memory Tcm, effector memory Tem) in cured mice. Figure 7 ru: Treatment outcome in the B16F10-MSLN lung metastasis model, including lung appearance (r), metastatic nodule count (s), flow cytometry analysis of residual lung tumor cells (t), and lung tissue weight (u).

[0041] Figure 8 This document presents a summary of the preparation process, engineering strategies, and overall protocol for combining induced pluripotent stem cell (iPSC)-derived exosomes with CAR-T cells for the treatment of lung cancer. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to embodiments and comparative examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0044] Example 1: Preparation, yield advantage, and verification of inherent antitumor activity of iPSC-derived exosomes

[0045] 1. iPSC Culture and Exosome Isolation and Purification: After resuscitation, iPSCs derived from C57BL / 6 mice were maintained on gelatin-coated culture plates and expanded using Oricell mESC serum-free medium (Cyagen). When the cell confluence reached 70%-80%, the culture supernatant was collected. Exosomes were isolated using differential ultracentrifugation: (1) The supernatant was centrifuged sequentially at 800×g for 5 minutes, 2,000×g for 10 minutes, and 10,000×g for 30 minutes to remove cells, dead cells, and cell debris; (2) The supernatant was filtered through a 0.22 μm sterile filter membrane; (3) The filtrate was ultracentrifuged at 100,000×g for 90 minutes at 4°C; (4) The supernatant was discarded, the precipitate was resuspended in PBS, and washed again by ultracentrifugation under the same conditions to finally obtain iPSC-derived exosomes (IEXO).

[0046] 2. Yield analysis used a nanoparticle tracking analyzer to determine exosome concentration. For example... Figure 2As shown in figure a, iPSCs exhibit superior exosome production capacity, with exosome production per 10^7 cells over 48 hours approximately 387.7 times that of Lewis lung cancer (LLC) cells, 537.2 times that of T cell-derived exosomes, and 14.8 times that of mesenchymal stem cell (MSC)-derived exosomes. This result confirms the scalability advantages of iPSCs as exosome production factories.

[0047] 3. Verification of Intrinsic Antitumor Activity This example systematically verified the intrinsic antitumor activity of IEXO for the first time: (1) Inhibition of Proliferation: LLC-MSLN cells were co-cultured with different concentrations of IEXO (0-100 μg / mL) for 24 hours. CCK-8 assay showed that IEXO significantly inhibited tumor cell proliferation in a dose-dependent manner (see...). Figure 2 bc). (2) Induction of apoptosis and inhibition of migration: Flow cytometry results showed that the apoptosis rate of tumor cells in the IEXO treatment group was significantly increased (see Figure 2 (de); Transwell migration assay and scratch assay showed that IEXO significantly inhibited the migration ability of LLC cells (see de); Figure 2 Western blot analysis further confirmed that IEXO treatment downregulated vimentin expression while upregulating the expression of the apoptosis marker cleaved caspase-3 (see fg). Figure 2 (3) Transcriptome regulation: Transcriptome sequencing analysis showed that IEXO treatment significantly downregulated the expression of genes related to proliferation and metastasis in tumor cells (see h). Figure 2 i). (4) In vivo efficacy: In the C57BL / 6 mouse subcutaneous LLC tumor model, intratumoral injection of IEXO significantly reduced tumor volume and tumor weight (see i). Figure 2 (5) In vivo antitumor effect of human IPSC exosomes: In a nude mouse subcutaneous A549 lung cancer model, intratumoral injection of human IEXO significantly inhibited tumor growth, and the tumor volume and weight were significantly reduced compared with the PBS control group (see jl). Figure 2 m,n).

[0048] Example 2: Construction and characterization of engineered bispecific exosomes (BIEXO)

[0049] 1. Genetically engineered lentiviral vectors expressing anti-PD-1 / MSLN bispecific single-chain antibody (scFv) were constructed. (1) Vector design: Anti-human MSLN scFv (derived from P4 antibody) and anti-mouse PD-1 scFv (derived from RMP1-14 antibody) were tandemly linked by a flexible linker (GGGGS)3. To achieve exosome surface display, the fusion gene was fused with the transmembrane domain of platelet-derived growth factor receptor (PDGFR), and a His tag was added to the C-terminus for detection (see [link]). Figure 1 a). (2) Virus preparation and transduction: The pCDH-EF1 expression vector constructed above and the packaging plasmids (pMD2.G and psPAX2) were transfected into 293T cells using PEI at a molar ratio of 4:1:3. Viral supernatant was collected and iPSCs were infected with MOI=5. The engineered iPSC strain (BiPSC) stably expressing bispecific antibodies was obtained by screening with puromycin (2 μg / mL). Three sequences were constructed, and their nucleotide sequences are shown in SEQ ID NO:1 to SEQ ID NO:3. SEQ ID NO:1 displays an anti-PD-1 single-chain antibody on its surface and is fused with mCherry fluorescent protein, which can be used to track the distribution, cell uptake and in vivo tracking of exosomes by fluorescence microscopy or flow cytometry. It stably displays scFv on the surface of exosomes through the PDGFR transmembrane domain, which is suitable for T cell tracking and exosome-immune cell interaction studies. SEQ ID NO: 2 is identical to SEQ ID NO: 1, displaying an anti-PD-1 single-chain antibody on its surface. The difference is that it carries a His tag for easier detection and purification: it can be detected by Western blotting, immunofluorescence, flow cytometry, or affinity purification using the anti-His antibody. SEQ ID NO: 3 is a bispecific targeting sequence, targeting PD-1 on the surface of T cells via anti-PD-1 scFv and mesothelin (MSLN) on the surface of tumor cells via anti-MSLN scFv. By simultaneously binding to T cells and tumor cells, it brings them closer together, promoting T cell recognition and killing of tumors.

[0050] 2. Preparation and Characterization of BIEXO: Engineered exosomes (BIEXO) were isolated from BiPSC supernatant according to the method described in Example 1. (1) Physicochemical Properties: Transmission electron microscopy (TEM) showed that BIEXO exhibited a typical saucer-like membrane vesicle structure (see Figure 1 b); NTA showed that its particle size distribution was between 30-150 nm (see...). Figure 1 c); Zeta potential measurements showed that it carried a negative charge (-20 to -30 mV), which facilitated penetration into lung mucus (see...). Figure 1e). (2) Surface modification verification: His-tagged protein was detected by both Western blotting and immunofluorescence staining, confirming that the bispecific scFv was successfully displayed on the surface of exosomes (see Figure 1 (3) Stability: After incubation in a buffer containing 10% serum for 7 days, flow cytometry analysis showed that >90% of scFv expression was still retained on the surface of BIEXO, and the particle size did not change significantly, indicating that it has excellent stability (see fg). Figure 1 hi).

[0051] Example 3: Preparation and drug release of BIEXO@IPA loaded with a metabolic regulator

[0052] 1. Electroporation loading process: The immunomodulator indole-3-propionic acid (IPA) was loaded into BIEXO using electroporation technology. (1) Procedure: BIEXO (100 μg / mL), IPA (100 μg / mL), and trehalose (25 mM) were mixed in PBS. The mixture was placed in an electroporation cuvette and electroporated using a Gene Pulser Xcell system (Bio-Rad) with the following parameters: voltage 350 V, capacitance 125 μF, and resistance 400 Ω. (2) Purification: After electroporation, the mixture was washed three times by ultracentrifugation to remove unencapsulated free IPA.

[0053] 2. Loading efficiency and release kinetics (1) Loading optimization: By UV spectrophotometry (276 nm), the optimal balance between encapsulation efficiency and drug loading was achieved when the IPA concentration was 100 μg / mL (see [reference]). Figure 1 (2) Release curve: In vitro release experiments showed that BIEXO@IPA exhibited sustained-release properties at physiological temperature (37°C), with approximately 70% of the IPA being released cumulatively within 50 hours (see [link]). Figure 1 (l) is beneficial for the sustained metabolic reprogramming effect in the tumor microenvironment.

[0054] Example 4: Targeted delivery of BIEXO@IPA and its in vivo anti-tumor mechanism

[0055] 1. Bispecific targeting and cell bridging (in vitro) (1) Binding ability: ELISA and flow cytometry confirmed that BIEXO can specifically bind to PD-1 and MSLN receptors, with signal intensity more than 10 times higher than the control group (see Figure 3 (2) Bridging effect: Confocal microscopy and FRET experiments show that BIEXO can effectively bring PD-1 closer together. + T cells and MSLN + The distance between tumor cells, and in a dose-dependent manner (see [link to relevant documentation]). Figure 3 (3) Functional verification: In the presence of PD-L1, BIEXO treatment effectively prevented T cell exhaustion and maintained the killing activity of T cells (LDH release assay, see eh). Figure 3 ij).

[0056] 2. Establishment of an LLC-MSLN-luciferase orthotopic lung cancer mouse model by nebulized inhalation. (1) Comparison of administration routes: In vivo imaging (IVIS) showed that intravenously injected exosomes were mainly enriched in the liver, while nebulized inhalation increased the accumulation in the lungs by more than 10 times (see Figure 4 (2) Cellular uptake specificity: Flow cytometry analysis showed that BIEXO@IPA had a specific uptake rate of up to 79.33% in lung tumor cells, which was significantly better than the liposome control group (Lipo@IPA, 47.89%) (see ad). Figure 4 This is thanks to the natural affinity of exosomes and the targeting effect of dual antibodies.

[0057] 3. Therapeutic effect in orthotopic lung cancer model: Mice were given nebulized treatment 6 days after tumor inoculation. (1) Tumor suppression: IVIS monitoring showed that the tumor burden in the BIEXO@IPA group was reduced by 87.9% compared with the PBS control group, which was significantly better than the single drug group (see Figure 5 (bc). (2) Survival benefit: The 80-day survival rate of mice in the BIEXO@IPA group reached 80%, while the median survival of the PBS group was only 29 days (see bc). Figure 5 e). (3) Immune microenvironment remodeling: Flow cytometry analysis on day 14 post-treatment showed that CD8+ in the tumor microenvironment... + The proportion of T cells increased significantly, and the expression of functional molecules (IFN-γ, TNF-α, Granzyme B) was upregulated; at the same time, the proportion of regulatory T cells (Tregs) decreased significantly (see [link to relevant documentation]). Figure 5 fm). In particular, the addition of IPA makes PD-1 + CD8 + The expression of the stem cell marker TCF-1 in T cells was significantly increased, suggesting that T cell exhaustion was reversed (see [link to relevant documentation]). Figure 5 j).

[0058] Example 5: Evaluation of BIEXO@IPA's Enhancement of CAR-T Therapy and Safety

[0059] 1. Biosafety assessment A comprehensive toxicological evaluation of BIEXO@IPA was conducted: (1) General condition: There was no significant change in mouse body weight during treatment (see Figure 6a). (2) Organ function: After 30 days of treatment, serum biochemical indicators (ALT, ALP, BUN, Cr) and blood routine (WBC, RBC, PLT) were all within the normal range (see Figure 6 (3) Pathology and tumorigenicity: No pathological damage was observed in the major organs by H&E staining (see ci). Figure 6 j); and serum tumor markers (AFP, CEA, MUC1, CA125, CA19-9) were not elevated, ruling out the tumorigenic risk of iPSC-derived products (see [link]). Figure 6 ko).

[0060] 2. Enhancing the therapeutic effect of CAR-T cells: A lentiviral vector co-expressing Myc tag anti-MSLN CAR and mCherry reporter gene was constructed and transduced into mouse spleen T cells to prepare CAR-T cells. In an orthotopic lung cancer model, a treatment regimen of BIEXO@IPA nebulized inhalation combined with CAR-T intravenous infusion was adopted. (1) Breakthrough in efficacy: The combined treatment group achieved a complete remission rate (CR) of 66.7%, and all mice (100%) were still alive on the experimental cutoff date (day 80); in contrast, the CAR-T monotherapy group had a survival rate of only 16.7% (see Figure 7 (2) Immune memory and relapse prevention: Tumor re-challenge was performed on cured mice on day 110. The results showed that 83.3% of the mice were completely resistant to the second tumor inoculation. Flow cytometry analysis confirmed the formation of long-lasting effector memory T cells in vivo (see df). Figure 7 (3) Anti-metastatic effect: In the B16F10-MSLN lung metastasis model, the combination therapy almost eliminated lung metastatic nodules, with an inhibition rate of 93% (see nq). Figure 7 rs).

[0061] The above embodiments confirm that, as Figure 8 The BIEXO@IPA platform constructed by the method of this invention not only utilizes the natural high yield and anti-tumor properties of iPSC exosomes, but also achieves bispecific targeting and metabolic regulator delivery through engineering means, which can safely and efficiently enhance the efficacy of CAR-T cells in solid tumors.

Claims

1. Application of induced pluripotent stem cell-derived exosomes in the preparation of agents for the prevention and / or treatment of tumors.

2. The application according to claim 1, characterized in that, The induced pluripotent stem cell-derived exosomes exhibit antitumor activity by directly inhibiting tumor cell proliferation, inducing apoptosis, and inhibiting migration.

3. The application according to claim 1, characterized in that, The induced pluripotent stem cell exosomes are obtained by the following method: culturing induced pluripotent stem cells in cell culture medium, and extracting the induced pluripotent stem cell exosomes from the supernatant of the induced pluripotent stem cell culture medium; the cell culture medium is a serum-free cell culture medium.

4. The application according to claim 1, characterized in that, The induced pluripotent stem cells are derived from mice or humans.

5. Application of engineered induced pluripotent stem cell-derived exosomes in the preparation of antitumor and / or therapeutic agents for tumors.

6. An engineered induced pluripotent stem cell-derived exosome, characterized in that, The surface of the exosomes displays an anti-PD-1 / MSLN bispecific single-stranded variable fragment, and the exosomes are internally loaded with indole-3-propionic acid. The anti-PD-1 / MSLN bispecific single-stranded variable fragment has a nucleotide sequence as shown in SEQ ID NO:

3.

7. A method for preparing exosomes derived from engineered induced pluripotent stem cells as described in claim 6, characterized in that, Includes the following steps: 1) Construct a lentiviral vector expressing PD-1 / MSLN bispecific scFv; 2) Lentiviral transduction of iPSCs, screening for stable expression strains; 3) Cultivate engineered iPSCs and collect the culture supernatant; 4) Centrifugation to separate and purify exosomes; 5) Electroporation loading of indole-3-propionic acid.

8. The use of the engineered induced pluripotent stem cell-derived exosomes as described in claim 6 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumor is a solid tumor.

10. The application according to claim 9, characterized in that, The antitumor drug is administered via nebulized inhalation.

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