Abstract:
Objective Chimeric antigen receptor T-cell (CAR-T) immunotherapy has made major breakthroughs in the treatment of blood tumors. However, current CAR-T therapies face several limitations: they require autologous cells, involve a lengthy and costly production process, and use lentiviral transduction that carry risk of insertional carcinogenesis due to random integration. Therefore, there is an urgent need to develop a universal cost-effective cancer immunotherapy method generating CAR-T cells for in vivo cancer immunotherapy.
Methods This study successfully established an exosome-mediated, T-cell targeted delivery system, demonstrating both precise design and functional efficacy for biomedical applications. To optimize CAR-T cell generation the transfection dose was adjusted, and the kinetics of CAR-T cell percentage were recorded. The cytotoxicity of the resulting CAR-T cells was evaluated in vitro by calcein-AM release. To test the tumor-killing in vivo of engineered exosomes, human PBMCs were injected into NPG mice via the tail vein to establish humanized mice, followed by intravenous injection of tumor cells to induce cancer.
Results To overcome the limitations of conditional autologous CAR-T cells, we developed a T cell-targeted exosome system capable of specifically targeting human CD3 +, CD4+, and CD8 +T cells. CAR-T production was dose-dependent, with transfection efficiency reaching upto 97.8% at 106 particles/cell. Both in vitro cytotoxicity assays and in vivo animal experiments demonstrated that exosome-incubated CAR-T cells effectively eliminated CD19-positive Raji cells, highlighting their specificity and therapeutic potential in antigen-directed applications.
Conclusions We successfully established a CD8-targeting exosome delivery system for CAR-T cell production capable of transforming CD8+T cells into functional CAR-T cells, which showed significant tumor-killing ability in vitro and in mice. Compared with the traditional lentiviral vector for the preparation of CAR-T cells in vitro, in vivo-reprogrammed CAR-T cells using our CD8-targeted exosome delivery system, with higher transfection efficiency, shorter production period, lower cost, and eliminated the risk of insertion carcinogenesis. This strategy promises to bring a new era of universal CAR-T medicine, which can improve cancer immunotherapy and may hold promise as a therapeutic platform to treat various diseases.
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