Enhancing Anti‐Tumor Effects of Engineered Extracellular Vesicles via Endocytosis Route Switching and Interferon Response Suppression

Engineered extracellular vesicles (EVs) represent a promising therapeutic strategy with many applications in cancer therapy. EVs derived from engineered tumor-targeting killer cells, such as chimeric antigen receptor (CAR)-T cells. However, the application of CAR-T-EVs is limited by several drawbacks. This study shows that engineered EVs with potent cancer-targeting and killing abilities can be generated from easily manipulable non-killer cells, providing a solution to overcome the limitations of CAR-T-EVs. It is found that EVs derived from non-killer cells such as CD19-targeting 293 cells possess target cell killing capacities comparable to those derived from CD19-CAR-T cells. A technique is developed to ensure the presence of sufficient targeting modules on the EV surface using a chimeric CD8-CD63/CD81 transmembrane region. Uptake of CD19-targeting EVs by target cells can be optimized by switching the route of CD19 endocytosis from clathrin-mediated endocytosis (CME) to aggregation-dependent endocytosis (ADE), leading to lysosomal degradation of the CD19/EVs complex. Degradation of the EVs leads to impairment in the IFN response and subsequent enhancement in EV uptake by target cells, creating a potent feedback cycle. CD19 depletion results in the disruption of the CD19-AKT-Myc pathway in the target cells, enhancing the killing capacity both in vitro and in vivo.