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

Abstract 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.