Introduction
Chimeric antigen receptor (CAR) cell-based therapies have transformed treatment paradigms for hematological malignancies. However, their application to solid tumors remains limited due to poor tumor vascularization, immunosuppressive microenvironments, and systemic toxicities such as cytokine release syndrome (CRS). CAR-engineered extracellular vesicles (CAR-EVs) offer a promising cell-free alternative, potentially overcoming these limitations. We developed a novel immunotherapy platform using CAR-EVs derived from natural killer (NK) cells engineered to target epidermal growth factor receptor (EGFR), for treatment of triple-negative breast cancer (TNBC).
Methods
NK cells were genetically modified to express EGFR-specific CARs and validated for CAR expression. These CAR-NK cells were expanded under serum-free, xeno-free conditions, and conditioned media were collected for EV isolation. CAR-EVs were purified using ion-exchange chromatography (EXO-ACE™) and characterized via nanoparticle tracking analysis (Zetaview), flow cytometry, and Western blotting to confirm EV identity and CAR protein presence. Anti-tumor efficacy was assessed in vitro using xCELLigence RTCA eSight and Incucyte systems on TNBC cell line (Hs578T).
Results
Purified CAR-EVs displayed characteristic EV morphology and size, expressed canonical EV markers (CD63, Flotillin), and contained functional CAR proteins and cytotoxic molecules such as granzyme B. Treatment with CAR-EVs significantly inhibited proliferation of both TNBC cell lines, while control EVs from HEK-293 cells showed no effect, confirming specificity and therapeutic potential.
Conclusion
CAR-EVs offer a promising, scalable, and safer alternative to conventional CAR-cell therapies for solid tumors. The demonstrated in vitro efficacy and specificity support further development toward in vivo validation and clinical translation. This platform represents a significant step forward in next-generation immunotherapies for aggressive breast cancers.