Enhancing Chimeric Antigen Receptor-Extracellular Vesicles (CAR-EV) Technology: The Future of Cancer Therapy.

CAR cell therapies have significantly advanced personalized treatment for several hematological malignancies. Currently, seven CAR- cell products are approved by the Food and Drug Administration (FDA) and six by the European Medicines Agency (EMA) for treating lymphoma, multiple myeloma, and chronic lymphocytic leukemia. Several challenges and limitations remain, with cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) being the most significant. Cell-free therapies, such as CAR-EVs, offer substantive advantages over their cellular counterparts. These include enhanced tumor infiltration and the potential for repeat administration while minimizing the risks of CRS, ICANS, and other adverse side effects. Additionally, the potency of CAR-EVs can be tuned by engineering the inclusion of cytotoxic agents and function-modifying ribonucleic acids (RNAs). Herein, we report on the development of a scalable CAR-EV platform for producing tunable CAR-EVs. This platform includes the engineering and pre-conditioning of EV producer cells (e.g., CAR-T and CAR-natural killer (CAR-NK) cells), isolation and enrichment of CAR-EVs using a Good Manufacturing Practice (GMP) grade ion-exchange chromatography (IEX) platform, fully automated high-throughput EV subpopulation analysis, and in vitro evaluation of CAR-EV functional cytotoxic activity. The platform has been validated using CAR-NK-EVs and CAR-T-EVs for both hematological and solid tumor cell lines. The CAR-EV platform represents a promising approach for the rapid development of off-the-shelf therapeutic CAR-EVs tailored to specific disease indications, with the potential to reduce adverse side effects associated with CAR-cell-based therapies.