CD19.CAR-T cell-derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy.

Chimeric Antigen Receptor (CAR) T cell-derived Extracellular Vesicles (EVs) might represent a new therapeutic tool for boosting CAR-T cell antileukemic effects. Here, a cohort of 22 patients infused with CD19.CAR-T cells was monitored for the presence of circulating CD19.CAR+-T cell-derived EVs (CD19.CAR+EVs), which were then separated and functionally characterized for their killing abilities. A GMP-compliant separation method was also developed. Results demonstrated that CD19.CAR+EVs were detectable in peripheral blood up to 2 years after infusion indicating long-lasting persistence of their parental cells. Notably, early decreases of circulating CD19.CAR+EVs concentrations correlated with failure of CAR-T therapy. Circulating CD19.CAR+EVs displayed a median size (SD) of 133.1±65.5 nm and carried a pro-apoptotic protein cargo. These EVs expressed higher CAR levels than their parental cells. Furthermore, CD19.CAR+EVs did not activate heterologous T cells and produced significant, specific and dose-dependent cytotoxic effects on CD19+ cell lines and on primary cells. The new GMP-compliant EV isolation method allowed a recovery of 63±5.7 % of CD19.CAR+EVs. A deeper analysis of the different protein cargoes carried by EVs derived from different CAR-T cell subpopulations identified a pro-apoptotic functional pathway linked to CD8+LAG-3+ EVs. Overall, our data indicate that CD19.CAR+EVs may be proposed as promising dynamic new biomarkers of CAR-T cell activity and, contributing to the direct killing of the leukemic target, represent a new product with a strong therapeutic potential that could be infused independently from CAR-T cells.