CD19.CAR T-cell–derived extracellular vesicles express CAR and kill leukemic cells, contributing to antineoplastic therapy
Paola Lanuti, Francesco Guardalupi, Giulia Corradi, Rosalba Florio, Davide Brocco, Serena Veschi, Elsa Pennese, Domenico De Bellis, Francesca D’Ascanio, Anna Piro, Laura De Lellis, Pasquale Simeone, Maria Concetta Cufaro, Serena Pilato, Isabella D’Amario, Ida Villanova, Barbara Di Francesco, Lucia Di Re, Fabio Verginelli, Damiana Pieragostino, Prassede Salutari, Fabrizia Colasante, Annalisa Natale, Maria Vittoria Mattoli, Simone Vespa, Antonella Fontana, Raffaella Giancola, Bianca Fabi, Stefano Baldoni, Stella Santarone, Francesco Restuccia, Nicola Tinari, Piero Del Boccio, Alessandro Cama, Mauro Di Ianni
Abstract
ABSTRACT: 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 who received infusion with CD19 CAR T cells were 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 good manufacturing practice (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+EV concentrations correlated with failure of CAR T-cell therapy. Circulating CD19.CAR+EVs displayed a median size (standard deviation) of 133.1 ± 65.5 nm and carried a proapoptotic 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 primary cells. The new GMP-compliant EV isolation method allowed for 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 proapoptotic 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, by contributing to the direct killing of leukemic targets, represent a new product with strong therapeutic potential that could be infused independently of CAR T cells.