CAR-T cell biology in hematologic malignancies: CAR⁺ extracellular vesicles as predictive biomarkers of ICANS

Predicting immune effector cell-associated neurotoxicity syndrome (ICANS) in patients receiving CAR-T cell therapy remains an unmet challenge. This complication, traditionally attributed to CAR-T cell activation, typically emerges several days after infusion, when circulating CAR-T cells are scarcely detectable and specific biomarkers of their in vivo activity are lacking. CAR+ extracellular vesicle (CAR+EV) release was assessed in human CD19.CAR-T cells cocultured with CD19+ target cells. A prospective cohort of 100 patients with B-cell lymphoma infused with approved CD19.CAR-T cell products was assessed for plasma CAR+EVs as biomarkers of in vivo CD19.CAR-T cell activation. Human induced pluripotent stem cell-derived (iPSC-derived) neural cells were used as a model for CAR+EV-induced neurotoxicity. In vitro, CAR⁺EV release occurred within one hour of target engagement. In patients, CAR⁺EVs became detectable in plasma as early as one hour after infusion. A CAR⁺EV concentration exceeding 132.8 EVs/μL at hour +1 and 224.5 EVs/μL at day +1 accurately predicted the onset of ICANS approximately four days in advance, with superior sensitivity and specificity compared to currently available predictive parameters. Moreover, exposure of iPSC-derived neural cells to CAR⁺EVs induced the release of neuron-specific Enolase positive (ENO2⁺) nanoparticles, which were also elevated in the plasma of patients who developed ICANS. Collectively, these data identify plasma CAR⁺EVs as immediate readout of CD19.CAR-T cell activation, robust early predictors of neurotoxicity, and potential mediators of ICANS pathogenesis.