Target antigen-displaying extracellular vesicles boost CAR T cell efficacy in cell and mouse models of neuroblastoma

Glypican-2 (GPC2) and the disialoganglioside GD2 are validated CAR T cell targets in neuroblastoma, but durable clinical responses remain limited. This modest chimeric antigen receptor T cell (CAR T cell) efficacy is in part due to suboptimal T cell persistence, antigen down-regulation, and a hostile tumor microenvironment, which includes immune cell-modulating extracellular vesicles (EVs). Neuroblastoma-derived EVs may contain CAR targets or other immunoregulatory elements that can modulate CAR T cell antitumor activity. Thus, we first profiled the surfaceome of neuroblastoma EVs and assessed their impact on both GPC2 and GD2 CAR T cell function. Neuroblastoma EVs displayed GPC2 and GD2, with minimal expression of programmed death-ligand 1 (PD-L1), and were detected in blood from tumor-bearing mice and patients. These EVs directly activated paired CAR T cells, suggesting a role for a peripheral source of CAR antigen. To exploit this therapeutically, we engineered nontumor-derived GPC2+ synthetic EVs (SyntEVs) as CAR T cell enhancers and armored them with either albumin-binding domains or GD2-binding domains. In mice harboring human neuroblastoma cell line-derived or patient-derived xenografts, serial infusion of armored SyntEVs after GPC2 CAR T cells enhanced tumor control by boosting peripheral CAR T cell persistence. Moreover, GD2-targeting SyntEVs decorated low-antigen tumor cells with GPC2, circumventing antigen down-regulation. This SyntEV platform offers a versatile system to address the therapeutic limitations of CAR T cells in solid tumors.