CD3ɛ Nanobody-Engineered Extracellular Vesicles Driving In Vivo Generation of TCE-secreting CAR-Ts for Solid Tumor Therapy With Memory Response and Minimal Immunogenicity.

In vivo generation of chimeric antigen receptor-T (CAR-T) cells offers an innovative approach to CAR-T therapy; however, current in vivo CAR-T technologies rely on synthetic carriers or viral particles, which raise immunogenicity and safety concerns in clinical applications. Extracellular vesicles (EVs) are cell-derived natural nano-platforms with improved biocompatibility and the potential to deliver the transgene for in vivo CAR-T generation. In this study, we pioneered a CD3ε nanobody (Nb)-CD63 chimeric construct and stably expressed it on HEK-293T cell-derived EVs to produce CD3ε-targeting EVs, which were further loaded with Nb-CAR.TCE (Nb-CAR with secretable bispecific T-cell engager) transgene through electroporation. The CD3ε-Nb EVs selectively delivered Nb-CAR.TCE transgene into CD3(+) cells in vivo and exerted robust antitumor activity against various solid tumors. The CD3-targeting property of CD3ε-Nb EVs combined with the characteristics of Nb-CAR.TCE construct may enhance memory CAR-T proportion, prolong anti-tumor immunity, and strengthen resilience against tumor antigen rechallenge. Notably, the CD3ε-Nb EVs exhibited minimal immunogenicity risks compared to lipid-based and lentiviral carriers, despite their comparable anti-tumor activity. Taken together, the CD3-targeting EVs could drive the in vivo generation of bispecific CAR-T cells to effectively eliminate cancers and improve memory response with minimal immunogenicity.