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

ABSTRACT 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.