Introduction Chimeric Antigen Receptor T-Cell (CAR-T cell) Therapy faces challenges related to low potency and poor in vivo persistence. Incorporating cytokine signals, such as interleukin-12 (IL-12), has emerged as a promising strategy to enhance CAR-T cell functionality due to its potent immunostimulatory effects. However, clinical application of IL-12 is hindered by pleiotropic toxicity and limited bioavailability. Therefore, alternative agents or delivery systems are desired to exploit the benets of IL-12 safely. This study investigated the therapeutic potential of a novel IL-12 delivery system utilizing extracellular vesicles (EVs) modied with the target antigen CD19 to specically deliver IL-12 to CD19 CAR-T cells, thus augmenting their anti-tumor effects. Method IL-12-anchored extracellular vesicles (IL-12 EVs) were generated from engineered 293T cells expressing IL-12 on the cell membrane via GPI structure. Additionally, EVs co-expressing IL-12 and CD19 (CD19/IL-12 EVs) were produced from 293T cells coexpressing CD19 and IL-12. The physicochemical characteristics of the EVs were analyzed using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB). Binding and uptake efciency of the EVs with CD19 CAR-T cells were validated using
ow cytometry and imaging
ow cytometry. In vitro experiments were conducted to investigate the effects of the EVs on the cytotoxic function, degranulation, cytokine secretion, and proliferation of CD19 CAR-T cells.