Synergically enhanced anti-tumor immunity of in vivo CAR by circRNA vaccine boosting

Abstract Chimeric Antigen Receptor (CAR) T cell therapy has shown promise in treating hematologic malignancies. However, it is limited to individualized cell therapy and faces challenges, including high costs, extended preparation time, and limited efficacy against solid tumors. Here, we generated circular RNAs (circRNAs) encoding Chimeric Antigen Receptor (CAR) transmembrane proteins, referred to as circRNA CAR , which mediated remarkable tumor killing in both T cells and macrophages. In addition, macrophages exhibited efficient phagocytosis of tumor cells and pro-inflammatory polarization induced by circRNA CAR in vitro . We demonstrated that circRNA CAR , delivered with immunocyte-tropic lipid nanoparticles (LNPs), significantly inhibited tumor growth, improved survival rates and induced a pro-inflammatory tumor microenvironment in mice. Importantly, the combination of circRNA Anti-HER2-CAR and circRNA-based cancer vaccines encoding the corresponding transmembrane HER2 antigen, termed circRNA HER2 , exhibited synergistically enhanced anti-tumor activity. This proof-of-concept study demonstrated that the combination of circRNA-based in vivo CAR and vaccines, termed in vivo CAR-VAC, holds the potential to become an upgraded off-the-shelf immunotherapy.