Novel fourth generation-like CAR^miR cells release therapeutic miRNA via exosomes and enhance glioblastoma cell killing activity

Fourth-generation chimeric antigen receptor (CAR) T-cells, using costimulatory domains to drive the co-expression of transgenic key cytokines, have been shown to significantly enhance the efficacy of CAR T cell therapy in the treatment of solid tumors. Recent studies identified several miRNAs, such as miR-34a-5p, miR-181a-5p, miR-218–5p and miR-433–3p, that are involved in regulating cancer cell proliferation, apoptosis, and metastasis. However, controlled delivery of miRNA to non-hepatic tissue remains a challenge. In this study, we developed novel CAR^miR cells as a proof-of-concept cell based therapeutic for in situ production and delivery of therapeutic miRNA. The system was developed by cloning a CAR molecule with modified interleukin 13 (IL13 E12Y), which has exhibited high binding affinity to glioblastoma cells expressing IL13Rα2, and pre-miR-34a under the NFAT-IL2 minimal promoter. When CAR cells are stimulated with the cancer antigen, upregulation and exosome exportation of miR-34a-5p is induced. The CAR^miR cells and their isolated exosomes showed enhanced toxicity towards U87 cells compared to cells expressing only CAR. To the best of our knowledge, this is the first report that shows the release of therapeutic miRNA through CAR activation, which could lead to significantly improved overall CAR T cell therapy for solid tumors.