A novel chimeric antigen receptor (CAR) system using an exogenous protease, in which activation of T cells is controlled by expression patterns of cell‑surface proteins on target cells.

Anti‑CD19 chimeric antigen receptor (CAR)‑T cell therapy against refractory B‑cell malignancies shows excellent therapeutic effects. However, there are some obstacles to be overcome in this treatment. Since current CAR‑T cells target a single cell‑surface protein on tumor cells, the CAR‑T cells also attack normal cells expressing the protein. This is one of the major adverse effects of this therapy. To improve target‑cell‑specificity of this therapy, we established a novel CAR system, in which T‑cell activation was controlled by expression patterns of proteins on target cells. Our novel CAR‑T cells had two distinct CARs consisting of a 'Signal‑CAR', recognizing a protein on tumor cells, and a 'Scissors‑CAR', recognizing another protein on normal cells. The signal‑CAR had a peptide sequence which was cleaved by the Scissors‑CAR, and functional domains for cellular activation. The Scissors‑CAR had a protease domain that cleaved its recognition peptide sequence in the Signal‑CAR. When tumor cells expressed only the protein recognized by the Signal‑CAR, the tumor cells were attacked. By contrast, normal cells expressing both the proteins induced inactivation of the Signal‑CAR through cleavage of the recognition site when getting in contact with the CAR‑T cells. To establish this system, we invented a Scissors‑CAR that was dominantly localized on cell membranes and was activated only when the CAR‑T cells were in contact with the normal cells. Using a T‑cell line, Jurkat, and two proteins, CD19 and HER2, as target proteins, we showed that the anti‑CD19‑Signal‑CAR was cleaved by the anti‑HER2‑Scissors‑CAR when the CAR‑T cells were co‑cultivated with cells expressing both the proteins, CD19 and HER2. Furthermore, we demonstrated that primary CAR‑T cells expressing both the CARs showed attenuated cytotoxicity againsT cells with both the target proteins. Our novel system would improve safety of the CAR‑T cell therapy, leading to expansion of treatable diseases by this immunotherapy.