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.