Differential susceptibility and role for senescence in CART cells based on costimulatory domains

Despite the success of chimeric antigen receptor T (CART) cell therapy in hematological malignancies, durable remissions remain low. Here, we report CART senescence as a potential resistance mechanism in 41BB-costimulated CART cell therapy. To mimic cancer relapse, we utilized an in vitro model with repeated CART cell activation cycles followed by rest periods. Using CD19-targeted CART cells with costimulation via 4-1BB-CD3? (BB?) or CD28-CD3? (28?), we showed that CART cells undergo functional, phenotypical, and transcriptomic changes of senescence, which is more prominent in BB?. We then utilized two additional independent strategies to induce senescence through MYC activation and irradiation. Induction of senescence impaired BB? activity but improved 28? activity in preclinical studies. These findings were supported by analyses of independent patient data sets; senescence signatures in CART cell products were associated with non-response to BB? but with improved clinical outcomes in 28? treatment. In summary, our study identifies senescence as a potential mechanism of failure predominantly in 41BB-costimulated CART cells. Overall design: RNA-seq profiling of T cells (T_cell_D0) collected from three different normal donors and chimeric antigen receptor T (CART) cell generated from those T cells. T cells are divided into three groups to generate Day 8 (D8) untransduced control (UTD_D8), D8 CD19 targeting 4-1BB costimulated CART cells (CART19-4-1BB_D8) and D8 CD19 targeting CD28 costimulated CART cells (CAR19-CD28_D8). D8 CART cells are recurrently activated/rested to generate UTD_D15, CART19-4-1BB_D15 and CAR19-CD28_D15.