Nonactivated and IL-7 cultured CD19-specific CAR T cells are enriched in stem cell phenotypes and functionally superior.

CD19-specific chimeric antigen receptor (CAR) T-cells have demonstrated impressive responses in patients with relapsed and refractory B-cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T-cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T-cells involve T-cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T-cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T-cells in the absence of activation. In these studies, we utilized IL-7 to generate CD19 CAR T-cells without stimulating CD3. Non-activated and IL-7 cultured (NICE) CD19 CAR T-cells were enriched with the memory T stem cell population (TSCM), retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T-cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B-cell lymphoma models. These results suggest that NICE CD19 CAR T-cells may improve outcomes for B-cell malignancies and warrant clinical evaluation.