Optimal pairing of binder and co-stimulatory domains improves dual CART cell efficacy.

We explore whether T cells expressing two chimeric antigen receptors (CARs) with distinct signaling motifs (dual CARs) improve CART cell (CART) efficacy against leukemia and lymphoma. Moreover, we investigate whether dual CARTs targeting two antigens (multi-targeted) are superior to dual CARTs targeting a single antigen (single targeted). Functional assays revealed that multi-targeted dual CARTs targeting both CD19 and CD22 were more potent than single-targeted dual CARTs targeting only CD19 or CD22. RNA expression profiling demonstrated that single-targeted dual CARTs augmented canonical nuclear factor κB (NF-κB), non-canonical NF-κB, and Th17 differentiation pathways equivalently upon target engagement. Interestingly, the transcriptional profile of multi-targeted dual CARTs favored the co-stimulatory domain linked to the binder of the more robustly tumor-expressed CD19 rather than one linked to the binder of the less tumor-expressed CD22. In vivo and T cell exhaustion assays found that multi-targeted dual CARTs led to greater durable control of B-ALL than single-targeted dual CARTs, with T cells co-expressing CD19.BBζ and CD22.28ζ being the most potent. These data indicate that optimal pairing of CAR binder domain with signaling cassette bolsters anti-tumor efficacy.