Abstract
The surface antigen landscape of acute myeloid leukemia (AML) displays significant heterogeneity and overlap with healthy hematopoietic cells. This imparts a substantial hurdle to the development of AML-targeting chimeric antigen receptor (CAR) T-cells that can avoid on-target, off-tumor toxicity. Here, we develop a dual-antigen targeting CAR-T against CD70 and the active conformation of integrin β2 (aITGB2), each previously reported as promising AML targets due to minimal off-tumor expression. We show an OR-gated approach for these antigens significantly increases the proportion of AML blasts that can be targeted, in part using a novel ex vivo co-culture method to restore surface protein homeostasis following a freeze-thaw cycle. We test dual-targeting CAR-T constructs with different combinations of costimulatory domains, identifying constructs with superior anti-tumor cytotoxicity in vitro against AML cell line and patient derived xenograft models. We further show significantly improved in vivo tumor clearance and survival for a dual targeting CAR in murine models of AML tumor heterogeneity. Finally, we show that this dual-targeting CAR does not increase off-tumor toxicity, especially against hematopoietic stem and progenitor cells. Together, these findings demonstrate a promising clinically-translatable approach for the treatment of AML without the notable toxicity liabilities associated with other leading CAR-T targets for this disease.