Rational Combinatorial Targeting by Adapter CAR T Cells (AdCAR-T) Prevents Antigen Escape in Acute Myeloid Leukemia (AML)

Abstract

Abstract Targeting AML by chimeric antigen receptor T cells (CAR-T) has been challenging due to the promiscuous expression of AML-associated antigens on healthy hematopoietic stem and progenitor cells as well as a high degree of inter- and intratumoral heterogeneity. Consequently, we did not yet see dramatic clinical successes, as in B-phenotypic malignancies treated with CD19- or BCMA-directed CAR-T cells. In the present study, we analyzed the inter- and intratumoral heterogeneity of AML-associated antigens in 30 primary pediatric AML samples at single-cell resolution. We identified CD33, CD38, CD371, IL1RAP and CD123 as the most frequently expressed. Notably, high variability was observed not only across the different patient samples but also among leukemic cells of the same patient, which indicates that multiplexed targeting approaches will be needed to target all leukemic cells. To address this need, we utilized our modular Adapter CAR (AdCAR) platform, enabling precise qualitative and quantitative control over CAR-T-cell function. We show highly efficient and target-specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. Importantly, we prove that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to monotargeted CAR-T therapy. For the first time, we demonstrate in a PDX model that rational combinatorial targeting by AdCAR-T cells can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting utilizing the AdCAR platform.