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

Targeting AML by chimeric antigen receptor T cells (CAR-T) has been challenging due to 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 the dramatic clinical successes as in B-phenotypic malignancies treated with CD19 or BCMA directed CAR-T.

In the present study, we analyzed 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 to be most frequently expressed. Notably, high variability was not only observed across the different patient samples, but also among leukemic cells of the same patient, which indicates that multiplexed targeting approaches will be required 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 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 mono-targeted CAR-T therapy. For the first time, we demonstrate in a PDX model that rational combinatorial targeting by AdCAR-T 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.