Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between T cell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) T cells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33^PAN antibodies). CD33^PAN CAR T cells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33^neg leukemia. Compared to CD33^V-set CAR T cells, CD33^PAN CAR T cells showed greater in vitro and in vivo efficacy against several human AML cell lines with differing levels of CD33 without increased expression of exhaustion markers. CD33^PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33^PAN CAR T cells had greater in vitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR T cells binding CD33 close to the cell membrane, providing the rationale to investigate CD33^PAN CAR T cells further toward possible clinical application.