The Art and Science of Selecting a CD123-Specific Chimeric Antigen Receptor for Clinical Testing

Chimeric antigen receptor (CAR) T cells targeting CD123, an acute myeloid leukemia (AML) antigen, hold the promise of improving outcomes for patients with refractory/recurrent disease. We generated five lentiviral vectors encoding CD20, which may serve as a target for CAR T cell depletion, and 2nd or 3rd generation CD123-CARs since the benefit of two costimulatory domains is model dependent. Four CARs were based on the CD123-specific single-chain variable fragment (scFv) 26292 (292) and one CAR on the CD123-specific scFv 26716 (716), respectively. We designed CARs with different hinge/transmembrane (H/TM) domains and costimulatory domains, in combination with the zeta (z) signaling domain: 292.CD8aH/TM.41BBz (8.41BBz), 292.CD8aH/TM.CD28z (8.28z), 716.CD8aH/TM.CD28z (716.8.28z), 292.CD28H/TM. CD28z (28.28z), and 292.CD28H/TM.CD28.41BBz (28.28.41BBz). Transduction efficiency, expansion, phenotype, and target cell recognition of the generated CD123-CAR T cells did not significantly differ. CAR constructs were eliminated for the following reasons: (1) 8.41BBz CARs induced significant baseline signaling, (2) 716.8.28z CAR T cells had decreased anti-AML activity, and (3) CD28.41BBz CAR T cells had no improved effector function in comparison to CD28z CAR T cells. We selected the 28.28z CAR since CAR expression on the cell surface of transduced T cells was higher in comparison to 8.28z CARs. The clinical study (NCT04318678) evaluating 28.28z CAR T cells is now open for patient accrual. [Display omitted] Acute myeloid leukemia (AML) remains a challenging disease to treat. Chimeric antigen receptor (CAR) T cells specific for CD123, an antigen expressed on AML blasts and to a lesser degree on normal hematopoietic progenitor cells (HPCs), have the potential to improve outcomes for patients with relapsed or refractory disease. Here we describe the generation of a panel of 5 lentiviral constructs encoding a CD20 safety switch and a 2nd or 3rd generation CD123-CAR. We perform a comprehensive analysis of T cells expressing this panel of CD123-CAR constructs including transgene expression, expansion kinetics, immunophenotype, and antitumor activity in vitro and in vivo. In addition, we demonstrate the functionality of the CD20 safety switch, which would allow for rapid elimination of T cells in the event of toxicity. This careful analysis allowed us to elucidate subtle differences between constructs that could be linked to differences in the antigen recognition, hinge/tran