High-affinity FRß-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRß)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRß-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRß CAR exhibited greatly enhanced antitumor activity against FRß(+) AML in vitro and in vivo compared with a low-affinity FRß CAR (54.3 nm KD). Using the HA-FRß immunoglobulin G, FRß expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRß CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRß CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRß CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.