ABSTRACT Antigen-specific immunotherapies have long been pursued to improve outcomes in acute myeloid leukemia (AML). So far most exploited for this purpose are antibodies targeting CD33, a glycoprotein displayed on the cell surface of leukemic blasts in almost all cases and possibly leukemia stem cells in some. Improved survival of some patients treated with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this approach, but many patients with CD33+ AML do not benefit from GO. This has prompted interest in developing improved CD33-directed therapeutics, with several investigational drugs recently advancing to clinical testing. Similar to GO, all these molecules recognize immune-dominant epitope(s) within the membrane-distal V-set domain of CD33. In our preliminary studies with CD33V-set/CD3 bispecific antibodies, however, we have observed enhanced immune-effector cell function and higher cytolytic efficacy with membrane-proximal binding of CD33. We therefore hypothesize antibodies against the membrane-proximal C2-set domain will provide a superior approach to CD33-targeted immunotherapy than existing therapeutics. Moreover, since the V-set but not C2-set domain is missing in some CD33 splice variants, C2-set domain-directed antibodies can recognize all naturally- occurring variants of CD33 (i.e. are “CD33PAN antibodies”). As a first step toward our long-term goal of developing this new form of CD33 immunotherapy, we have recently generated murine and – with use of humanized (“Trianni”) mice – fully human antibodies against the C2-set domain of human CD33. Proof of concept studies with a murine CD33PAN/CD3 bispecific molecule demonstrated potent anti-leukemia activity and effective T-cell engagement. We now propose to develop a new therapy that is based on T-cells expressing chimeric antigen receptors (CARs) with human CD33PAN antibody binding sequences for patient use. We plan to rigorously test the anti-leukemia properties of these CAR T-cells in vitro and in vivo, while at the same time studying how CD33 gene-edited normal hematopoietic stem and progenitor cells (HSPCs) can mitigate toxicity of our adoptive cell therapy to normal blood cells. To accomplish these goals, we have assembled a multidisciplinary team of investigators with complementary expertise in CD33-directed immunotherapies, CAR T-cell technology, and transplantation of gene-modified HSPCs. Expected results will guide the further development of CD33PAN CAR T-cells. Alone or together with engineered hematopoietic cells to widen their therapeutic window, these cells may offer a new treatment option for AML and other CD33+ neoplasms for which outcomes continue to be unsatisfactory.