ABSTRACT There is an urgent and critical need for the development of leukemia stem cell (LSC)-directed therapeutic approaches for the treatment of acute myeloid leukemia (AML). One such strategy is targeting antigens that are specific to LSCs but absent from normal hematopoietic stem cells (HSCs). CD371 (CLEC12A, CLL-1), which is present on mature myeloid cells, has been described as one such targetable disease marker given its presence on both bulk AML cells and LSCs. Although not ubiquitously expressed on all AML cells, it is expressed in up to 95% of AML patients, is enriched on LSCs and chemoresistant AML subpopulations, and most importantly, is absent on HSCs. We have successfully developed and validated a fully-human CD371-targeted chimeric antigen receptor (CAR) T cell product which also secretes IL-18. Given that our CD371-targeting motif is entirely human, it is expected to have reduced immunogenicity and thus minimizes host-mediated CAR T cell-directed immune elimination in the context of constitutive IL18 secretion. In addition, IL18 secretion is predicted to enhance CAR T cell persistence and modulation the tumor microenvironment (TME) by increasing and activating immune cell infiltrates, leading to the induction of an endogenous T cell mediated anti-tumor immune-response capable of eradicating antigen-negative tumor cell subpopulations. Our central hypothesis is that CD371-targeted IL18-secreting CAR T cells will lead to eradication of antigen- positive chemoresistant and LSC subpopulations, and the induction of an endogenous AML-reactive T cell response that will lead to elimination of antigen-negative disease, without long-term HSC toxicity. This will be tested in AML patient-derived xenograft models of heterogeneously antigen-positive disease (Aim 1) and a phase I clinical trial with CD371-targeted IL18-secreting CAR T cells in patients with relapsed/refractory AML (Aim 2). This effort will therefore assess the safety and efficacy of this novel CAR-T cell approach in both preclinical and clinical settings, and will also address biomarkers of response and efficacy in numerous correlative studies (Aim 3).