Acute myeloid leukemia (AML) is a clinically heterogeneous disease characterized by the expansion and accumulation of immature myeloid cells in the bone marrow and peripheral blood. Advances in the AML genetics have led to a better understanding of disease progression, paving the way for the development of novel targeted therapies. Still, the mainstay of AML treatment is chemotherapy, which has remained mostly unchanged for several decades, and fewer than one-third of adult AML patients experience a durable remission. Reduced- intensity hematopoietic stem cell transplantation (HSCT) for AML is potentially curative, but relapse rates exceed 50%. We have shown that allogeneic natural killer (NK) cell infusions after high-dose lymphodepletion (LD) can induce remission in 30-40% of patients with relapsed/refractory AML. However, this approach is limited by the toxicity of high-dose LD, NK cell numbers within haploidentical blood products, inadequate donor NK cell persistence, lack of NK cell antigen specificity, allogeneic rejection and challenges of exporting adoptive NK cell therapy beyond specialty centers. To overcome these barriers, we have developed a scalable GMP-compliant platform for the differentiation of induced pluripotent stem cells (iPSCs) into highly potent NK cells (termed iNK). With our system, we can generate stable gene-edited lines for tailored approaches to NK cell immunotherapy. In the past year, we have safely treated patients with up to 6 cryopreserved doses of off-the-shelf iNK cell products. In this project we will test the central hypothesis that triple gene-modified iNK cells (FT538) represent a novel and effective immunotherapy for the treatment of AML. FT538 is a cell product with three functional modifications: a high affinity, non-cleavable CD16 Fc receptor to augment antibody-dependent cellular cytotoxicity; a membrane-bound IL-15/IL-15 receptor fusion molecule (IL-15RF) that acts intrinsically to enhance NK cell activity; and knockout of CD38 to mitigate NK cell fratricide in the presence of the anti-CD38 monoclonal antibody, daratumumab. The central hypothesis will be tested in three independent aims. In Aim 1, we will test the hypothesis that FT538 iNK cells will exhibit sustained persistence and function after adoptive transfer for targeting of CD38+ AML cells when combined with daratumumab in an FDA-approved and enrolling phase I clinical trial. Because CD38 is highly expressed on AML, immune suppressor cells, and activated CD8+ T cells, our clinical trial will test whether daratumumab dosing before FT538 iNK cell treatment enhances the persistence of iNK through selective immunodepletion. In Aim 2, we will test the hypothesis that iNK cell specificity, potency, and persistence can be enhanced by optimizing IL-15 signaling in the context of CD38 knockout and that a novel anti-CD5-ADC will provide effective and safer LD to enhance persistence. In Aim 3, we will test the hypothesis that dual antigen-specific target...