The long-term goals of this project are to translate novel findings in the field of immunology into early phase immunotherapy clinical trials for patients with leukemia. Allogeneic hematopoietic cell transplantation (allo HCT) is a standard treatment for high-risk or relapsed AML and is potentially curative. However, patients who relapse after allo HCT have a dismal prognosis, and there remains no clearly effective standard therapy. Mechanisms contributing to failure of the graft versus leukemia (GVL) effect include alterations in the AML cells to avoid immune recognition, compromised antigen presentation, and immune cell exhaustion. Thus, post-HCT relapse AML patients have a high unmet need for safe and innovative immunotherapies. Ideally, new treatments will eliminate AML and act in concert with donor T cells to boost GVL. We hypothesize that donor memory-like (ML) NK cellular therapy will provide 1) immediate NK-GVL effect that will induce AML remissions, and 2) cytokines (IFN-γ) that rescue T cell-mediated GVL, thereby achieving short-term disease control and long-term remissions. We discovered brief cytokine (IL-12, IL-15, and IL-18) activation results in differentiation of human ML NK cells, which respond robustly upon restimulation (to AML) and have multiple anti-leukemic advantages compared to conventional (c)NK cells. Safety (no CRS/ICANS/GVHD) and preliminary AML responses were observed in our phase 1 clinical trial in relapsed/refractory (rel/ref) AML. In our previous SPORE clinical trial project, we demonstrated that augmenting allo HCT with immune compatible HCT-donor ML NK cells early (Day+7) resulted in early AML remissions and donor ML NK cell expansion and functional persistence for >3 months after a single NK product dose. Here, we propose to test ML NK cells as therapy for adult AML patients who have relapsed after HCT in an open and accruing phase 2 trial cohort. Our prior work also identified NKG2A as a predominant inhibitory checkpoint for ML NK cell responses to AML, but this has not been thoroughly tested in vivo, nor in the post-HCT relapse setting. While NK activating receptors recognize many AML blasts, this detection is imperfect, and thought to result in AML resistance. Leveraging our prior work with ML NK cell chimeric antigen receptor (CAR) engineering, we are positioned to test CAR ML NK cells for enhanced ability to eliminate AML. In this proposal, we will 1) perform a phase 2 study of same-donor ML NK cell immunotherapy for patients with AML relapsed after allo HCT, 2) define donor ML NK cell expansion, functionality, and longevity, mechanisms of AML resistance to NK cell therapy, and impact on T cells in the post-HCT relapse setting, and 3) evaluate NKG2A blockade and CAR engineering combined with ML NK cells. These studies will lead to a new understanding of mechanisms whereby NK cells effectively attack AML, and whereby AML resists NK cell therapy, and hence strategies to improve memory-like NK cell anti-AM...