Summary: Allogeneic hematopoietic transplantation (HSCT) is an effective treatment for AML. Relapse of the malignancy remains the major cause of treatment failure and improved strategies to eradicate leukemia are required. Natural killer (NK) cells are a unique class of lymphocytes, with cytotoxic, and immunoregulatory function which can mediate potent antileukemia effects against myeloid leukemias while simultaneously preventing GVHD in preclinical models. Relatively few NK cells are present in peripheral blood or bone marrow transplant cell products and the NK cells recovering early post transplant have functional defects impairing cytotoxicity. We have developed robust clinical procedures for the ex vivo expansion and activation of cord blood (CB)-derived NK cells. The long-tem objective of this research is to develop novel cell based therapies to harness the antileukemic potential of NK cells against AML, and to further enhance their effector function by both redefining their specificity and/or enhancing their potency Aim 1 tests the hypothesis that the addition of optimally selected, ex vivo expanded and activated CB-derived NK cells will improve the outcomes of patients with AML after cord blood transplantation (CBT). We have found that patients with AML whose HLA type is homozygous for group C2 have a significantly higher risk of relapse following CBT compared to those who are homozygous or heterozygous for HLA group C1. Mechanistically, we have shown that the increased risk of disease progression in the C2/C2 risk group is related to delayed emergence of immunocompetent C2-specific KIR2DL1/S1+ NK cells post-CBT. This suggests that such patients would benefit from the post-CBT infusion of activated mature CB-NK cells that express KIR2DL1/S1. Extensive biologic correlates will examine the phenotype, function and homing of adoptively infused CB-NK cells, and their impact on immune reconstitution post-transplant. Aim 2 capitalizes on the finding that the baseline cytotoxicity of NK cells can be augmented by expression of a chimeric antigen receptor (CAR) against the target cells. CD123, the α chain of the interleukin-3 receptor, is an attractive target for cellular immune therapy in AML, but cytotoxicity against normal hematopoietic cells would preclude safe delivery in the absence of HSCT. We will test the hypothesis that NK cells transduced with a CAR to CD123, and coexpressing CD28 and IL15 to mediate long term persistence, and inducible caspase 9 (IC9) to manage potential toxicity will provide enhanced activity against AML. Aim 3 examines if blocking the TGF-beta pathway by retrovirally transducing NK cells with a dominant negative TGF-beta type II receptor can render NK cells resistant to the immunosuppressive AML microenvironment. Optimal strategies formulated in aims 2 and 3 will be translated into human clinical trials, based on the optimal platform protocol determined in aim 1.