PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) accounts for ~80% of adult acute leukemias and ~20% of childhood leukemias. Among patients who initially achieve a complete remission with standard induction therapy, most will ultimately relapse and die of disease-related complications. Allogeneic hematopoietic cell transplantation (HCT) has reduced relapse rates and can be curative for many high-risk patients. However, HCT regimens can cause considerable morbidity and mortality, and many high-risk AML patients are not candidates for HCT, due to age, comorbidity and/or lack of an available donor. Adoptively transferred autologous T cells engineered to express a high affinity T cell receptor (TCR) that recognizes the AML-associated antigen Wilms’ Tumor Antigen 1 (WT1) represents a strategy to provide a numerically potent immune response in the absence of HCT. WT1-specific T cells in vitro and in vivo can recognize and kill leukemia cells due to the expression of abnormally high levels of WT1, without affecting normal cells including hematopoietic stem cells that express lower levels of WT1. In two previous clinical trials, we targeted the HLA A+02-restricted immunoproteasome (IP)-dependent WT1126-134 epitope, with CD8 T cell clones or CD8 ‘TCRC4’-transduced T cells, documenting direct anti-leukemic activity and prevention of post-HCT relapse. However, we noted AML escape after loss of IP expression and IP-dependent epitope presentation, and/or limited persistence of transferred T cells in patients with abundant AML. To overcome these obstacles, we now propose to utilize a high-affinity ‘TCRE50’ that targets the IP-independent WT137-46 epitope and will be inserted in a lentiviral vector encoding CD8?? chains to produce WT137-46-targeting CD4 and CD8 T cells. Moreover, autologous CD62L-expressing naïve and central/stem memory CD8 T cells will be engineered, as these cells are associated with longer persistence in vivo. We plan a Phase I/II trial for patients at high risk of AML relapse who are not HCT candidates. TTCR-E50 will be infused after induction therapy. Shortly after the last infusion of TCR E50-engineered T cells (TTCR-E50), patients will receive azacitidine, which can increase WT1 epitope presentation. We will use high dimensional analytic methods to identify TTCR- E50 and AML cell parameters pre- and post-infusion that correlate with clinical responses vs. AML progression. As the efficacy of transferred T cells has been shown to in part reflect the lack of tumor-associated costimulatory ligands, we will also use in vitro and murine models to explore the pre-clinical safety and efficacy of providing co-stimulatory/survival signals by co-transducing ‘switch’ receptors.