PROJECT SUMMARY It is essential to study improved therapies for the treatment of advanced acute myeloid leukemia (AML), as nearly 30% of children will relapse or have refractory disease, bringing the survival down to a mere 20%. Hematopoietic stem cell transplantation (HSCT) is curative for relapsed AML; however, the genotoxicity of bone marrow (BM) conditioning represents a substantial barrier in its use. Current BM conditioning regimens consist of a combination of alkylating chemotherapeutic agents such as busulfan and high doses of total body irradiation (TBI). Similar to the non-targeted chemotherapeutics used as first-line treatment for AML, these conditioning agents are also highly genotoxic and have several harmful toxicities outside of the hematopoietic compartment, such as organ failure and secondary malignancies. In contrast, cellular therapies provide strong support for the use of targeted therapies in the relapsed setting, as evident by five total FDA approved chimeric antigen receptor (CAR) products for the treatment of relapsed cancers, three of which have been approved within the last year. Therefore, we hypothesize that the development of non-genotoxic conditioning regimens that specifically target hematopoietic stem cells (HSCs) and AML will be transformative to the treatment of the disease. The use of c- kit (CD117) as a target for non-genotoxic conditioning has been explored by our lab and others due to its expression on HSCs. Up to 90% of AML patients have c-kit expression, and this expression correlates with poor prognosis and resistance to chemotherapy. We hypothesize that specifically targeting c-kit for AML treatment will reduce tumor burden in addition to serving as a non-genotoxic conditioning regimen prior to HSCT. This proposal seeks to explore a novel ligand-based c-kit directed CAR (SCF CAR) to target HSCs (Specific Aim 1) and AML (Specific Aim 2) by utilizing the c-kit receptor’s natural ligand stem cell factor (SCF) as the recombinant antigen binding domain of our CAR. Importantly, ligands offer a greater understanding of receptor-ligand interactions and can potentially reduce tonic CAR signaling through enhanced protein stability, leading to less T cell exhaustion, among other key advantages. Furthermore, our lab has optimized the use of γδ T cells as a cytotoxic alternative to ⍺β T cells. γδ T cells are uniquely beneficial for this setting since they i) are innate immune cells that do not form memory T cell phenotypes to the same extent as ⍺β T cells, ii) do not cause graft-vs-host disease (GvHD) when transplanted across major histocompatibility (MHC) barriers, iii) contribute to the innate killing of leukemia cells via graft-vs-leukemia (GvL) interactions, as γδ T cells can recognize cancer cell stress antigens, and iv) have been shown to enhance survival and decrease leukemia relapse post-HSCT. We therefore propose utilizing the cytotoxic abilities of γδ T cells to capitalize on these effects, as our lab has op...