PROJECT SUMMARY Each year, nearly 20,000 patients in the United States are diagnosed with acute myeloid leukemia (AML), and 5-year overall survival rates remain dismal despite high intensity chemotherapy and, in many cases, stem cell transplant. Success of chimeric antigen receptor (CAR) T cell therapy in patients with B cell malignancies has prompted intense interest in applying this revolutionary type of immunotherapy to AML. However, clinical translation has been limited by overlap of AML target expression on indispensable, healthy tissues, which increases their susceptibility to CAR T cell-mediated cytotoxicity. Endothelial cell toxicity is of particular concern for some AML targets under investigation. To advance CAR T cell therapy for AML, it is critically important to devise strategies to preserve anti-leukemic efficacy while simultaneously sparing normal tissue from on-target, off-tumor toxicity. The central hypothesis of this proposal is that antigen-specific inhibitory CARs (iCARs) can be engineered to modulate CAR T cell activation signals and will decrease on-target, off-tumor toxicity of AML CAR T cells. Guided by strong preliminary data, this hypothesis will be tested with three specific aims: 1) Identifying optimal inhibitory motifs to incorporate into iCAR-containing NOT-gated CAR T cells; 2) Defining mechanisms by which those inhibitory motifs within iCARs can interrupt CAR T cell signaling using advanced proteomic techniques; and 3) Determining endothelial-specific NOT-gate surface targets by integrating transcriptomic and proteomic data. Key innovations of this proposal include implementation of a targeted screen to identify a best- in-class iCAR that will be generalizable across targets and application of mass cytometry (CyTOF) to interrogate fundamental inhibitory signaling mediators in CAR T cells. While the immediate focus of this proposal is designing a NOT-gated CAR for AML, principles defined by these experiments will provide the framework for applying this technology broadly to other tumor types. The proposed research activities are part of a comprehensive career development plan that will build on past expertise of the applicant and are crucial to her development as an independent investigator focused on translational immunotherapy. In particular, the applicant will gain expertise in high dimensional proteomics and bioinformatics by taking advantage of the world class scientific and mentorship environment at Stanford University. The applicant will be mentored by Dr. Crystal Mackall, renowned for her work on fundamental CAR T cell immunology and translational immunotherapy, and co-mentored by Dr. Ravi Majeti, an expert on therapeutic targeting of AML stem cells. Under their guidance, along with the assembled advisory committee (including Drs. Kara Davis, Ansuman Satpathy, and Kenneth Weinberg) and specific educational plan, the applicant will receive the necessary support and resources to accomplish the proposed aims an...