Project Summary Engineered T cells that express a chimeric antigen receptor (CAR) specific to tumor antigens have shown exceptional efficacy in hematologic malignancies. However, their success in treating solid tumors remains limited. Our understanding of the primary barriers faced by CAR T cells in solid tumors is hindered by a lack of tissue specimens and suitable high-depth tumor microenvironment (TME) profiling technologies. Model systems have largely been unsuccessful in accurately predicting human CAR T cell biology. To understand the barriers faced by CAR T cells in solid tumors, Andrew J. Rech proposes an integrated spatial multi-omic analytical platform. Dr. Rech plans to apply this platform to tissue specimens taken before and after CAR T cell infusion from five CAR T cell clinical trials. These trials focus on pancreatic cancer, prostate cancer, and highly active IL-18-secreting CAR T cells in non-Hodgkin lymphoma. Dr. Rech will leverage the significant differences in the TME in these diseases to understand the principles of response and resistance to therapy. Dr. Rech’s overarching hypothesis is that CAR T cell function and therapeutic efficacy are constrained by specific, actionable interactions between tumor, CAR T cells, and tumor-infiltrating myeloid cells. His preliminary data suggest that paracrine interactions between CAR T cells and myeloid cells could limit CAR T cell function and be linked to hyperinflammatory toxicity. In Aim 1, Dr. Rech will define the tumor and stromal features that influence CAR T cell tumor infiltration and function. In Aim 2, he will determine the impact of CAR T cell functional profile on myeloid tumor stroma. Lastly, in Aim 3, Dr. Rech will determine the impact of CAR T cell functional profile on tumor cell plasticity and antigenicity. In each Aim, he will also develop a relevant murine model to validate results mechanistically and enable a path to future phase I clinical trials. In summary, the spatial multi-omic profiling Dr. Rech proposes will identify response, resistance, and toxicity pathways that impact CAR T cell therapy in patients and that can be targeted to improve therapy. In this proposal, Dr. Rech leverages his background as an interdisciplinary wet lab scientist, computational scientist, and board-certified pathologist. He has developed a new line of investigation that is distinct from the research of current and previous mentors and has already achieved major landmarks of scientific independence. Dr. Rech has also garnered substantial institutional support at the University of Pennsylvania, a globally recognized leader in the field of T cell engineering. Along with his mentors and department, Dr. Rech has designed a six-component career development plan to facilitate his transition to independence. In summary, the proposed studies will identify TME response mechanisms to CAR T cell therapy in unprecedented detail in humans, guiding the development of next-generation CAR T cell ther...