PROJECT SUMMARY/ABSTRACT Chimeric Antigen Receptors (CAR) are engineered receptors that direct the killing activity of T cells to targets of interest and have had dramatic results in the treatment of B cell leukemia. However, the application of this technology to lung tumor remains challenging in part due to inhibitory tumor microenvironments (TME). An immunosuppressive TME is seen even at the earliest stages of human lung cancer. These tumors are infiltrated with immunosuppressive cells including macrophages and T regulatory cells, and have a reduction in effector T cells. Clinical trials using large doses of solid-tumor directed CAR T cells did not find clear radiographic responses. While numerous approaches to improve CAR T cell persistence and killing of solid tumors have been developed, to-date, it remains unclear why clinical responses to second-generation CAR T cells have not reproduced the dramatic success seen in the treatment of B cell malignancies. Evidently, this challenge requires mechanistic studies that are only possible in clinically meaningful solid tumor models. This proposal aims to define the factors what have limited CAR T cell efficacy against solid tumors by building an immunocompetent tumor model and testing novel therapeutic strategies. We propose to develop the first autochthonous solid tumor model targeted by CAR T cells. This model will allow us to test CAR T cells on tumors that faithfully recapitulate the immunosuppressive TME. Prior studies have suggested that radiation can alter the TME. We will use this model to determine if and how radiotherapy can augment CAR T cell killing of solid tumors. Furthermore, recent gene editing and delivery advances in CAR T cells have created new avenues to develop novel CAR T cell therapies that require immunocompetent models for preclinical validation and safety testing. We will use this platform to test different therapeutic strategies to overcome the immunosuppressive TME that include tumor- directed radiotherapy, CAR T cell expansion, and gene editing of CAR T cells with CRISPR/Cas9 to avoid immunosuppressive interactions or reverse them. These experiments will aid in the advancement of CAR T cell therapy for solid tumors and provide a foundation to determine the applicability of TME-directed approaches to other targetable solid malignancies.