Abstract The use of T cells engineered to express specific chimeric antigen receptors (CARs) to treat cancer has generated durable cures for many types of cancer and resulted in the first FDA approved CAR-T cell therapy to treat childhood acute lymphoblastic leukemia in 2017. Despite this success, CAR-T immunotherapies have been much less effective at targeting solid tumors. Part of this limited success stems from the solid tumor microenvironment, which forms a physical barrier to immune cell infiltration and produces soluble factors that downregulate T cell activity and accelerate T cell exhaustion. While immunotherapies targeting solid tumors are initially effective, the tumor microenvironment’s inhibition of T cells prevents these treatments from producing durable responses. In this application, we propose a novel CAR-T cell therapy aimed to improve outcomes for patients with advanced stage pancreatic cancer by overcoming the deficiencies that plague current CAR-T cell therapies. To this end, we will engineer T cells to express multiple CARs, enabling these cells to target tumor cells and cells in the immune-suppressive tumor microenvironment. Specifically, we will leverage the non-viral, Tc Buster DNA transposon system to insert a large multicistronic genetic construct containing multiple CARs and a selection marker into T cells. Using this platform, we will generate T cells with CARs targeting mesothelin (MSLN), a protein expressed by 80-85% of pancreatic cancer tumors, and fibroblast activation protein (FAP), a marker of cancer associated fibroblasts in the tumor microenvironment. We will then select a pure population of T cells expressing MSLN- and FAP-CARs and determine the activity and specificity of these cells in vitro. We expect that engineered T cells will generate a specific and robust response, eliciting cytotoxic functions only against cells expressing their target antigen. We will then determine the efficacy of engineered T cells in vivo using a xenograft mouse model to generate MSLN and FAP positive pancreatic carcinomas followed by adoptive transfer of T cells. We expect immunotherapeutic delivery of bispecific T cells expressing FAP-CARs and MSLN- CARs will elicit a robust and long-lasting T cell response against MSLN+/FAP+ solid tumors resulting in tumor shrinkage and increased survival. Furthermore, we expect the development of a flexible, efficient, and reliable process to generate bispecific T cells with a single, non-viral gene delivery approach will facilitate the emergence of novel therapies to overcome many issues facing engineered T cell therapy today, including antigen escape and target specificity.