The use of T cells engineered to express specific chimeric antigen receptors (CARs) to treat cancer hasgenerated durable cures for many types of cancer and resulted in the first FDA approved CAR-T cell therapy totreat childhood acute lymphoblastic leukemia in 2017. Despite this success, CAR-T immunotherapies have beenmuch less effective at targeting solid tumors. Part of this limited success stems from the solid tumormicroenvironment, which forms a physical barrier to immune cell infiltration and produces soluble factors thatdownregulate T cell activity and accelerate T cell exhaustion. While immunotherapies targeting solid tumors areinitially effective, the tumor microenvironment's inhibition of T cells prevents these treatments from producingdurable responses. In this application, we propose a novel CAR-T cell therapy aimed to improve outcomes forpatients with advanced stage pancreatic cancer by overcoming the deficiencies that plague current CAR-T celltherapies. To this end, we will engineer T cells to express multiple CARs, enabling these cells to target tumorcells 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 CARsand 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), amarker of cancer associated fibroblasts in the tumor microenvironment. We will then select a pure population ofT cells expressing MSLN- and FAP-CARs and determine the activity and specificity of these cells in vitro. Weexpect that engineered T cells will generate a specific and robust response, eliciting cytotoxic functions onlyagainst cells expressing their target antigen. We will then determine the efficacy of engineered T cells in vivousing a xenograft mouse model to generate MSLN and FAP positive pancreatic carcinomas followed by adoptivetransfer 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 tumorshrinkage and increased survival. Furthermore, we expect the development of a flexible, efficient, and reliableprocess to generate bispecific T cells with a single, non-viral gene delivery approach will facilitate the emergenceof novel therapies to overcome many issues facing engineered T cell therapy today, including antigen escapeand target specificity. Project Narrative
This proposal describes a novel chimeric antigen receptor (CAR)-T cell therapy that aims to improve outcomes
for patients with advanced stage pancreatic cancer. We propose to overcome pancreatic cancer's resistance to
immunotherapy by engineering T cells to express multiple CARs, enabling these cells to target tumor cells and
the immune-suppressive tumor microenvironment. We expect immunotherapeutic delivery of these bispecific T
cells will improve patient outcomes and reduce recurrence in advanced stage pancreatic cancer by increasing
intratumor T cell infiltration and diminishing T cell exhaustion. 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