Abstract Despite recent advances in cancer treatment, patients with pancreatic cancer still do not have effective options for treatment and fewer than 10% survive more than five years. T cell receptor (TCR) based therapies have achieved positive responses in a subset of patients with advanced solid tumors, representing a promising approach for pancreatic cancer. However, low level antigen expression and the immunosuppressive tumor microenvironment impair antigen recognition and limit T cell persistence and function, impeding consistent success against solid tumors. To surmount these challenges, we have devised a strategy to improve anti-tumoral responses in a T cel- based platform. Mesothelin (MSLN) is expressed by 80-85% of pancreatic tumors, making it an attractive TCR target for the treatment of pancreatic cancer. We have validated the cytolytic activity of T cells engineered with human HLA-A2 restricted MSLN specific TCRs against MSLN expressing pancreatic tumor cells in vitro. Previous work from our group with murine Msln specific TCRs in a mouse model of pancreatic cancer further demonstrated engineered T cells reduced tumor burden in vivo but lacked persistence. We thus seek to develop a novel immunotherapy that combines our MSLN specific TCRs with the uniquely potent co- stimulatory properties of a synthetic CD8α:MyD88 fusion protein. In transgenic mice, tumor reactive T cells expressing the CD8α:MyD88 fusion protein demonstrate antigen specific increases in cytokine production, proliferation, and cytotoxicity, eliciting durable tumor regression compared to controls. To evaluate this approach as a clinically relevant immunotherapy platform for pancreatic cancer we will utilize a novel non-viral genetic engineering approach to co-express MSLN specific TCRs with the CD8α:MyD88 fusion protein in primary human T cells. Vector configuration will be optimized by evaluating stable TCR and CD8α:MyD88 fusion surface expression, as well as T cell phenotype, function, and cytotoxicity against pancreatic cancer cells in vitro. Optimized vector configurations will be carried forward and tested in preclinical animal models to evaluate safety and efficacy in support of follow-on IND enabling studies. If successful, our efforts will lead to an innovative new immunotherapeutic strategy to overcome challenges that have prevented meaningful improvement in pancreatic cancer patient outcomes.