Project summary/abstract Engineering trafficking circuits that drive therapeutic T cell infiltration into immune-excluded tumors Engineered chimeric antigen receptor (CAR) T cells have yet to achieve efficacy against solid cancers. Particularly challenging are immune-excluded “cold” tumors, which fail to accumulate large numbers of infiltrating T cells. In such cases, even if therapeutic T cells recognize and kill tumor cells in vitro, they will fail in vivo if they cannot infiltrate the tumor. We propose to engineer synthetic circuits that regulate T cell trafficking as a general strategy to drive therapeutic T cell infiltration into immunologically cold tumors. Immune cells naturally rely on complex trafficking behaviors. They patrol the body to surveil for diseases. Once diseased tissue is identified, they establish local residence and focally expand. Cell trafficking programs largely rely on regulation of three core cellular functions: 1) chemotaxis (modulating cell ingress and egress), 2) cell-cell adhesion (reducing cell egress), and 3) local proliferative signaling (cytokine signaling). While these mechanisms are naturally exploited by T cells, the evolved pathways are susceptible to suppression by numerous tumoral mechanisms. We hypothesize that synthetic regulatory circuits that directly wire tumor antigen signals to control therapeutic T cell chemotaxis, adhesion, and local proliferative signaling will improve targeted infiltration of immune excluded tumors. We propose to develop synthetic trafficking circuit designs through cycles of in silico modeling and in vitro experiments. We will test if synthetic trafficking circuits can improve CAR T cell efficacy, in vivo, using an immunocompetent murine model of immune-excluded pancreatic cancer. The resulting cell trafficking circuits should be applicable to a broad range of solid cancers, as well as other diseases. AIM 1. Design and characterize synthetic T cell trafficking circuits that coordinately regulate chemotaxis, adhesion and local proliferation in response to tumor antigen recognition 1.A. Use multi-scale computational modeling to explore design space of possible T cell trafficking circuits. Use model to identify circuit architectures and parameters that robustly increase tumor-selective infiltration 1.B. Construct a toolbox of modular trafficking circuits using synNotch receptors to control chemotaxis, adhesion, and proliferation in response to tumor antigen recognition; Construct combinatorial library of circuits. 1.C. Test synthetic trafficking circuits in vitro using multicompartment tissue models that measure T cell trafficking and migration. Evaluate circuits in vivo by measuring T cell trafficking in bilateral tumor xenograft mouse models. AIM 2. Use engineered trafficking circuits to improve anti-tumor efficacy in an immune excluded immunocompetent murine model of pancreatic ductal adenocarcinoma. Leverage synthetic trafficking circuits to improve murine α-Mesothelin CAR-T ...