UC San Diego

Protein engineering through directed evolution has been extensively used to improve and modify the structure and function of proteins for wide range of applications. In this dissertation, I utilized the conventional directed evolution through yeast surface display library to engineer a single molecular module, i.e., monobody to have high binding affinity to R-Phycoerythrin. This engineered monobody was then applied to 1) a FRET biosensor with improved spatial resolution to monitor biological process of cancer cell invasion, and 2) a universal chimeric antigen receptor (CAR) for cancer immunotherapy. To further extend the power of directed evolution, I combined it with other technologies, including mammalian cell library, functional screening by FACS-based FRET, high-throughput DNA sequencing, and sequence-function analysis, to systematically optimize the sensitivity, specificity, and dynamic range of the FRET biosensors for monitoring kinase activities with high spatiotemporal resolution in living cells. These optimized biosensors can be used to study the cell signaling of CAR T cells upon engagement with cancer cells to improve the efficiency of CAR T cell immunotherapy. Overall, this dissertation illustrates the potential of directed evolution as a tool to engineer the molecular modules/sensors for biomedical diagnosis and immunotherapy.