Project summary: Protein kinases are key regulators of cellular function and important therapeutic targets. Fluorescent protein-based biosensors have revolutionized the way we study these signaling enzymes and enabled direct interrogation of protein kinases in their native biological contexts. The goal of this proposal is to take this technology to the next frontier by creating a series of ultrasensitive, high-performance biosensors and developing multiplexed imaging approaches to be used in elucidating coordinated spatiotemporal signaling in cancer-immune interactions. We have assembled a strong interdisciplinary team with complementary expertise, including Dr. Jin Zhang, an expert in biosensor technologies and signal transduction, and Dr. Yingxiao Peter Wang, a renowned bioengineer whose lab focuses on engineering fluorescent biosensors, optogenetic tools, and chimeric antigen receptor (CAR) T technologies. In our preliminary studies, we developed single-FP kinase activity reporters that enable sensitive and multiplexed imaging of signaling activities in living cells. One of the newly evolved kinase biosensor achieved very high sensitivity and allowed high-resolution imaging in live mice. We have also developed an innovative technology platform that integrates directed evolution with high-throughput screening and next-generation sequencing to develop high- performance fluorescent biosensors directly in mammalian cells. In this project, we propose to integrate these technology platforms to engineer novel single-fluorophore biosensors for multiplexed imaging of PKA, CaMKII, Lck, and ZAP70 activities in living cells and perform parallel live-cell imaging to probe their dynamic activities during the CAR T and target tumor cell engagement. Cell-based immunotherapy has revolutionized cancer treatment, but still faces significant challenges. Understanding the mechanisms of cancer-immune interactions is critical for the development of enhanced therapeutic strategies. Protein kinases Lck, ZAP70, PKA, and CaMKII play essential roles in T cell activation, immunological synapse formation and cancer-immune evasion. We expect that parallel examination of these key node regulators simultaneously in cancer-immune interacting environments should reveal novel insights into the systems behaviors and identify essential links for therapeutic manipulation. While PKA, CaMKII, Lck, and Zap70 have been chosen as first-pass targets to develop and implement our systematic biosensor optimization approach, in principle, our platform can be readily extended to generate other kinase biosensors and develop biosensors capable of monitoring other posttranslational modification events in live cells. We believe that the success of this project will revolutionize biosensor engineering and kinase imaging to have a transformative impact on the treatment of cancer and other diseases.