Ultrasensitive kinase biosensors for multiplex imaging of coordinated spatiotemporal signaling in cancer-immune interactions

项目概要： 蛋白激酶是细胞功能的关键调节剂和重要的治疗靶点。荧光 基于蛋白质的生物传感器彻底改变了我们研究这些信号酶的方式， 蛋白激酶在其天然生物学背景下的询问。这项提案的目的是 通过创造一系列超灵敏、高性能的生物传感器， 开发用于阐明协调的时空信号的多路复用成像方法， 癌症免疫相互作用我们组建了一支强大的跨学科团队， 我们的专家包括生物传感器技术和信号转导专家张进博士， 著名生物工程师王颖晓（Yingxiao Peter Wang），其实验室专注于工程荧光生物传感器， 光遗传学工具和嵌合抗原受体（CAR）T技术。在初步研究中，我们 开发了单FP激酶活性报告基因，能够对信号传导进行敏感和多重成像 活细胞中的活动。一种新开发的激酶生物传感器实现了非常高的灵敏度， 高分辨率成像在活体小鼠。我们还开发了一个创新的技术平台， 将定向进化与高通量筛选和下一代测序相结合， 直接在哺乳动物细胞中进行荧光生物传感器。在这个项目中，我们建议将这些 技术平台，以工程设计新型单荧光团生物传感器，用于PKA，CaMKII， Lck和ZAP 70在活细胞中的活性，并进行平行的活细胞成像以探测它们的动态活性 在CAR T和靶肿瘤细胞接合期间。 基于细胞的免疫疗法彻底改变了癌症治疗，但仍面临重大挑战。 了解癌症-免疫相互作用的机制对于开发增强的 治疗策略蛋白激酶Lck、ZAP 70、PKA和CaMK II在T细胞活化中起重要作用， 免疫突触形成和癌症免疫逃避。我们希望对这些问题的平行审查 同时在癌症免疫相互作用的环境中的关键节点调节器应该揭示新的见解 进入系统行为，并确定治疗操作的基本环节。PKA、CaMKII、Lck、 和Zap 70被选为开发和实现我们的系统化生物传感器的首过目标 原则上，我们的平台可以很容易地扩展到生成其他激酶生物传感器 并开发能够监测活细胞中其他翻译后修饰事件的生物传感器。我们 我相信，该项目的成功将彻底改变生物传感器工程和激酶成像， 对癌症和其他疾病的治疗产生变革性影响。

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.