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

用于癌症-免疫相互作用中协调时空信号传导多重成像的超灵敏激酶生物传感器

• 批准号: 10445685
• 负责人: Yingxiao Wang
• 金额: $ 64.91万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445685
• 关键词: Address; Behavior; Benchmarking; Biological; Biomedical Engineering; Biosensor; Cell Line; Cell model; Cell physiology; Cells; Cellular immunotherapy; Color; Cyclic AMP-Dependent Protein Kinases; Development; Directed Molecular Evolution; Disease; Engineering; Environment; Enzymes; Event; Evolution; Face; Fluorescence Resonance Energy Transfer; Goals; Image; Immune; Immunotherapy; In Situ; In Vitro; Knowledge; Life; Link; Lymphocyte-Specific p56LCK Tyrosine Protein Kinase; Malignant Neoplasms; Mammalian Cell; Mediating; Molecular; Monitor; Mus; Nature; Noise; Performance; Phosphotransferases; Play; Post-Translational Protein Processing; Protein Kinase; Proteins; Reporter; Resolution; Role; Series; Signal Transduction; Specificity; System; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Technology; Therapeutic; Time; Toxic effect; Tumor Escape; Variant; Visualization; ZAP-70 Gene; base; calmodulin-dependent protein kinase II; cancer cell; cancer therapy; cellular imaging; chimeric antigen receptor; chimeric antigen receptor T cells; comparative; cytokine release syndrome; design; experimental study; fluorophore; frontier; high resolution imaging; high throughput screening; imaging approach; imaging platform; immunological synapse formation; improved; innovative technologies; insight; live cell imaging; multiplexed imaging; neoplastic cell; new technology; next generation sequencing; novel; optogenetics; prototype; sensor; spatiotemporal; success; therapeutic target; tool; tumor

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.

项目概要： 蛋白激酶是细胞功能的关键调节因子和重要的治疗靶点。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，并能够直接 在其天然生物环境中询问蛋白激酶。该提案的目标是采取这一措施 通过创建一系列超灵敏、高性能生物传感器和 开发多重成像方法用于阐明协调的时空信号传导 癌症-免疫相互作用。我们组建了一支强大的跨学科团队，优势互补 生物传感器技术和信号转导专家张进博士和 Yingxiao Peter Wang，著名生物工程师，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。在我们的初步研究中，我们 开发了单 FP 激酶活性报告基因，可实现信号的灵敏和多重成像 活细胞中的活动。新开发的激酶生物传感器之一实现了非常高的灵敏度，并允许 活体小鼠的高分辨率成像。我们还开发了一个创新技术平台 将定向进化与高通量筛选和下一代测序相结合，开发高通量 直接在哺乳动物细胞中发挥荧光生物传感器的作用。在这个项目中，我们建议整合这些 用于设计新型单荧光团生物传感器的技术平台，用于 PKA、CaMKII、 Lck 和 ZAP70 在活细胞中的活动，并进行并行活细胞成像以探测其动态活动 在 CAR T 和靶肿瘤细胞接触过程中。 基于细胞的免疫疗法彻底改变了癌症治疗，但仍然面临重大挑战。 了解癌症-免疫相互作用的机制对于增强型癌症的开发至关重要 治疗策略。蛋白激酶 Lck、ZAP70、PKA 和 CaMKII 在 T 细胞激活中发挥重要作用， 免疫突触形成和癌症免疫逃避。我们希望对这些内容进行并行审查 在癌症免疫相互作用环境中同时关键节点调节器应该揭示新的见解 深入了解系统行为并确定治疗操作的重要环节。而 PKA、CaMKII、Lck、 和 Zap70 已被选为开发和实施我们的系统生物传感器的首过目标 优化方法，原则上，我们的平台可以很容易地扩展以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 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。