Engineering Smart Antibody-like Protein Scaffolds with precision switches

项目摘要/摘要 这一重点技术研发方案的目标是开发和应用模块化和通用性 制备高精度智能抗体样蛋白支架的工程方法 开关，可以由光或药物控制，以远程控制内源蛋白质和 多种生物系统中的细胞生理学。在过去的十年里，各种化学发生和 光遗传工具已被设计用于可视化、非定域化、修饰和降解感兴趣蛋白(POI)。 然而，这些工程工作往往需要对目标POI有广泛的先验知识。至 调节活细胞或生物体中的内源性POI，人们必须用光或化学敏感的标记POI 模块通过基因敲入或基因组工程，从而使这一过程相当耗时和资源- 在消费。此外，一些现有的化学/光遗传工具仍然受到相对缓慢的影响 激活动力学，部分不可逆性，有限的化学开关选择，和狭窄的动态范围的线索- 引发的变化。为了应对这些挑战，跨学科团队建议设计模块化 Precision切换为单域抗体样蛋白支架，而不是内源性靶点本身， 对POI和相关的生物活动或途径进行严格控制。特别是，团队将 将精选的七个APSS模板(包括纳米体、单体体和贴合体)与创新的 开发新一代光或化学可控APSS的光遗传和化学遗传方法 (分别命名为LiAPSS和ChiAPSS)。优先选择的交换机包括：(I)光子 在蓝色、远红和近红外(NIR)范围(400-800 nm)发射，使现有的曲目多样化 LiAPSS并显著改善其动力学和动力学性质(具体目标1)；(2)FDA批准的药物 (抗病毒药物)和饮料(咖啡因及其代谢物)承诺减少翻译障碍 应用(具体目标2)。这些可切换的APS将允许团队远程控制抗体-抗原 在较高的时间和/或空间上以可逆的方式识别和操纵内源目标 决议。同时，该团队将演示工程智能APSS在急性和慢性疾病中的应用 精确启动和终止纤维素酶中的生物学过程，以及远程体内免疫 人类疾病的啮齿动物和果蝇模型中的神经调节。令人信服的初步数据显示 以证明提议的新方法的高度可行性，以及小组的 掌握应用程序中描述的方法、分析和模型的全部内容。创新的 将从该项目生成的分子工具包将提供广泛的精密开关选择，以实现 许多未来的生物学问题，并对生物医学领域产生高度和可持续的影响。

PROJECT SUMMARY / ABSTRACT The goal of this Focused Technology R&D proposal is to develop and apply modular and generalizable engineering approaches to generate smart antibody-like protein scaffolds (APSs) equipped with precision switches, which can be controlled by light or drugs to confer remote control over endogenous proteins and cellular physiology in multiple biological systems. Over the past decade, a variety of chemogenetic and optogenetic tools have been designed to visualize, delocalize, modify, and degrade proteins of interest (POIs). These engineering efforts, nonetheless, often require extensive prior knowledge on the targeted POIs. To regulate endogenous POIs in living cells or organisms, one has to tag POIs with light- or chemical-sensitive modules via genetic knock-in or genome engineering, thereby making the process rather time- and resource- consuming. Furthermore, some of the existing chemo/optogenetic tools still suffer from relatively slow activation kinetics, partial irreversibility, limited choices of chemoswitches , and narrow dynamic ranges of cue- induced changes. To address these challenges, the transdisciplinary team proposes to engineer modular precision switches into single-domain antibody-like protein scaffolds, rather than the endogenous target itself, to confer tight control over POIs and the associated biological activities or pathways. Specially, the team will combine seven selected APSs templates (including nanobody, monobody and affibody) with innovative optogenetic and chemogenetic approaches to develop new generations of light- or chemical-controllable APSs (named as LiAPSs and ChiAPSs, respectively). The prioritized choices of switches include: (i) photons emitting in the blue, far-red, and near infrared (NIR) range (400-800 nm) to diversify the existing repertoire of LiAPSs and significantly improve their kinetic and dynamic properties (Specific Aim 1); (ii) FDA-approved drugs (antivirals) and beverages (caffeine and its metabolites) that promise to reduce barriers for translational applications (Specific Aim 2). These switchable APSs will allow the team to remotely control antibody-antigen recognition and to manipulate endogenous targets in a reversible manner at high temporal and/or spatial resolution. In parallel, the team will demonstrate the applications of engineered smart APSs for acute and precise initiation and termination of biological processes in cellulo, as well as remote in vivo immuno- or neuromodulation in both rodent and Drosophila models of human diseases. Compelling preliminary data have been provided to demonstrate the high feasibility of the proposed new approaches, as well as the team’s mastery of the repertoire of methods, assays, and models described in the application. The innovative molecular toolkit to be generated from the project will offer a wide choices of precision switches to enable many future biological questions and impose a high and sustainable impact to the biomedical field.