Engineering Smart Antibody-like Protein Scaffolds with precision switches

项目总结/摘要 本聚焦技术研发提案的目标是开发和应用模块化和可推广的 工程方法，以产生智能抗体样蛋白质支架（APS）配备了精确的 开关，其可以通过光或药物控制，以赋予对内源性蛋白质的远程控制， 多种生物系统中的细胞生理学。在过去的十年里，各种化学遗传和 已经设计了光遗传学工具来可视化、离域、修饰和降解目的蛋白（POI）。 然而，这些工程工作通常需要关于目标POI的广泛的先验知识。到 为了调节活细胞或生物体中的内源性POI，必须用光敏或化学敏感的标记POI， 模块通过基因敲入或基因组工程，从而使这一过程，而不是时间和资源， 消耗。此外，一些现有的化学/光遗传学工具仍然遭受相对缓慢的生物学效应。 激活动力学、部分不可逆性、有限的化学开关选择和窄的提示动态范围， 诱发的变化。为了应对这些挑战，跨学科团队建议设计模块化 精确转换成单域抗体样蛋白支架，而不是内源性靶点本身， 以赋予对POI和相关生物活性或途径的严格控制。特别是，团队将 联合收割机将七种精选的APS模板（包括纳米抗体、单抗体和双抗体）与创新的 开发新一代光或化学可控APS的光遗传学和化学遗传学方法 （分别命名为LiAPS和ChiAPS）。开关的优先选择包括：（i）光子 在蓝色、远红色和近红外（NIR）范围（400-800 nm）内发射，以使现有的 LiAPS并显著改善其动力学和动力学性质（具体目标1）;（ii）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， 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 在高时间和/或空间上以可逆的方式识别和操纵内源性靶标 分辨率与此同时，该团队将展示工程智能APS在急性和慢性疾病中的应用， 精确启动和终止细胞内的生物过程，以及远程体内免疫或 啮齿动物和果蝇的人类疾病模型中的神经调节。令人信服的初步数据 已提供，以证明所提出的新方法的高度可行性，以及该小组的 掌握申请中描述的方法、测定和模型的全部知识。创新 该项目产生的分子工具包将提供多种精密开关， 许多未来的生物学问题，并施加一个高的和可持续的影响，生物医学领域。

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.