Engineering Smart Antibody-like Protein Scaffolds with precision switches

项目概要/摘要 该重点技术研发提案的目标是开发和应用模块化和通用化 生成配备精度的智能抗体样蛋白支架（APS）的工程方法 开关，可以通过光或药物控制，以远程控制内源性蛋白质和 多个生物系统中的细胞生理学。在过去的十年中，各种化学遗传学和 光遗传学工具旨在可视化、离域、修改和降解感兴趣的蛋白质 (POI)。 然而，这些工程工作通常需要对目标兴趣点具有广泛的先验知识。至 调节活细胞或生物体中的内源性 POIs，必须用光或化学敏感的标记 POIs 通过基因敲入或基因组工程模块，从而使该过程相当耗时和资源 消耗。此外，一些现有的化学/光遗传学工具仍然存在速度相对缓慢的问题。 激活动力学、部分不可逆性、化学开关的有限选择以及提示的动态范围狭窄 引起的变化。为了应对这些挑战，跨学科团队建议设计模块化 精确切换到单域抗体样蛋白质支架，而不是内源性靶标本身， 严格控制 POI 和相关的生物活动或途径。特别的是，团队将 将七个选定的 APS 模板（包括纳米抗体、单体和亲和体）与创新的 光遗传学和化学遗传学方法开发新一代光或化学可控 APS （分别命名为 LiAPS 和 ChiAPS）。开关的优先选择包括： (i) 光子 发射蓝光、远红光和近红外 (NIR) 范围（400-800 nm），使现有的光谱多样化 LiAPS 并显着改善其动力学和动态特性（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）有望减少转化障碍 应用程序（具体目标 2）。这些可切换的 APS 将使团队能够远程控制抗体-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目标 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。 细胞中生物过程的精确启动和终止，以及远程体内免疫或 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 提供了证明所提出的新方法的高度可行性以及团队的 掌握申请中描述的所有方法、测定和模型。创新的 该项目生成的分子工具包将提供多种精密开关选择，以实现 许多未来的生物学问题，并对生物医学领域产生重大且可持续的影响。

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.