Engineering Smart Antibody-like Protein Scaffolds with precision switches

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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.