Design, build and test of a targeted synthetic protein strategy.

INTRODUCTION: Incorporating minimal antibody regions into engineered proteins is an effective strategy for creating targeted therapeutics and diagnostics. However, a major barrier to clinical translation is the lack of efficient methods to track their performance in vivo. Bioluminescence imaging using targeted reporter proteins offers a promising solution to this challenge. The aim of this study was to develop a novel in vivo imaging strategy using computationally engineered targeted synthetic proteins and to establish a validated pipeline for their design, construction, and testing. METHODS: Multi-part synthetic proteins were designed and screened using in silico tools. These constructs targeted either an example bacterial target (S. aureus ClfA) or a cancer biomarker (MUC1). The top candidates, featuring antibody fragments (ScFv) and a luciferase reporter, were subsequently produced and tested in vitro and in murine models. RESULTS: Following computational design, select proteins were successfully produced and showed specific binding to their intended targets in vitro. Subsequent in vivo studies demonstrated that systemically administered proteins specifically accumulated at target cell locations. This was confirmed by localized bioluminescence that was dependent on both target cell number and protein quantity. DISCUSSION: This study demonstrates the use of targeted reporter proteins for real-time in vivo imaging and validates an effective workflow integrating computational design with wet-lab experimentation. Ultimately, this establishes a target-specific reporter protein platform that can be adapted for sensitive in vivo imaging of diverse targets, from bacterial infections to tumors.