Affibody-targeted fluorogen activating protein for in vivo tumor imaging

Molecular imaging using near-infrared (NIR) fluorescence is useful for intraoperative imaging and real-time margin identification. Directly conjugated IR dyes possess useful properties for in vivo imaging, but conjugation often substantially alters the circulation dynamics of targeting moieties. We developed and characterized a new tumor-targeting probe, affiFAP, which consists of a protein that specifically binds EGFR (affibody) and a fluorogen activating protein (FAP). This compact molecular recognition reagent can reversibly bind and activate fluorescence of otherwise nonfluorescent dyes and allows tumor visualization with low nonspecific tissue staining. We demonstrate molecular pre-targeting of affiFAPs and subsequent systemic or topical application of fluorogenic dye to achieve high contrast, fast clearance, and good tissue penetration that may be used in clinical settings to molecularly define tumor margins.