Engineering a Bicyclic Peptide-Based Positron Emission Tomography Probe Targeting Urokinase-Type Plasminogen Activator Receptor Enables Early Sensing and Therapeutic Monitoring of Pulmonary Fibrosis.

Pulmonary fibrosis remains challenging to detect at an early stage because current clinical imaging relies primarily on high-resolution computed tomography (HRCT), which detects established structural damage rather than active fibrotic remodeling. To address this sensing gap, we engineered and evaluated [68Ga]Ga-HT448, a positron emission tomography (PET) probe based on a high-affinity bicyclic peptide targeting the urokinase-type plasminogen activator receptor (uPAR), a receptor upregulated across multiple cell types during fibrogenesis. Probe performance was assessed through longitudinal PET/CT imaging in two mechanistically distinct murine models of pulmonary fibrosis (bleomycin-induced and radiation-induced), benchmarked head-to-head against the clinically translated fibroblast activation protein-targeted probe [68Ga]Ga-FAPI-04, and validated against HRCT, histopathology, and immunohistochemistry, including pharmacodynamic assessment during nintedanib therapy. [68Ga]Ga-HT448 exhibited high uPAR binding affinity (KD = 7.85 nM), favorable pharmacokinetics, and low pulmonary background, enabling sensitive detection of increased lung uptake as early as Day 7 post-induction in both models, preceding HRCT-detectable structural changes and correlating strongly with fibrosis severity and uPAR expression. Compared with [68Ga]Ga-FAPI-04, the uPAR-targeted probe [68Ga]Ga-HT448 produced earlier, higher, and more sustained signals during disease progression and demonstrated a significantly greater reduction in signal following antifibrotic treatment (p < 0.001 vs p > 0.05). These findings establish uPAR-targeted PET as a sensitive and pharmacodynamically responsive approach for early sensing and therapeutic monitoring of pulmonary fibrosis.