Exploration and Evaluation of PEGylation and Aromatic Substitution of Covalent FAP-Targeted Radiopharmaceuticals: Preclinical and Preliminary Clinical Studies

Abstract Fibroblast activation protein (FAP) is an attractive target for cancer theranostics; however, currently available FAP-targeted radiopharmaceuticals often exhibit rapid clearance and insufficient tumor retention. In this study, we designed two covalently modified FAP-targeted radiotracers, [68Ga]Ga-T1 and [68Ga]Ga-T2, by incorporating a sulfonyl fluoride (SuFEx) covalent warhead together with PEGylation and naphthalene substitution strategies. Both tracers were synthesized successfully and radiolabeled with 68Ga at high radiochemical purity (>98%). Molecular docking demonstrated favorable binding to FAP, with docking scores of −7.9 kcal/mol for T1 and −7.1 kcal/mol for T2, and both tracers showed high binding affinity in U87MG cells (IC50 = 4.46 nM and 6.63 nM, respectively). In U87MG tumor-bearing mice, both tracers showed specific tumor targeting and prolonged tumor retention, achieving a tumor uptake of 9.21 ± 0.26 %ID/g and 10.48 ± 1.80 %ID/g at 1 h post injection for [68Ga]Ga-T1 and [68Ga]Ga-T2, respectively, and FAP expression in the xenografts was confirmed by immunohistochemistry. No treatment-related toxicity was observed at doses up to 37 MBq. Preliminary clinical PET imaging further confirmed effective tumor visualization with [68Ga]Ga-T1 in two patients with lung cancer. Notably, increased uptake in glandular tissues, particularly the thyroid and pancreas, was observed in humans but was not predicted by preclinical biodistribution studies. Collectively, these findings demonstrate the feasibility of applying a covalent targeting strategy to FAP-directed molecular imaging and provide initial insights into the pharmacokinetic and biodistribution characteristics of covalent FAP-targeted radiopharmaceuticals. The results further suggest that optimization principles established for noncovalent FAP inhibitors may not be directly transferable to covalent systems, offering considerations for a future probe design.