Synthesis and preclinical evaluation of FAP-targeting radiotracers for PET and optical imaging

Successful treatment of solid cancers relies on precise diagnosis, e.g. using noninvasive molecular imaging, followed by surgical removal and/or chemo/immunotherapy. Despite advances in pre-operative imaging, real-time intraoperative tools remain limited, which often results in high rates of tumor-positive margins and recurrence after tumor resection. To address this limitation, we aimed to develop multifunctional fibroblast activation protein alpha (FAP) targeting tracers for bimodal medical imaging, enabling both pre-operative noninvasive molecular imaging via positron emission tomography (PET) and optical visualization during intraoperative fluorescence-guided surgery.

NODAGA-FAP647 and NODAGA-FAP800 targeting human FAP (hFAP) were synthesized bearing a (R)-NODAGA chelator and a fluorophore (AlexaFluor647 or IRDye800CW, respectively). Binding affinities and binding kinetics of both unlabeled and ^67/68Ga-labeled compounds were evaluated in vitro using HT1080 cells (hFAP-expressing and wild type, WT) along with respective frozen xenograft tissue sections. Using real-time binding, both compounds exhibited picomolar binding affinities to hFAP via radioactive/fluorescent detection. This was primarily driven by low dissociation rate constants in vitro. Pharmacokinetics and tumor uptake were evaluated via PET and fluorescence imaging in mice bearing xenografts from the same cells. In vivo, both compounds were rapidly distributed and accumulated in hFAP-expressing but not WT-HT1080 tumors within 10-20 min post-injection. Fluorescence imaging showed a similarly good and selective tumor uptake in the first two hours and a qualitatively visible difference compared to WT-HT1080 beyond 24 h. Both compounds were quickly cleared from normal tissue and excreted renally.

Two FAP-targeting bimodal ligands were synthesized and evaluated in vitro and in vivo, showing high specificity and selectivity, along with rapid and selective tumor accumulation. Their long tumor retention and high imaging contrast make them promising candidates for clinical translation.