In vitro and in vivo analyses of eFAP: a novel FAP-targeting small molecule for radionuclide theranostics and other oncological interventions

Abstract Background Fibroblast activation protein (FAP), a transmembrane serine protease overexpressed by cancer-associated fibroblasts in the tumor stroma, is an interesting biomarker for targeted radionuclide theranostics. FAP-targeting radiotracers have demonstrated to be superior to 18 FFDG PET/CT in various solid cancers. However, these radiotracers have suboptimal tumor retention for targeted radionuclide therapy (TRT). We aimed to develop a novel FAP-targeting pharmacophore with improved pharmacokinetics by introducing a substitution at the 8-position of (4-quinolinoyl)-glycyl-2-cyanopyrrolidine, which allows for conjugation of a chelator, dye, or other payloads. Results Here we showed the synthesis of DOTA-conjugated eFAP-6 and sulfo-Cyanine5-conjugated eFAP-7. After chemical characterization, the uptake and specificity of both tracers were determined on FAP-expressing cells. In vitro, 111 InIn-eFAP-6 demonstrated a superior affinity and a more rapid, although slightly lower, peak uptake than gold standard 111 InIn-FAPI-46. Confocal microscopy demonstrated a quick FAP-mediated internalization of eFAP-7. Studies with HT1080-huFAP xenografted mice confirmed a more rapid uptake of 177 LuLu-eFAP-6 vs. 177 LuLu-FAPI-46. However, tumor retention at 24 h post injection of 177 LuLu-eFAP-6 was lower than that of 177 LuLu-FAPI-46, hereby currently limiting its use for TRT. Conclusion The superior affinity and faster tumor accumulation of eFAP-6 over FAPI-46 makes it a suitable compound for radionuclide imaging. After further optimization, the eFAP series has great potential for various oncological interventions, including fluorescent-guided surgery and effective targeted radionuclide theranostics.