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

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 [¹⁸F]FDG 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, [¹¹¹In]In-eFAP-6 demonstrated a superior affinity and a more rapid, although slightly lower, peak uptake than gold standard [¹¹¹In]In-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 [¹⁷⁷Lu]Lu-eFAP-6 vs. [¹⁷⁷Lu]Lu-FAPI-46. However, tumor retention at 24 h post injection of [¹⁷⁷Lu]Lu-eFAP-6 was lower than that of [¹⁷⁷Lu]Lu-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.