Head-to-Head Comparison of Monomeric, Homodimeric, and Heterodimeric Fibroblast Activation Protein-Targeting...

Fibroblast activation protein (FAP) has been identified as a promising biomarker for tumor-targeting radioligands. Among these, a small molecule FAP inhibitor (FAPI), [⁶⁸Ga]-Ga-FAPI-46, and a cyclic peptide-based FAP-targeting radioligand, [¹⁷⁷Lu]-Lu-FAP-2286, are recognized as promising agents. To date, multiple strategies to further improve the potential of FAPI-46 and FAP-2286 have been employed for the development of FAP-targeting radioligands. Multimerization of FAP-targeting moieties is widely utilized as a promising strategy to improve tumor accumulation. In this study, we newly designed and synthesized three types of FAP-targeting dimers based on FAPI-46 and FAP-2286: heterodimer FAP-PID, small molecule-based homodimer FAP-ID, and cyclic peptide-based homodimer FAP-PD. To identify preferable dimeric structures, we conducted a head-to-head comparison of the three dimers and their monomeric counterparts (FAPI-46 and FAP-2286). All compounds were successfully radiolabeled with ¹¹¹In. Each dimer exhibited comparable stability in murine plasma and in vitro FAP-selectivity and specificity. FAP-binding affinities of [^natIn]-In-FAP-PID and [^natIn]-In-FAP-ID were comparable to or higher than those of the monomeric counterparts. In the biodistribution assays, [¹¹¹In]-In-FAP-PID demonstrated the highest tumor accumulation and tumor/blood ratio among the five radioligands. Furthermore, [¹¹¹In]-In-FAP-PID allowed clear visualization of tumors in the SPECT/CT studies. In conclusion, [¹¹¹In]-In-FAP-PID achieved a favorable tumor-to-background ratio and higher tumor accumulation than homodimers, suggesting the utility of the heterodimeric structure based on both small molecule FAPI and cyclic peptide for FAP-targeting radioligands in tumor imaging and therapy.