Novel Covalent FAP-Targeted [⁶⁸Ga]Ga-DOTA-mFS-KERERG-FAP-2286 for Improved Pharmacokinetics and Enhanced Tumor Uptake

Preclinical and clinical investigations have revealed the promising potential of various fibroblast-activating protein inhibitor (FAPI) radiopharmaceuticals. One strategy of particular interest involves the covalent targeting of these FAPI-based agents to fibroblast-activating protein (FAP) itself, which has been shown to enhance tumor uptake and retention. This study aims to optimize ligand structures through covalent binding strategies, developing an optimal radionuclide-based tracer for tumors with high FAP expression. All precursors were synthesized with DOTA-FAP-2286 as the molecular skeleton. The physicochemical properties, imaging, and biodistribution of radioactive ligands were investigated by ⁶⁸Ga-labeling to evaluate their pharmacokinetic properties, as well as their affinity and specificity for FAP. The clinical transformation of [⁶⁸Ga]Ga-DOTA-mFS-KERERG-FAP-2286 PET imaging was performed. Two novel covalent FAP-targeted ligands, DOTA-mFS-FAP-2286 and DOTA-mFS-KERERG-FAP-2286, were successfully synthesized. The K_D values for DOTA-FAP-2286, DOTA-mFS-FAP-2286, and DOTA-mFS-KERERG-FAP-2286 are 2.8 nM, 84 nM, and 0.21 nM, respectively. In the HEK293huFAP tumor model, the tumor uptake values of [⁶⁸Ga]Ga-DOTA-FAP-2286, [⁶⁸Ga]Ga-DOTA-mFS-FAP-2286, and [⁶⁸Ga]Ga -DOTA-mFS-KERERG-FAP-2286 at 2 h were 15.21 ± 2.53% ID/g, 13.65 ± 1.64% ID/g, and 23.33 ± 1.96% ID/g, respectively. The biodistribution and imaging studies showed that [⁶⁸Ga]Ga-DOTA-mFS-FAP-2286 was excreted through both the hepatic-biliary and urinary systems, while [⁶⁸Ga]Ga-DOTA-mFS-KERERG-FAP-2286 was excreted solely through the urinary system. Micro-PET/CT imaging studies revealed that [⁶⁸Ga]Ga-DOTA-mFS-KERERG-FAP-2286 was observed to exhibit increased tumor uptake and retention along with reduced off-target accumulation relative to [⁶⁸Ga]Ga-DOTA-FAP-2286. In tumor patients' clinical translation imaging, [⁶⁸Ga]Ga-DOTA-mFS-KERERG-FAP-2286 PET/CT demonstrated higher uptake and identified more small lymphatic and distant metastases than [⁶⁸Ga]Ga-DOTA-FAP-2286 PET/CT. The potential diagnostic value of [⁶⁸Ga]Ga-DOTA-mFS-KERERG-FAP-2286 lies in its design as a derivative of the DOTA-FAP-2286 scaffold, incorporating a sulfur(VI) fluoride exchange (SuFEx)-based covalent warhead and a hydrophilic polypeptide. This modification is anticipated to alter the pharmacokinetic profile, leading to enhanced tumor uptake and retention, thereby improving clinical diagnostic accuracy.