Heterobivalent fibroblast activation protein-targeted Radiotheranostic ligands integrating cyclic peptide and small-molecule motifs for enhanced tumor retention.

Fibroblast activation protein (FAP), which is highly expressed in cancer-associated fibroblasts, has emerged as an attractive target for tumor imaging and radionuclide therapy. However, FAP-targeted radioligands suffer from rapid tumor washout, limiting effective dose delivery. The heterobivalent design strategy effectively enhances target engagement and tumor retention, addressing a key limitation of current FAP-targeted radioligands. In this study, six heterobivalent FAP-targeted radioligands (1-6) were rationally designed by incorporating small-molecule FAP inhibitor motifs, including 2-cyanopyrrolidine and 2-pyrrolidinylboronic acid derivatives, into the cyclic peptide scaffold via systematic optimization of the linker structures and conjugation sites. All ligands were successfully radiolabeled with gallium-68, achieving radiochemical purities exceeding 95% and exhibiting favorable hydrophilicity and high in vitro stability. The heterobivalent ligands demonstrated significantly enhanced FAP binding affinity compared with monomeric ligand FAP-2286. The ligands also exhibited increased cellular uptake, pronounced internalization, and reduced efflux in U87MG cells, relative to their monovalent counterparts. In vivo PET/CT imaging in U87MG xenograft-bearing mice revealed rapid clearance from nontarget tissues and substantial tumor accumulation for all tracers, with [68Ga]Ga-5 exhibiting the highest tumor uptake and superior tumor-to-background contrast. Notably, the therapeutic analog [177Lu]Lu-5 displayed the most favorable tumor-to-kidney area under the curve ratio and achieved significantly improved tumor growth inhibition compared with [177Lu]Lu-FAP-2286. Overall, ligand 5 shows promise as an FAP-targeted radiotheranostic agent and demonstrates potential for further translational development.