FAP-Inhibitor Homodimers with Engineered Linker-Spacer Units: Toward Optimized Radiopharmaceuticals

Monomeric fibroblast activation protein inhibitors (FAPi) based PET tracers have demonstrated very suitable imaging characteristics preclinically in various cancer models. More recently, homodimeric FAP inhibitors (FAPi dimers), that consist of two identical FAPi targeting vectors, have been developed, offering prolonged tumor retention and thus enhancing the potential of targeted radioligand therapy (TRT) with long-lived therapeutic radionuclides such as ¹⁷⁷Lu. Building on the promising results of DOTAGA.Glu.(FAPi)₂ in preclinical and patient studies, we synthesized a series of novel homodimeric compounds incorporating different linker and spacer units (NPyr-, Glu₂-, and PEG₂.Glu-linked dimers), each coupled to either DOTAGA or DO3A as the chelator. Radiolabeling with ⁶⁸Ga and ¹⁷⁷Lu resulted in high to quantitative radiochemical conversion and purity. All FAPi homodimers exhibited high hydrophilicity, excellent stability in both human serum and PBS and showed subnanomolar FAP affinity with high selectivity over PREP (prolyl endopeptidase) and DPP4 (dipeptidyl peptidase 4). In parallel, saturation binding studies were performed to determine the affinity of the corresponding ⁶⁸Ga-labeled Glu₂- and PEG₂.Glu-linked DOTAGA derivatives and exhibited high FAP affinity (K _d: 1.1-1.2 nM). Internalization was rapid, with up to 95% of total cell-bound activity internalized within 30 min in CAFs (cancer-associated fibroblasts). Metabolic studies confirmed that ⁶⁸Ga-labeled radioligands remained stable in plasma, and PET quantification demonstrated favorable pharmacokinetics. Overall, these findings highlight the new FAPi homodimers as promising candidates for efficient ¹⁷⁷Lu-based TRT. Further in vivo studies are warranted to comprehensively assess their pharmacological properties for therapeutic application.