Optimizing FAPI homodimers for theranostic applications: The impact of the linker design in DOTAGA.Glu2.(FAPI)2 and DOTAGA.PEG2.Glu.(FAPI)2

Purpose This study evaluated the imaging performance, tumor retention, and therapeutic potential of two new FAPI homodimers, DOTAGA.Glu 2 .(FAPI) 2 and DOTAGA.PEG 2 .Glu.(FAPI) 2 , specifically designed to improve FAP-targeted tumor retention. Methods DOTAGA.Glu 2 .(FAPI) 2 and DOTAGA.PEG 2 .Glu.(FAPI) 2 were radiolabeled with gallium-68 and lutetium-177. In vitro studies included lipophilicity, protein binding, saturation binding, internalization and externalization using FAP + CAFs. In vivo evaluation in PC3-mice comprised biodistribution, metabolic stability, blood and organ clearance, PET/SPECT/CT imaging, and autoradiography. Therapeutic efficacy was assessed using fractionated radioligand therapy with [ 177 Lu]Lu-DOTAGA.Glu 2 .(FAPI) 2 , as monotherapy or combined with everolimus. Results All radioligands showed > 99% radiochemical purity, high FAP affinity (K d : 0.9–1.5 nM), hydrophilic profiles (LogD octanol/PBS ≤-3), and low protein binding (< 10%). They exhibited high internalization and low externalization (~ 20% for [ 177 Lu]Lu-DOTAGA.Glu 2 .(FAPI) 2 and ~ 35% for [ 177 Lu]Lu-DOTAGA.PEG 2 .Glu.(FAPI) 2 ). The 68 Ga-labeled radiotracers displayed sustained tumor uptake up to 3 h p.i. (> 14%I.A./g) with increasing tumor-to-organ ratios. For the 177 Lu-labeled analogues the initial uptake was comparable (~ 15%I.A./g at 4 h p.i.), with [ 177 Lu]Lu-DOTAGA.Glu 2 .(FAPI) 2 , showing even improved retention compared to [ 177 Lu]Lu-DOTAGA.PEG 2 .Glu.(FAPI) 2 (4.2 ± 0.5 vs. 1.2 ± 0.02%I.A./g at 96 h). Blood clearance was rapid and biphasic, with faster kinetics for DOTAGA.Glu 2 .(FAPI) 2 . For the same compound, tumor half-life was significantly prolonged (44.5 vs. 19 h), resulting in 2-fold higher tumor exposure (AUC: 971 ± 74 vs. 542 ± 28%I.A.×h/g). Imaging confirmed high tumor-to-background ratios, while autoradiography revealed heterogeneous intratumoral FAP distribution. Fractionated therapy significantly inhibited tumor growth and improved survival, further enhanced by everolimus. Conclusion DOTAGA.Glu 2 .(FAPI) 2 demonstrates superior tumor retention, favorable pharmacokinetics, and enhanced therapeutic efficacy, supporting its potential for clinical translation.