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.Glu2.(FAPI)2 and DOTAGA.PEG2.Glu.(FAPI)2, specifically designed to improve FAP-targeted tumor retention.

METHODS

DOTAGA.Glu2.(FAPI)2 and DOTAGA.PEG2.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 [177Lu]Lu-DOTAGA.Glu2.(FAPI)2, as monotherapy or combined with everolimus.

RESULTS

All radioligands showed > 99% radiochemical purity, high FAP affinity (Kd: 0.9-1.5 nM), hydrophilic profiles (LogDoctanol/PBS ≤-3), and low protein binding (< 10%). They exhibited high internalization and low externalization (~ 20% for [177Lu]Lu-DOTAGA.Glu2.(FAPI)2 and ~ 35% for [177Lu]Lu-DOTAGA.PEG2.Glu.(FAPI)2). The 68Ga-labeled radiotracers displayed sustained tumor uptake up to 3 h p.i. (> 14%I.A./g) with increasing tumor-to-organ ratios. For the 177Lu-labeled analogues the initial uptake was comparable (~ 15%I.A./g at 4 h p.i.), with [177Lu]Lu-DOTAGA.Glu2.(FAPI)2, showing even improved retention compared to [177Lu]Lu-DOTAGA.PEG2.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.Glu2.(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.Glu2.(FAPI)2 demonstrates superior tumor retention, favorable pharmacokinetics, and enhanced therapeutic efficacy, supporting its potential for clinical translation.