A heterodimeric radioligand labeled with gallium-68 targeting fibroblast activation protein.

Background: Fibroblast activation protein (FAP) targeting radiotracers have emerged as promising agents for cancer imaging and therapy. Recent advancements have focused on optimizing these agents for better tumor targeting and enhanced theranostic efficacy. In this study, we introduced a novel heterodimeric radioligand labeled with gallium-68, which targets FAP. We aimed to evaluate its in vitro and in vivo performance, comparing its efficacy with monomeric FAPI derivatives.

Results: The heterodimeric ligand BiFAPI was synthesized by conjugating a cyclic peptide with a quinoline-based motif via a DOTA chelator. [⁶⁸ Ga]Ga-BiFAPI demonstrated high radiochemical purity (> 95%) and exceptional stability in physiological conditions, as well as in both PBS and serum. In vitro studies revealed that the binding affinity of BiFAPI was comparable to that of FAP2286 and FAPI-04. Notably, [⁶⁸ Ga]Ga-BiFAPI exhibited superior cellular uptake, with rapid internalization and slower efflux rates. Micro-PET/CT imaging in tumor-bearing mice demonstrated significantly higher tumor uptake than [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. Co-injection with a FAP inhibitor reduced tumor uptake, confirming the tracer's FAP specificity. In vitro autoradiography, immunohistochemistry, and Western blotting confirmed the correlation between radioactive tracer accumulation and FAP-positive regions. Biodistribution studies revealed high tumor-to-blood ratios and rapid clearance from non-target tissues, further supporting the tracer's favorable pharmacokinetics.

Conclusion: [⁶⁸ Ga]Ga-BiFAPI demonstrated superior tumor-targeting properties, higher tumor uptake, and favorable pharmacokinetics compared to [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. Its promising performance in preclinical models positioned it as a potentially valuable agent for FAP-targeted PET imaging and cancer theranostics.