A new ⁶⁸Ga-labeled dimeric FAP ligand to advance targeted PET imaging of cancer

Background: Fibroblast activation protein (FAP) has emerged as a critical biomarker in the tumor microenvironment of various cancers. Radioligands targeting FAP have shown promise for cancer theranostics. To advance cancer imaging, we synthesized a dimeric radioligand based on a 4-quinolinoyl-glycyl-2-cyanopyrrolidine scaffold and conducted studies in cells and mouse tumor models to evaluate its target-binding affinity and PET imaging performance. Results: OncoFAP, a commercially available FAP ligand, was conjugated via amide coupling with 1,4-butanediamine to generate a monomeric intermediate, hmFAP₁. A homodimeric molecule, hmFAP₂, was synthesized by tethering two hmFAP₁ moieties into a single construct. In enzymatic inhibition assays, both hmFAP₁ and hmFAP₂ demonstrated specific antagonist activity against FAP, with hmFAP₂ exhibiting a 14-fold increase in inhibitory potency compared to hmFAP₁. Cy5.5 fluorescent derivatives of hmFAP₁ and hmFAP₂ were generated for cell-binding assays in HeLa cells and xenografted tumors with positive FAP expression, revealing enhanced targeting efficacy of Cy5.5-hmFAP₂. The IC₅₀ values derived from cell-binding curves were 130 nM for hmFAP₁ and 8 nM for hmFAP₂ (P < 0.001). Using DOTA as the chelator, both ligands were radiolabeled with ⁶⁸Ga, yielding stable products [⁶⁸Ga]Ga-DOTA-hmFAP₁ and [⁶⁸Ga]Ga-DOTA-hmFAP₂ for PET imaging. Consistently, [⁶⁸Ga]Ga-DOTA-hmFAP₂ demonstrated superior tumor uptake with high specificity in mice bearing HeLa, MDA-MB-231, and HEK293T xenografts with variable levels of FAP expression. The liver and intestinal radioactivity showed no difference between the groups of mice imaged with [⁶⁸Ga]Ga-DOTA-hmFAP₂ and [⁶⁸Ga]Ga-DOTA-hmFAP₁. Conclusions: hmFAP₂ markedly enhances FAP-targeting efficiency, providing higher binding affinity, improved tumor uptake, and reduced nonspecific distribution compared with its monomeric counterpart. The favorable imaging properties of hmFAP₂ position it as a promising candidate for translation into clinical PET imaging and as a potential scaffold for developing FAP-targeted theranostic agents.