A New 68Ga-labeled Heterodimer 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 heterodimeric 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.