Development of [Cu-64]RTX-1363S, a high-affinity ligand for FAP-targeted PET imaging

Aim/Introduction: Fibroblast activation protein-α (FAP) is recognized as a pivotal target in molecular imaging, important in oncology and inflammatory and fibrotic diseases due to its presence in cancer-associated fibroblasts and activated fibroblasts. RTX-1363S, a tri-functional small molecule identified using the Trillium™ platform, integrates a FAP-binding ligand, a pharmacokinetic-modulating albumin-binding moiety, and a chelator for incorporation of radiometals. This high affinity FAP ligand, radiolabeled with Cu-64, Ga-68, and [F-18]AlF, enables specific PET imaging of FAP expressing tissues.
Materials and Methods: The affinity of RTX-1363S and [Cu-nat]RTX-1363S for human FAP was determined by surface plasmon resonance (SPR). Specificity was evaluated against related prolyl peptidases in enzyme assays. Biodistribution of [Cu-67]RTX-1363S in U-87 MG xenograft mice was determined by SPECT/CT imaging and ex vivo gamma counting at 1, 3, and 24 hours post-injection. Biodistribution of [Cu-64]RTX-1363S in male and female Sprague Dawley rats (n=3/sex/time point) was investigated to project human tissue radiation absorbed dosimetry using the %kg/g scaling method. Toxicity was assessed in Sprague-Dawley rats and off-target interactions were assessed with the SafetyScan47 panel.
Results: RTX-1363S and [Cu-nat]RTX-1363S displayed high affinity for human FAP by SPR with dissociation constants (KD) of 0.0123 ± 0.003 nM and 0.0211 ± 0.004 nM, respectively. IC50 values for [Cunat]RTX-1363S were >10 µM for human DPPIV and 2.76 ± 0.79 nM for human PREP, demonstrating minimal cross-reactivity with other prolyl peptidases. SPECT/CT imaging showed significant tumor retention in mice, with [Cu-67]RTX-1363S achieving peak tumor accumulation at 1 h post-injection. Tumor:blood, tumor:kidney and tumor:muscle ratios at 1 h were 15.4, 6.3 and 14.1, respectively. In rats, [Cu-64]RTX-1363S demonstrated rapid clearance through the kidneys, with >80% decrease in blood concentration by 24 h. The projected effective doses for [Cu-64]RTX-1363S in human males and females was estimated to be 31.2 and 17.1 µSv/MBq, respectively. At >100x the anticipated human dose, RTX-1363S produced no toxic side effects in rats. No significant results (defined as >50% inhibition at 10 µM concentration) were observed across a functional panel of 47 targets/pathways commonly implicated in adverse drug reactions. Conclusion: RTX-1363S is a selective, high affinity FAP inhibitor that demonstrates favorable biodistribution to the tumor and rapid background clearance allowing for early tumor imaging. These data, combined with the clean safety profile, underscore the potential of [Cu-64]RTX-1363S for clinical PET imaging of FAP-expressing tissues to provide insights into both cancerous and fibrotic diseases.