"Imaging Triplets on Two Wheels": 61Cu-, 64Cu-, and 68Ga-labeled Bicyclic Peptides Targeting Nectin-4 Enable High-Contrast PET Imaging of Urothelial Carcinoma and Triple-Negative Breast Cancer.

Nectin-4 is a validated target for theranostic approaches toward urothelial carcinoma, and radiolabeled ligands for PET and SPECT imaging are gaining increasing interest for noninvasive quantification of Nectin-4 prior to a targeted therapy. The goal of this study was to develop bicyclic peptides that allow radiolabeling with three different radionuclides (61Cu, 64Cu, and 68Ga) and to evaluate their suitability for high-contrast PET imaging in urothelial carcinoma (UC) and triple-negative breast cancer (TNBC). For this purpose, NECT-330, NECT-334, and NECT-346 were designed based on our previously developed ligand NECT-224 but harbor additional linker units between the bicyclic scaffold and the radiometal chelator. Binding kinetics to recombinant Nectin-4 and related isoforms were assessed by surface plasmon resonance (SPR) interaction analysis. Nectin-4-specific cell binding of the radiolabeled probes was determined using UC and TNBC cell lines, and PET/CT imaging was performed in respective tumor xenograft models. All novel ligands showed potent and selective binding to Nectin-4, with improved koff values compared to NECT-224. In small-animal PET/CT imaging studies, the most favorable properties were observed for [64Cu]Cu-NECT-334 (tumor-to-muscle of 20.8 and tumor-to-heart of 11.2) and [64Cu]Cu-NECT-346 (tumor-to-muscle of 15.5 and tumor-to-heart of 8.7) 1-2 h postinjection using a HT-1376 tumor model. Although the novel radioligands provided superior imaging properties to the previously developed radioligand [64Cu]Cu-NECT-224, a first-in-human application of the latter radioligand was initiated to assess the general suitability of PET/CT imaging in humans with a 64Cu-labeled bicyclic peptide directed to Nectin-4. Prospectively, the improved tumor-to-background contrasts achieved with the novel radioligands might further improve diagnostic accuracy and facilitate effective theranostic options in Nectin-4-expressing malignancies.