Kisspeptins (KPs) and their receptor (KISS1R) promote metastasis and tumor progression in various cancers such as triple-negative breast cancer (TNBC). Targeting KISS1R holds great promise for molecular imaging and targeted radionuclide therapy of aggressively disseminated cancers. First ligand-based approaches using Ga-68/Lu-177-labeled KPs (KP-10, KP-54) have demonstrated feasibility but suffer from proteolytic degradation and low uptake in KISS1R positive tumors. However, lead structure optimization alone is insufficient, as KISS1R biology remains unexplored in a radiotheranostic context. In this study, N-terminally functionalized conjugates of KP-10, KP-54, and the hybrid peptide KiSS-34 (AMBA-2-Nal-Gly-Leu-Arg-Trp-NH2), including scrambled controls, were synthesized in high purity (≥95%) for comparative studies. The conjugation to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and Alexa-Fluor-488 (AF-488) functionalities preserved biological activity, confirmed by (sub)nanomolar EC50-values (0.05-0.85 nM) in calcium mobilization assays in transfected CHO-KISS1R cells. Conventional target detection methods using antibodies (Abs) and AF-488-KPs failed to visualize KISS1R in both model (CHO-KISS1R) and native cancer cell lines, likely due to unspecific Abs and rapid KISS1R internalization upon agonist stimulation. However, rapid KISS1R internalization was successfully visualized via live-cell imaging using AF-488-KP-10 and novel analogue AF-488-KiSS-34. Furthermore, DOTA-KPs were radiolabeled with Lu-177 in high efficiencies (≥95%) and examined in internalization assays, showing highest uptake (4.8%) and internalization rate (45.9%) for 177LuLu-DOTA-KiSS-34 in CHO-KISS1R cells compared to its KP-10 analogue (total uptake: 1.3%; internalization rate: 37.6%). Higher uptakes likely derive from faster binding kinetics, improved KISS1R targeting, and/or slower dissociation as evidenced by oil-based kinetics assays showing higher total uptake for 177LuLu-DOTA-KiSS-34 (15.3%) compared to KP-10 (3.8%) and KP-54 (4.5%) counterparts after 30 min. Positron emission tomography/computerized tomography (PET/CT) imaging, urine analysis, and all in vitro studies indicate that Ga-68/Lu-177-labeled DOTA-KiSS-34 exhibits superior pharmacodynamics, pharmacokinetics, and in vivo stability compared to its KP-10 and KP-54 analogues, which are critically suffering from rapid in vivo degradation. These results position DOTA-KiSS-34 as a strong structural lead for KISS1R-based radiotheranostics. Nevertheless, the dynamics between KPs and KISS1R need to be further investigated to fully harness the radiotheranostic potential of KISS1R for TNBC and other cancers.
Taş et al. (Tue,) studied this question.