Optimizing the Dissociation Rate constant of ⁶⁴Cu-labeled Nectin-4-Targeting Bicyclic Peptides to Prolong Tumor Retention

Nectin-4 is a cell adhesion protein with a high abundance in various tumor entities, in particular urothelial carcinoma (UC) including bladder cancer. Its suitability as a therapeutic target has been proven by the clinically approved antibody-drug conjugate enfortumab vedotin. Recently, radiolabeled peptidic probes for molecular imaging emerged, but their potential for radioligand therapy remains limited due to insufficient tumor retention. The present study aimed at reducing the dissociation rate constant (k_off) of our previously developed ligand NECT-224 to potentially prolong the tumor retention.

Starting from the peptidic scaffold of NECT-224, a series of analogs bearing a single amino acid substitution in position 3 (dAsp), was synthesized via solid-phase peptide synthesis. Binding constants toward recombinant human Nectin-4 were determined by surface plasmon resonance interaction analysis. Peptides with the lowest k_off values were chosen for conjugation of the radiometal chelator (R)-NODAGA. The resulting compounds NECT-458 (dPhe³) and NECT-460 (dTrp(5OH)³) were radiopharmacologically characterized upon labeling with ⁶⁴Cu using the HT-1376 urothelial cancer model in vitro and in vivo by small-animal PET/CT imaging up to 24 h p.i.

Compared to NECT-224 with a k_off value of , NECT-460 and NECT-458 exhibited reduced k_off values (). This resulted in lower dissociation constants (K_d) towards recombinant Nectin-4 () and comparable on HT-1376 cells () as compared to NECT-224 (). PET/CT imaging in HT-1376 tumor-bearing mice revealed a significantly longer tumor retention for both ⁶⁴Cu-labeled peptides compared to [⁶⁴Cu]Cu-NECT-224, which enabled even a clear tumor visualization at 24 h p.i.

These findings support our hypothesis that lowering the dissociation rate constant is an effective strategy for improving the tumor retention within this series of Nectin-4-targeting bicyclic peptides and suggest that k_off can guide the selection of candidates for further theranostic radioligand development.