[⁶⁴Cu]Cu-NOTA-EV-F(ab')₂ Enables Same-Day Immuno-PET Imaging of Nectin-4 in Triple-Negative Breast and Urothelial...

Our study aimed to develop and evaluate [⁶⁴Cu]Cu-NOTA-EV-F(ab')₂ for immuno-PET imaging of nectin-4 expression in triple-negative breast cancer (TNBC) and urothelial bladder cancer (UBC) models, with the goal of achieving rapid, specific tumor targeting and high tumor-to-background contrast. Methods: Bivalent antibody fragments were generated from enfortumab vedotin (EV) using IdeS protease and conjugated with NOTA for radiolabeling with ⁶⁴Cu. In vitro binding and uptake studies were performed using TNBC and UBC cell lines. Immuno-PET imaging and biodistribution studies were conducted in athymic nude mice bearing subcutaneous xenografts with varying nectin-4 expression. Results: [⁶⁴Cu]Cu-NOTA-EV-F(ab')₂ exhibited rapid tumor accumulation and high specificity in nectin-4-positive tumors, with peak uptake observed at 4 h after injection. EV-F(ab')₂ demonstrated superior tumor-to-background ratios compared with the full-length EV antibody, particularly in nectin-4-expressing models (MDA-MB-468, BT474, SW780, and HT-1376). Blocking studies confirmed nectin-4-specific targeting. Favorable pharmacokinetics of EV-F(ab')₂ allowed for same-day imaging and reduced radiation exposure relative to intact antibodies. Conclusion: [⁶⁴Cu]Cu-NOTA-EV-F(ab')₂ is a promising immuno-PET probe for assessing nectin-4 expression in TNBC and UBC.