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

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.