Payload-Free [89Zr]Zr-DFO*-sq-Enfortumab ImmunoPET for Imaging Nectin-4 Expression in Bladder Cancer

Abstract Nectin-4–targeted imaging has emerged as a promising strategy for patient stratification in urothelial carcinoma, yet currently available radiotracers remain limited by rapid renal clearance, urinary background activity, or potential pharmacologic confounding associated with antibody-drug conjugate (ADC)-based constructs. To overcome some of these limitations, we developed a payload-free immunoPET radiotracer based on the native enfortumab antibody conjugated with the bifunctional chelator DFO*-sq and radiolabeled with zirconium-89 (89Zr). The resulting [89Zr]Zr-DFO*-sq-Enfortumab preserved Nectin-4–specific targeting while demonstrating high radiochemical stability in human serum (>99% through Day 12). In vitro studies showed efficient antigen-specific cellular internalization (80–85%), prolonged intracellular retention, and evidence consistent with partial recycling of antibody-associated species. In vivo, [89Zr]Zr-DFO*-sq-Enfortumab exhibited sustained tumor uptake reaching 17.4 ± 2.8% ID/g in high Nectin-4–expressing xenografts and correlated with differential Nectin-4 expression across multiple bladder cancer models. Importantly, the full-length antibody platform enabled high-contrast visualization of pulmonary metastatic lesions and orthotopic bladder tumors despite some urinary background activity. Together, these findings demonstrate that payload-free [89Zr]Zr-DFO*-sq-Enfortumab provides a biologically relevant immunoPET platform for noninvasive assessment of variable Nectin-4 expression and support its further evaluation as a companion imaging strategy for Enfortumab Vedotin–based therapies.