Abstract 2579: A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma

Abstract Introduction: Enfortumab vedotin (EV), an antibody-drug conjugate targeting nectin-4, offers hope for urothelial carcinoma patients (UC) but prolonged use leads to treatment-related toxicities often necessitating dose adjustments. How EV engages nectin-4 at the tumor and its relevance to efficacy is unknown. To understand the pharmacodynamics (target engagement) of EV at the tumor across FDA approved dosing levels, we developed a nectin-4 specific peptide-based radiotracer, 68GaAJ647, for positron emission tomography (PET) imaging. Methods: Six UC cell lines of luminal (HT-1376, UC-14, RT-112) and basal (UC-9, SCaBER, T24) morphological type were selected for in vitro and in vivo testing of 68GaAJ647 and nectin-4 target engagement by EV. Cell uptake and competitive binding assays were conducted to test 68GaAJ647 specificity for nectin-4 in the presence or absence of EV. Flow cytometry derived receptor density was used to correlate 68GaAJ647 uptake in cells with nectin-4 expression. Pharmacokinetics, biodistribution and specificity of 68GaAJ647 for nectin-4 expression was evaluated in six xenograft models by PET imaging and biodistribution studies. Target engagement by EV was quantified by measuring unoccupied nectin-4 levels by 68GaAJ647-PET in HT-1376 xenografts at different doses (15, 9, and 6 mg/kg) and time (1, 3, and 6 days). Results: 68GaAJ647 was generated in 20-30% radiochemical yield with 95% purity. In vitro assays showed distinctive uptake in cell lines (HT-1376 UC-14 RT-112; UC-9 SCaBER T24) that correlated with nectin-4 density (R2=0. 85). PET and biodistribution studies showed tractable pharmacokinetics of 68GaAJ647 enabling high contrast images in 60 min and exhibiting nectin-4 dependent uptake in all the six xenografts. Uptake of 68GaAJ647 in UC cells reduced in a dose dependent manner in the presence of EV, indicating that 68GaAJ647 measures unoccupied nectin-4, and paving the way for EV target engagement evaluation. For target engagement evaluation, 68GaAJ647 accumulation in HT-1376 tumors in saline treated mice was assumed as 100% unoccupied nectin-4 levels. In mice treated with EV for 24 hrs (n=3-5), unoccupied nectin-4 levels were 21. 1±11. 6, 23. 2±14. 5, and 24. 3±18. 8 for 15, 9, and 6 mg/kg doses, respectively. Whereas on day 6, unoccupied nectin-4 levels showed significant differences at 29. 5±15. 8, 64. 2±21. 0, and 89. 5±18. 9 for 15, 9, and 6 mg/kg dose, respectively. These data indicate that FDA approved doses of EV show higher target engagement at 24 h but a dramatic reduction at lower doses over time. Conclusion: 68GaAJ647-PET quantifies variable nectin-4 expression in tumors. Furthermore, 68GaAJ647-PET quantifies EV therapy induced pharmacodynamics at the tumor. Such real-time ADC target engagement assessment could inform patient selection and dosing strategies to enhance efficacy and minimize toxicities. Citation Format: Akhilesh Mishra, Ajay K. Sharma, Kuldeep Gupta, Burles A. Johnson, David J. McConkey, Jeannie Hoffman-Censits, Sridhar Nimmagadda. A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 2579.