Abstract 2579: A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma
Authors: Mishra Akhilesh , Sharma Ajay K. , Gupta Kuldeep , Johnson Burles A. , McConkey David J. , Hoffman-Censits Jeannie , Nimmagadda Sridhar 
Published: March 2024

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, [68Ga]AJ647, 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 [68Ga]AJ647 and nectin-4 target engagement by EV. Cell uptake and competitive binding assays were conducted to test [68Ga]AJ647 specificity for nectin-4 in the presence or absence of EV. Flow cytometry derived receptor density was used to correlate [68Ga]AJ647 uptake in cells with nectin-4 expression. Pharmacokinetics, biodistribution and specificity of [68Ga]AJ647 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 [68Ga]AJ647-PET in HT-1376 xenografts at different doses (15, 9, and 6 mg/kg) and time (1, 3, and 6 days).

Results: [68Ga]AJ647 was generated in 20-30% radiochemical yield with &gt;95% purity. In vitro assays showed distinctive uptake in cell lines (HT-1376 &gt; UC-14 &gt; RT-112; UC-9 &gt; SCaBER &gt; T24) that correlated with nectin-4 density (R2=0.85). PET and biodistribution studies showed tractable pharmacokinetics of [68Ga]AJ647 enabling high contrast images in 60 min and exhibiting nectin-4 dependent uptake in all the six xenografts. Uptake of [68Ga]AJ647 in UC cells reduced in a dose dependent manner in the presence of EV, indicating that [68Ga]AJ647 measures unoccupied nectin-4, and paving the way for EV target engagement evaluation. For target engagement evaluation, [68Ga]AJ647 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: [68Ga]AJ647-PET quantifies variable nectin-4 expression in tumors. Furthermore, [68Ga]AJ647-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(6_Suppl):Abstract nr 2579.