Abstract A090: Vepugratinib (LY3866288), a potent highly isoform-selective FGFR3 inhibitor, enhances the activity of enfortumab vedotin (EV) in FGFR3-altered metastatic urothelial cancer (mUC) models

Abstract Background: Vepugratinib is an oral, potent, and highly isoform-selective FGFR3 inhibitor targeting oncogenic FGFR3 alterations that was designed to limit the off-target toxicity associated with approved pan FGFR inhibitors1. Here, we report results from in vitro and in vivo UC models when vepugratinib was combined with EV (NECTIN4 ADC), with or without pembrolizumab (P). Materials and Methods: Immunohistochemistry was performed using a commercially available NECTIN4 antibody. Cell surface protein levels were analyzed by flow cytometry and total NECTIN4 by western blot, in cells/tumoroids treated with vepugratinib at clinically relevant doses. Soluble NECTIN4 was measured by MSD assay. NECTIN4 mRNA levels were determined using RNAseq. Cell viability was measured using CellTiter-Glo and apoptosis by annexin V dye assays. For in vivo studies, CDX or PDX were used in NCG, athymic nude and NOG EXL mouse backgrounds. In select models, mice were humanized by CD34+ progenitor reconstitution. When average tumor size reached 200 mm3, mice were treated with vepugratinib (BID); EV (QW); pembrolizumab (BIW). Results: As previously reported2 we found that NECTIN4 levels were heterogenous in tissue micro arrays from patients (n=176) with UC (stage III/IV and metastatic); high expression (H-score 200-300) in 4%, moderate expression (H-score 100-99) in 35% and low expression (H-score 15-99) in 25%. We also investigated the impact of FGFR3 inhibition with vepugratinib on NECTIN4 expression in bladder cancer cell lines and tumoroids harboring the oncogenic FGFR3 S249C mutation or FGFR3-TACC3 fusion. In UC cell lines, the levels of NECTIN4 mRNA, and NECTIN4 protein (total, cell surface, and soluble) increased up to 3-fold 24 hrs post vepugratinib treatment. Similarly, in UC-derived tumoroids, total NECTIN4 increased up to 2-fold over a 14-day study. NECTIN4 protein levels remained unchanged in an FGFR3 WT bladder cancer cells and in primary human keratinocytes treated with vepugratinib, consistent with this finding being mediated by oncogenic FGFR3 signaling. Moreover, vepugratinib enhanced EV activity on cell growth inhibition and apoptosis compared with either as a single agent (∼70% vs ∼30% annexin V staining). Additionally, significant tumor regression was observed in vivo when vepugratinib was combined with EV or EV+P in UC models harboring FGFR3 alterations; efficacy was greater than additive when compared to single agent, and additive compared to EV+P. Conclusions: Vepugratinib upregulates NECTIN4 in FGFR3-altered mUC models, enhancing the activity of standard of care EV+P and supporting the investigation of this combination clinically. FORAGER-1 is investigating the combination of vepugratinib with EV+P in patients with untreated locally advanced or mUC with an FGFR3 genetic alteration (NCT05614739). References: 1Ballard et al. 2021 Mol Cancer Ther 20 (12_Supplement): P141. 2Kluemper et al. 2023 Clin Cancer Res 29: 1496–1505. Citation Format: Eva Goetz, Viviana Volta, Haniyeh Eyvani, Robert John . Evans, Bojana Jovanovic, Wei-Jen Chung, Marie Mutryn, Supreet Agarwal, Franny Hough, Audrey Au, Wei Zheng, Timothy Bailey, Marguerita O'Mahony, Abhinav Achreja, Sarah M. Bogner, Joseph Puccini, Cindy Lee, Jason Manroe, Brent D. Butts, Yu-Huan Shih, Farideh Ordikhani, Rowena Almonte-Baldonado, Chun Ping Yu, Wei Guo Xu, Andrew Capen, Veena Coothan Kandaswamy, Vanessa Rodrik-Outmezguine, Karsten Boehnke, Loredana Puca. Vepugratinib (LY3866288), a potent highly isoform-selective FGFR3 inhibitor, enhances the activity of enfortumab vedotin (EV) in FGFR3-altered metastatic urothelial cancer (mUC) models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A090.