The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor cells by delivering MMAE, a potent microtubule disrupting agent, to induce cell death. EV has demonstrated single agent activity and encouraging activity (71% ORR) when combined with pembrolizumab (anti-PD-1) in the 1L setting of cis-ineligible metastatic urothelial carcinoma (mUC) (EV-103, NCT03288545). Here we demonstrated that EV may promote multiple mechanisms of action including bystander cell killing and hallmarks of immunogenic cell death (ICD) including ER stress, immune cell recruitment and activation. Two urothelial carcinoma models, T-24 and UM-UC-3, were engineered to express Nectin-4, and both were sensitive to EV in vitro and in vivo. In these Nectin-4 expressing cell lines, EV internalized with Nectin-4, trafficked to lysosomal vesicles, and released intracellular MMAE as shown by intracellular MMAE accumulation. In addition, EV demonstrated a bystander effect by release of the cell permeable MMAE from Nectin-4 positive cells to kill Nectin-4 negative cancer cells in an admixed cellular assay. Furthermore, EV treated cells exhibited early markers of ICD such as induction of the ER stress response through phosphorylation of JNK in Nectin-4 expressing cells. The ER protein calreticulin and protein chaperones like HSP70 are translocated from the intracellular compartments and exposed on the cell surface to signal for immune cell recruitment upon EV treatment. Additional hallmarks of ICD were observed with EV included extracellular release of ATP and HMGB1 in both the T-24 and UM-UC-3 Nectin-4 expressing cell lines. Immune cell recruitment and activation in the T-24 Nectin-4 xenograft model that demonstrated potent anti-tumor activity were measured in vivo using IHC, RNA-seq, flow cytometry, and immune cytokine paneling. Immune profiling assessment showed an enhancement of the immune cell markers in 6 of 7 tumors compared to untreated animals or non-binding ADC control treated animals. Analysis of gene signatures using RNA-seq support our qualitative IHC analysis of immune cell recruitment. Additional gene signature analyses identified altered gene transcripts associated with microtubule disruption, mitotic arrest, and ER stress. In addition, Luminex assessment of mouse cytokines showed coincident changes in RNA expression of genes associated with macrophage activation such MIP1α and MIPβ, suggesting EV treatment can promote APC activation. This mechanistic data and ongoing research by which EV induces cell death to promote immune cell recruitment and activation provides a potential mechanism underpinning the clinical benefit observed with EV as a monotherapy or in combination with pembrolizumab in mUC.

Citation Format: Bernard A. Liu, Devra Olson, Katie Snead, John Gosink, Elena-Marie Tenn, Margo Zaval, Anthony Cao, Disha Sahetya, Albina Nesterova, Kelly Hensley, Julia Cochran, Shyra Gardai, Timothy S. Lewis. Enfortumab vedotin, an anti-Nectin-4 ADC demonstrates bystander cell killing and immunogenic cell death anti-tumor activity mechanisms of action in urothelial cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5581.