Background Enfortumab vedotin (EV) is a first-in-class Nectin-4-directed antibody-drug conjugate (ADC) with demonstrated improved overall survival in patients with previously treated advanced-stage urothelial carcinoma.¹ EV is comprised of a fully human Nectin-4-directed monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) by a protease cleavable maleimidocaproyl-valine-citrulline linker. EV has a multifaceted mechanism of action. Previously, we demonstrated that EV induces antitumor activity in vitro via direct cytotoxicity on Nectin-4-expressing malignant cells and indirect bystander activity on neighboring Nectin-4­ negative cells, both of which are mediated by MMAE release within target cells. Here, we expand upon the mechanism of action and show EV induces tumor cell killing in a manner leading to immunogenic cell death (ICD) and improves antitumor responses when combined with checkpoint inhibitors.

Methods The ability of EV to induce hallmarks of ICD was evaluated in vitro in Nectin-4-expressing human urothelial carcinoma cell lines. Immune activation associated with ICD was assessed in vitro in monocytes co-cultured with EV-treated tumor cells and in vivo by immunohistochemistry, RNA-seq, flow cytometry, and immune cytokine profiling. The effects of EV plus anti-PD-1 on tumor growth inhibition, the tumor microenvironment, and immune memory were evaluated in syngeneic mouse models engineered to express human Nectin-4. Antitumor immune memory was also assessed in mice vaccinated with EV-treated cells.

Results In vitro, EV induced ICD via MMAE-mediated microtubule disruption and concomitant endoplasmic reticulum (ER) stress, as evidenced by increased phosphorylation of JNK, extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. Xenograft tumors treated with EV demonstrated upregulation of MHC genes as well as genes involved in ER stress, autophagy, and type I interferon response. Additionally, there were noted increases in both macrophages and dendritic cells along with cytokines involved in chemoattraction and T-cell stimulation. Consistent with ICD induction, vaccination with EV-treated Nectin-4-expressing tumor cells promoted antitumor immunity and provided protection against tumor rechallenge. Lastly, the combination of EV with PD-1 inhibition improved antitumor activity and durable immunity in vivo, consistent with complementary modes of action of these two anticancer agents.

Conclusions These data provide insight into the clinical activity observed with EV and bolster the scientific rationale to combine EV with checkpoint inhibitors, which is currently an area of active clinical investigation across multiple studies.^2-6

References

1. Powles T, Rosenberg JE, Sonpavde GP, Loriot Y, Duran I, Lee JL, et al. Enfortumab Vedotin in Previously Treated Advanced Urothelial Carcinoma. N Engl J Med. 2021;384(12):1125–35. Epub 2021/02/13. doi:10.1056/NEJMoa2035807. PubMed PMID: 33577729; PubMed Central PMCID: PMCPMC8450892.

2. Friedlander TW, Milowsky MI, Bilen MA, Srinivas S, McKay RR, Flaig TW, et al. Study EV-103: Update on durability results and long term outcome of enfortumab vedotin + pembrolizumab in first line locally advanced or metastatic urothelial carcinoma (la/mUC). Journal of Clinical Oncology 2021;39(15_suppl):4528. doi: 10.1200/JCO.2021.39.15_suppl.4528.

3. Galsky MD, Necchi A, Shore ND, Plimack ER, Jia C, Sbar E, et al. KEYNOTE-905/EV-303: Perioperative pembrolizumab or pembrolizumab plus enfortumab vedotin (EV) and cystectomy compared to cystectomy alone in cisplatin-ineligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol. 2021;39(6_suppl):TPS507. doi: 10.1200/JCO.2021.39.6_suppl.TPS507.

4. Heijden MSVD, Gupta S, Galsky MD, Derleth CL, Lee S, Kataria RS, et al. Study EV-302: A two-arm, open-label, randomized controlled phase 3 study of enfortumab vedotin in combination with pembrolizumab versus chemotherapy in previously untreated advanced urothelial carcinoma (aUC) (trial in progress). J Clin Oncol 2022;40(6_suppl):TPS589. doi: 10.1200/JCO.2022.40.6_suppl.TPS589.

5. Hoimes CJ, Bedke J, Loriot Y, Nishiyama H, Fang X, Kataria RS, et al. KEYNOTE-B15/EV-304: Randomized phase 3 study of perioperative enfortumab vedotin plus pembrolizumab versus chemotherapy in cisplatin-eligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol 2021;39(15_suppl):TPS4587. doi: 10.1200/JCO.2021.39.15_suppl.TPS4587.

6. ClinicalTrials.gov [Internet] Bethesda (MD): U.S. National Library of Medicine. 2000 – . ClinicalTrials.gov Identifier: NCT04960709. Treatment Combination of Durvalumab, Tremelimumab and Enfortumab Vedotin or Durvalumab and Enfortumab Vedotin in Patients With Muscle Invasive Bladder Cancer Ineligible to Cisplatin or Who Refuse Cisplatin (VOLGA). 2021 Jul 14 [cited 2022 Jul 22]. Available from: https://clinicaltrials.gov/ct2/show/NCT04960709.

Ethics Approval All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen, Astellas, or the external testing facilities that conducted the studies.