Cancer Therapy: Preclinical An Antibody–Drug Conjugate Targeting the Endothelin B Receptor for the Treatment of Melanoma Jyoti Asundi, Chae Reed, Jennifer Arca, Krista McCutcheon, Ronald Ferrando, Suzanna Clark, Elizabeth Luis, Janet Tien, Ron Firestein, and Paul Polakis Abstract Purpose: To identify and evaluate targets amenable to antibody therapy in melanoma. Experimental Design: We searched for mRNA transcripts coding for cell-surface proteins with expres- sion patterns similar to that of the melanoma oncogene MITF. One such candidate, the endothelin B receptor (EDNBR), was first analyzed for a functional contribution to tumor growth by conditional induction of shRNA. Second, antibodies were raised to the receptor, conjugated with monomethyl auristatin E, and tested for efficacy against melanoma tumor models generated from cell lines. Results: Conditional knockdown of the receptor in tumor xenograft models resulted in only a modest impact on tumor growth. A monoclonal antibody reactive with the N-terminal tail of EDNBR was found to internalize rapidly into melanoma cells. When conjugated with monomethyl auristatin E, the antibody– drug conjugate (ADC) showed remarkable efficacy against human melanoma cell lines and xenograft tumor models that was commensurate with levels of receptor expression. Comparative immunohisto- chemistry revealed a range of EDNBR expression across a panel of human melanomas, with the majority expressing levels equivalent to or greater than that in the models responsive to the ADC. Conclusion: An ADC targeting the EDNBR is highly efficacious in preclinical models of melanoma. Clin Cancer Res; 17(5); 1–11. Ó2011 AACR. Introduction Melanoma is an aggressive form of skin cancer that has recently undergone an alarming increase in incidence (1). Although cures can be achieved with surgical resection of localized lesions, the advanced stages of melanoma are only poorly responsive to currently approved therapies. The 5-year survival rate for stage IV metastatic melanoma is approximately 10% (1). New therapeutic approaches, including antisense to Bcl2, antibodies to CTLA4, small molecule RAF kinase inhibitors, and adoptive immu- notherapy, are currently in clinical testing for metastatic melanoma (2). The results from some of these recent studies seem to be encouraging, but a durable impact on overall survival will likely require therapeutic combina- tions including additional new agents. More than 20 years ago, endothelin-1 (ET-1) was isolated from aortic endothelial cells and found to have potent vasoconstrictive activity (3). The receptors for endothelins were cloned shortly thereafter (4, 5) and their expression in various cell types, including melanocytes and melanoma cells, pointed to functions independent of their role in endothelium. It is now well recognized that the endothelin B receptor (EDNBR) is critical for the faithful derivation of melanocytic cells emanating from the neural crest during embryonic development (6, 7). Melanocyte precursors rely on EDNBR activity to proliferate and migrate from the neural tube to their final destinations (8, 9). Mice with defective genes coding for either EDNBR or endothelin-3 (ET-3) exhibit a pigmentation deficit in their coats and a shortage of enteric ganglion cells, also derived from the neural crest. These characteristics strongly resemble those associated with the WS4 variant of Waardenburg syndrome in humans, which has been attributed to germline muta- tions in either ET-3 or EDNBR (10–12). An additional variant of this syndrome, WS2, has been mapped to heri- table mutations in the microphthalmia-associated tran- scription factor (MITF), a key regulator of melanocyte development and a melanoma proto-oncogene (13–15). The strong genetic evidence linking EDNBR activity to the fate of melanoblasts underscores a potential role for this receptor in the progression of melanoma. The expres- sion of EDNBR mRNA and protein was reported to increase during disease progression from dysplastic nevi to meta- static melanoma (16). Blockade of EDNBR activity by 2 independent small molecule inhibitors interfered with growth and survival of melanoma cells and tumor xeno- grafts (17–19). These preclinical studies implicate EDNBR as a potential driver of melanoma progression. However, Authors' Affiliation: Genentech, Inc., South San Francisco, California Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Paul Polakis, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Phone: 650-225-5327; Fax: 650-225-6127; E-mail: ppolakis@gene.com doi: 10.1158/1078-0432.CCR-10-2340 Ó2011 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org OF1 Research. on June 9, 2017. © 2011 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst January 18, 2011; DOI: 10.1158/1078-0432.CCR-10-2340