Therapeutics, Targets, and Chemical Biology RG7116, a Therapeutic Antibody That Binds the Inactive HER3 Receptor and Is Optimized for Immune Effector Activation Christian Mirschberger 1 , Christian B. Schiller 3 , Michael Schr€ aml 2 , Nikolaos Dimoudis 1 , Thomas Friess 1 , Christian A. Gerdes 4 , Ulrike Reiff 1 , Valeria Lifke 1 , Gabriele Hoelzlwimmer 1 , Irene Kolm 1 , Karl-Peter Hopfner 3 , Gerhard Niederfellner 1 , and Birgit Bossenmaier 1 Abstract The EGF receptor (EGFR) HER3 is emerging as an attractive cancer therapeutic target due to its central position in the HER receptor signaling network. HER3 amplifies phosphoinositide 3-kinase (PI3K)–driven tumorigenesis and its upregulation in response to other anti-HER therapies has been implicated in resistance to them. Here, we report the development and characterization of RG7116, a novel anti-HER3 monoclonal antibody (mAb) designed to block HER3 activation, downregulate HER3, and mediate enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) via glycoengineering of the Fc moiety. Biochemical studies and X-ray crystallography revealed that RG7116 bound potently and selectively to domain 1 of human HER3. Heregulin binding was prevented by RG7116 at concentrations more than 1 nmol/L as was nearly complete inhibition of HER3 heterodimerization and phosphorylation, thereby preventing downstream AKT phosphorylation. In vivo RG7116 treatment inhibited xenograft tumor growth up to 90% relative to controls in a manner accompanied by downregulation of cell surface HER3. RG7116 efficacy was further enhanced in combination with anti-EGFR (RG7160) or anti-HER2 (pertuzumab) mAbs. Furthermore, the ADCC potency of RG7116 was enhanced compared with the nonglycoengineered parental antibody, both in vitro and in orthotopic tumor xenograft models, where an increased median survival was documented. ADCC degree achieved in vitro correlated with HER3 expression levels on tumor cells. In summary, the combination of strong signaling inhibition and enhanced ADCC capability rendered RG7116 a highly potent HER3-targeting agent suitable for clinical development. Cancer Res; 73(16); 5183–94. Ó2013 AACR. Introduction HER3 (ERBB3) is a member of the HER family of transmem- brane receptors. By homo- and heterodimerization, these receptors form a complex overlapping signaling network that regulates cell differentiation, migration, proliferation, and survival (1, 2). HER monomers are predominantly in an inactive "closed" conformation. Activation occurs following ligand binding by receptor dimerization and subsequent transpho- sphorylation within the dimerized receptor pair. HER3 is unique in that it lacks significant kinase activity (3) and does not form stable homodimers (4); thus it depends on HER1 [EGF receptor (EGFR)], HER2 (ERBB2), or HER4 (ERBB4) as hetero- dimerization partners. Within the HER family, HER3 is the most potent in activating the phosphoinositide 3-kinase (PI3K)/AKT pathway, as its intracellular domain contains multiple tyrosine phosphorylation sites that recruit the regu- latory p85 subunit of PI3K. HER2–HER3 heterodimers consti- tute a high-affinity coreceptor pair for heregulin [HRG (NRG1)], the predominant ligand for HER3 (5–7), and form the most potent dimeric HER signaling complex—strongly activating diverse cellular pathways such as the RAS/RAF/ mitogen-activated protein kinase (MAPK) and the PI3K/AKT pathways (8, 9). Deregulation of HER signaling is a key mechanism by which tumor cells can evade normal growth constraints. Several anti- HER agents are widely used clinically, including the anti-EGFR monoclonal antibody (mAb) cetuximab, the anti-HER2 mAb trastuzumab, and the EGFR tyrosine kinase inhibitors erlotinib and gefitinib. The importance of HER3 in cancer has only recently emerged; upregulation of HER3 is an adverse prog- nostic factor in many tumor types and is associated with worse survival (10–14). HER3 has also been implicated in the development of resistance to anti-EGFR or anti-HER2 therapy (15–17). A shift from HER1 homodimer to HER1/HER3 heterodimer signaling in response to sustained treatment with EGFR inhibitors leads to reactivation of the PI3K/AKT pathway and bypasses the Authors' Affiliations: 1 Pharma Research and Early Development (pRED), 2 Roche Diagnostics GmbH, Penzberg; 3 Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, Munich, Germany; and 4 Roche Glycart AG, pRED, Schlieren, Switzerland Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). C. Mirschberger, C.B. Schiller, and M. Schr€ aml contributed equally to this work and should be considered as co-first authors. Corresponding Author: Birgit Bossenmaier, Pharma Research and Early Development, Roche Diagnostics GmbH, Nonnenwald 2, DE-82377 Penz- berg, Germany. Phone: 49-8856-60-2635; Fax: 49-8856-60-3896; E-mail: birgit.bossenmaier@roche.com doi: 10.1158/0008-5472.CAN-13-0099 Ó2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org 5183 on February 5, 2016. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst June 18, 2013; DOI: 10.1158/0008-5472.CAN-13-0099