Cancer Therapy: Preclinical Pan-HER, an Antibody Mixture Simultaneously Targeting EGFR, HER2, and HER3, Effectively Overcomes Tumor Heterogeneity and Plasticity Helle J. Jacobsen,Thomas T. Poulsen, Anna Dahlman, Ida Kjær, Klaus Koefoed, Jette W. Sen, Dietmar Weilguny, Bolette Bjerregaard, Christina R. Andersen, Ivan D. Horak, Mikkel W. Pedersen, Michael Kragh, and Johan Lantto Abstract Purpose: Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epider- mal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members. Experimental Design/Results: We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing com- pensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly effica- cious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting. Conclusions: Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER repre- sents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic. Clin Cancer Res; 21(18); 4110–22. Ó2015 AACR. See related commentary by Yarden and Sela, p. 4030 Introduction The human epidermal growth factor receptor (HER) family consists of four members: EGFR/HER1, ErbB-2/HER2, ErbB-3/ HER3, and ErbB-4/HER4. These receptors play an important role in normal cell growth, metabolism, proliferation, survival, and differentiation. However, deregulation through mutation, overexpression, or gene amplification of the HER family is commonly associated with development, progression, or acquired resistance of many human cancers (1). Homo- or hetero- dimerization induced by binding of ligands within the EGF family of growth factors results in cross-phosphorylation of the dimerization partners, ultimately triggering intracellular signaling (2, 3). EGFR and HER2 are clinically validated targets in squamous cell carcinoma of the head and neck, colorectal, breast, gastric, and non–small cell lung cancers, and growing evidence suggests that HER3 will prove to be a clinically relevant target as well (2, 4–6). In contrast, the role of HER4 in cancer pathogenesis is less clear. Both small-molecule tyrosine kinase inhibitors (TKI), targeting individual or multiple members of the HER family, and thera- peutic monoclonal antibodies have been approved for treatment of various cancers. However, currently approved antibody ther- apeutics only address individual receptors (e.g., cetuximab and panitumumab against EGFR and trastuzumab and pertuzumab against HER2), although pertuzumab was selected to prevent HER2 from dimerizing with other HER family members (7). Bispecific antibodies and combinations of antibodies that target two members of the HER family are also in clinical development (8, 9). Accumulating evidence shows that the HER family displays a high degree of plasticity and provides compensatory signaling leading to acquired resistance in response to therapeutic inter- vention (10, 11). Examples include HER family TKI (gefitinib and erlotinib) resistance in HER2-driven breast cancer due to increased HER3 expression (12), anti-HER2 trastuzumab resis- tance in breast cancer due to overexpression of EGFR and EGFR ligands (13) or increased EGFR and/or HER3 expression (14), EGFR/HER2 TKI lapatinib resistance in breast cancer due to HER3 upregulation (15), and anti-EGFR cetuximab resistance in colo- rectal cancer mediated by upregulation of HER2 or HER3 ligand neuregulin (heregulin; ref. 16). Simultaneous targeting of more than one HER family receptor is frequently able to reverse the resistance to the initial drug and is often more efficacious than single-receptor targeting alone (15–17). Inhibiting more than one HER family member may thus be critical to limit acquired resistance and more effectively treat cancer. Symphogen A/S, Ballerup, Denmark. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). H.J. Jacobsen and T.T. Poulsen contributed equally to this article. Corresponding Author: Johan Lantto, Symphogen A/S, Pederstrupvej 93, DK- 2750 Ballerup, Denmark. Phone: 45-4526-5050; Fax: 45-4526-5060; E-mail: jol@symphogen.com doi: 10.1158/1078-0432.CCR-14-3312 Ó2015 American Association for Cancer Research. Clinical Cancer Research Clin Cancer Res; 21(18) September 15, 2015 4110 on April 13, 2017. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst April 23, 2015; DOI: 10.1158/1078-0432.CCR-14-3312