Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies Young-Hun Lee a,b , Mariko Iijima a , Yuji Kado c , Eiichi Mizohata c , Tsuyoshi Inoue c , Akira Sugiyama a , Hirofumi Doi a,⇑ , Yoshikazu Shibasaki a,⇑ , Tatsuhiko Kodama a a Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan b Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan c Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan article info Article history: Received 25 October 2013 Available online 12 November 2013 Keywords: Epiregulin Humanization Antibody engineering Resurfacing Antibody-dependent cellular cytotoxicity abstract Growth factors are implicated in several processes essential for cancer progression. Specifically, epider- mal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that frame- work region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized anti- body. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The epidermal growth factor (EGF) signaling system consists of at least seven ligands, that is, EGF, amphiregulin, transforming growth factor a, heparin-binding EGF, betacellulin, epiregulin (EREG), and epigen [1]. These ligands bind to the extracellular re- gion of the EGF receptor (EGFR) and induce a conformational change in EGFR, leading to its dimerization and activation. Subse- quently, activated EGFR stimulates many intracellular signaling pathways, such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways, and promotes proliferation, cell survival, and angiogenesis [1,2]. In many differ- ent cancer cells, EGFR ligands are produced either by the cancer cells themselves or by surrounding stromal cells, leading to consti- tutive EGFR activation [3]. Of the EGF ligands, EREG is produced as a transmembrane precursor and exerts mitotic activity in various primary cell types, such as rat hepatocytes, and various types of human cancer cells, particularly epithelial tumor cells [4,5]. Inter- estingly, EREG is expressed at relatively low levels in most adult normal tissues, but is highly expressed in various human cancers, including colon, breast, prostate, and ovary cancers [6–9]. Many studies have demonstrated the possible involvement of EREG in tumorigenesis and the oncogenic effects of cancer-specific overex- pression of EREG. Thus, EREG is likely to be involved in the devel- opment of a variety of human cancers, and its potential use as a therapeutic target is being intensely investigated. To this end, sev- eral anti-EREG murine monoclonal antibodies (mAbs) have been successfully tested in vivo (unpublished data). However, one of the primary problems in developing monoclonal antibodies as drugs is the human anti-mouse antibody response (HAMA), which limits the administration of murine antibodies [10]. In this study, we have described the construction and expression of humanized anti-EREG antibodies with high-affinity targeted cytotoxicity and decreased immunogenicity through resurfacing the variable region and recombination. 2. Materials and methods 2.1. Materials 9E5, a human anti-EREG antibody, was a member of a panel of murine monoclonal antibodies (mAbs) generated using a subtrac- tive immunization protocol in hybridoma cells and was provided 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.11.014 ⇑ Corresponding authors. Fax: +81 3 5452 5232. E-mail addresses: doi-h@lsbm.org (H. Doi), shibasaki@lsbm.org (Y. Shibasaki). Biochemical and Biophysical Research Communications 441 (2013) 1011–1017 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc