Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies

• Published in: Biochemical and biophysical research communications 2013-11, Vol.441 (4), p.1011-1017
• Main Authors: Lee, Young-Hun, Iijima, Mariko, Kado, Yuji, Mizohata, Eiichi, Inoue, Tsuyoshi, Sugiyama, Akira, Doi, Hirofumi, Shibasaki, Yoshikazu, Kodama, Tatsuhiko
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal, Humanized - biosynthesis; Antibodies, Monoclonal, Humanized - chemistry; Antibodies, Monoclonal, Humanized - immunology; Antibody engineering; Antibody-dependent cellular cytotoxicity; Epidermal Growth Factor - antagonists & inhibitors; Epidermal Growth Factor - immunology; Epiregulin; HCT116 Cells; Humanization; Humans; Immunoglobulin Variable Region - biosynthesis; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - immunology; Jurkat Cells; Luciferases - biosynthesis; Models, Molecular; Molecular Sequence Data; NFATC Transcription Factors - metabolism; Protein Engineering; Receptors, IgG - biosynthesis; Resurfacing
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2013.11.014

•Epiregulin may be highly relevant for therapeutic applications.•We generated humanized anti-epiregulin antibodies by resurfacing the variable region.•Our method identified residues that critically affect antigen binding.•Our HM1 antibody had high-affinity targeted cytotoxicity against colon cancer cells. Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal 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 framework 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 antibody. 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.