Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies.
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.
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; Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Young-Hun
YH
Lee
Mariko
M
Iijima
Yuji
Y
Kado
Eiichi
E
Mizohata
Tsuyoshi
T
Inoue
Akira
A
Sugiyama
Hirofumi
H
Doi
Yoshikazu
Y
Shibasaki
Tatsuhiko
T
Kodama
D000595:Amino Acid Sequence; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000276:immunology*; D066260:Epiregulin; D045325:HCT116 Cells; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D019169:Jurkat Cells; D008156:Luciferases / Q000096:biosynthesis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000378:metabolism; D015202:Protein Engineering; D017452:Receptors, IgG / Q000096:biosynthesis
D061067:Antibodies, Monoclonal, Humanized; C587880:EREG protein, human; D066260:Epiregulin; C496124:FCGR3A protein, human; D007135:Immunoglobulin Variable Region; D050778:NFATC Transcription Factors; D017452:Receptors, IgG; D004815:Epidermal Growth Factor; D008156:Luciferases
Antibody engineering; Antibody-dependent cellular cytotoxicity; Epiregulin; Humanization; Resurfacing