Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization.
Glypican 3 (GPC3), a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, is expressed in a majority of hepatocellular carcinoma tissues. The murine monoclonal antibody GC33 that specifically binds to the COOH-terminal part of GPC3 causes strong antibody-dependent cellular cytotoxicity against hepatocellular carcinoma cells and exhibits strong antitumor activity in the xenograft models. To apply GC33 for clinical use, we generated a humanized GC33 from complementarity-determining region grafting with the aid of both the hybrid variable region and two-step design methods. The humanized antibody bound to GPC3 specifically and induced antibody-dependent cellular cytotoxicity as effectively as a chimeric GC33 antibody. To improve stability of the humanized GC33, we further optimized humanized GC33 by replacing the amino acid residues that may affect the structure of the variable region of a heavy chain. Substitution of Glu6 with Gln in the heavy chain significantly improved the stability under high temperatures. GC33 also has the risk of deamidation of the -Asn-Gly- sequence in the complementarity-determining region 1 of the light chain. As substitution of Asn diminished the antigen binding, we changed the neighboring Gly to Arg to avoid deamidation. The resulting humanized anti-GPC3 antibody was as efficacious as chimeric GC33 against the HepG2 xenograft and is now being evaluated in clinical trials.
Nakano
Kiyotaka
K
Department of Genome Antibody Product Research, Chugai Pharmaceutical Co. Ltd., Gotemba, Japan.
Ishiguro
Takahiro
T
Konishi
Hiroko
H
Tanaka
Megumi
M
Sugimoto
Masamichi
M
Sugo
Izumi
I
Igawa
Tomoyuki
T
Tsunoda
Hiroyuki
H
Kinoshita
Yasuko
Y
Habu
Kiyoshi
K
Orita
Tetsuro
T
Tsuchiya
Masayuki
M
Hattori
Kunihiro
K
Yamada-Okabe
Hisafumi
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000276:immunology* / Q000627:therapeutic use; D000920:Antibody-Dependent Cell Cytotoxicity; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000473:pathology / Q000628:therapy*; D022801:Complementarity Determining Regions / Q000276:immunology; D015195:Drug Design; D053673:Glypicans / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008113:Liver Neoplasms / Q000276:immunology* / Q000473:pathology / Q000628:therapy*; D051379:Mice; D055550:Protein Stability; D023041:Xenograft Model Antitumor Assays
D058846:Antibodies, Monoclonal, Murine-Derived; D022801:Complementarity Determining Regions; C105937:GPC3 protein, human; D053673:Glypicans; D007135:Immunoglobulin Variable Region