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Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization

Nakano, Kiyotakaa; Ishiguro, Takahirob; Konishi, Hirokoc; Tanaka, Megumic; Sugimoto, Masamichib; Sugo, Izumic; Igawa, Tomoyukic; Tsunoda, Hiroyukia; Kinoshita, Yasukob; Habu, Kiyoshia; Orita, Tetsuroa; Tsuchiya, Masayukia; Hattori, Kunihiroa; Yamada-Okabe, Hisafumid

doi: 10.1097/CAD.0b013e32833f5d68

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.

Departments of aGenome Antibody Product Research

bPharmaceutical Research

cPreclinical Research

dResearch Planning and Coordination, Chugai Pharmaceutical Co. Ltd., Gotemba, Japan

Correspondence to Hisafumi Yamada-Okabe, PhD, Research Planning and Coordination Department, Gotemba Research Laboratories, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan

Tel: +81 550 87 6730; fax: +81 550 87 3637;


Received 18 April 2010 Revised form accepted 13 August 2010

© 2010 Lippincott Williams & Wilkins, Inc.