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J. Biol. Chem., Vol. 283, Issue 2, 1156-1166, January 11, 2008

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Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528^*

Koki Makabe¹², Takeshi Nakanishi¹, Kouhei Tsumoto³, Yoshikazu Tanaka³, Hidemasa Kondo, Mitsuo Umetsu, Yukiko Sone, Ryutaro Asano, and Izumi Kumagai⁴

From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11-606, Aoba-ku, Sendai 980-8579, Japan and the Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Received for publication, July 27, 2007 , and in revised form, September 21, 2007.

The atomic coordinates and structure factors (code 1WT5 and 2Z4Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (to K. T. and I. K.). Additional support was provided through Grants-in-Aid for Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan and through the Proposal-based R&D Promotion Program and the Industrial Technology Research Grant Program in 2003 from the New Energy and Industrial Technology Development Organization of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Fig. S1.

¹ Both authors contributed equally to this work.

² Present address: Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637.

³ Present address: Dept. of Medical Genome Sciences, Graduate School of Engineering, University of Tokyo, Kashiwanoha, Kashiwa 277-8562, Japan.

⁴ To whom correspondence should be addressed. Tel.: 81-22-795-7274; Fax: 81-22-795-6164; E-mail: kmiz{at}kuma.che.tohoku.ac.jp.

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