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J. Biochem, 2001, Vol. 129, No. 1 5-12
© 2001 Japanese Biochemical Society

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Expression of Humanized Fab Fragments That Recognize the IgE-Binding Domain of Human Fc{varepsilon}RI{alpha} in COS and CHO Cells

Toshiro Takai*,{dagger},1, Ko Okumura{dagger}, Chisei Ra{dagger},{ddagger}, Toyokazu Yokota* and Yasushi Okumura*

*Bioscience Research & Development Laboratory, Asahi Breweries, Ltd 1-21 Midori 1-chome, Moriya-machi, Kitasoma-gun, Ibaraki 302-0106
{dagger}Department of Immunology, Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421
{ddagger}Division of Molecular Biology, Allergy Research Center, Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

1To whom correspondence should be addressed. Bioscience Research & Development Laboratory, Asahi Breweries, Ltd., 1-21 Midori 1-chome, Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan. Tel: +81-297-46-1503. Fax: +81-297-46-1505. E-mail: toshiro.takai{at}asa-hibeer.co.jp

Interfering with the binding of IgE to high-affinity IgE receptor {alpha} chain (Fc{varepsilon}RI{alpha}) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human Fc{varepsilon}RI{alpha} inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human Fc{varepsilon}RI{alpha} Fabs without papain-digestion of the whole antibody. Four Fabs with different C-termini of CH1 were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-Fc{varepsilon}RI{alpha} binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human Fc{varepsilon}RI{alpha} will provide useful information for the design of therapeutic reagents for allergy and asthma.


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S. Hashiguchi, T. Nakashima, A. Nitani, T. Yoshihara, K. Yoshinaga, Y. Ito, Y. Maeda, and K. Sugimura
Human Fc{varepsilon}RI{alpha}-Specific Human Single-Chain Fv (scFv) Antibody with Antagonistic Activity toward IgE/Fc{varepsilon}RI{alpha}-Binding
J. Biochem., January 1, 2003; 133(1): 43 - 49.
[Abstract] [Full Text] [PDF]


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