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Engineered Antibodies of IgG1/IgG3 Mixed Isotype with Enhanced Cytotoxic Activities

¹ Antibody Research Laboratories, Pharmaceutical Research Center and ² Antibody Business Office, Pharmaceuticals Business Unit, Kyowa Hakko Kogyo Co. Ltd., Tokyo, Japan; ³ Toxicological Research Laboratories, Ube Branch Pharmaceutical Research Center, Kyowa Hakko Kogyo Co. Ltd., Yamaguchi, Japan; and ⁴ Pharmacokinetic Research Laboratories, Pharmaceutical Research Center, Kyowa Hakko Kogyo Co. Ltd., Shizuoka, Japan

Requests for reprints: Rinpei Niwa, Antibody Research Laboratories, Pharmaceutical Research Center, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan. Phone: 81-42-725-2555; Fax: 81-42-725-2689; E-mail: rinpei.niwa{at}kyowa.co.jp.

Key Words: Antibodies • Complement-dependent cytotoxicity • Antibody-dependent cellular cytotoxicity • Antibody engineering • Immunotherapy

Enhancement of multiple effector functions of an antibody may be a promising approach for antibody therapy. We have previously reported that fucose removal from Fc-linked oligosaccharides greatly enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies. Here, we report a unique approach to enhance complement-dependent cytotoxicity (CDC), another important effector function of antitumor antibodies, by using engineered constant region of human IgG1/IgG3 chimeric isotypes. We systematically shuffled constant domains of IgG1 and IgG3 to generate a comprehensive set of mixed chimeric isotypes of anti-CD20 antibodies. Among these, the variant 1133, consisting of the CH1 and the hinge each from IgG1 and the Fc from IgG3, was unexpectedly found to exhibit markedly enhanced CDC that exceeded wild-type levels. However, it lacked protein A–binding capacity, an important feature for the industrial production. To eliminate this deficiency, a portion in COOH-terminal CH3 domain of 1133 was substituted with IgG1, resulting in full recovery of protein A binding without compromising the enhanced CDC and ADCC activities. The CDC-enhancing effect using a chimeric isotype was also shown in CD52 antigen/antibody system. The ADCC activity of the variants was also maximized by the absence of fucose from its carbohydrate structure, a phenomenon that has previously been observed for wild-type antibodies. Enhanced cytotoxicity of a variant was confirmed in a cynomolgus monkey model. These findings suggest that the variant antibodies with IgG1/IgG3 chimeric constant regions and nonfucosylated oligosaccharides that possess dual-enhanced cytotoxic functions may be an improvement for the next generation of therapeutic antitumor antibodies. [Cancer Res 2008;68(10):3863–72]