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J. Biochem, 2003, Vol. 133, No. 1 59-66
© 2003 Japanese Biochemical Society

BIOTECHNOLOGY

Development and Characterization of a Novel Fusion Protein Composed of a Human IgG1 Heavy Chain Constant Region and a Single-Chain Fragment Variable Antibody against Venezuelan Equine Encephalitis Virus

Wei-Gang Hu1,2, Azhar Z. Alvi1, Damon Chau1, Jeffrey E. Coles1, R. Elaine Fulton1, Mavanur R. Suresh2 and Les P. Nagata+,1

1 Chemical Biological Defence Section, Defence Research & Development Canada-Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada T1A 8K6; and 2 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8

Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy. In order to minimize the immunogenicity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed. It encoded a human IgG1 heavy chain constant region and a single-chain fragment variable antibody of 1A4A1. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recognition by protein G and human complement C1q binding. On non-reducing and reducing gel electrophoresis analysis of proteolytic fragments of the recombinant antbody, disulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recognition, and retained several functional activities. This work forms the basis for characterization of the recombinant antibody as to efficacy in vivo.

+ To whom correspondence should be addressed. Tel: +1-403-544-4628, Fax: +1-403-544-3388, E-mail: les.nagata{at}drdc-rddc.gc.ca


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