Development and Characterization of a Novel Fusion Protein Composed of a Human IgG sub 1 Heavy Chain Constant Region and a Single-Chain Fragment Variable Antibody Against Venezuelan Equine Encephalitis Virus

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Authors
  1. Nagata, L.P.
  2. Fulton, R.E.
  3. Hu, W-G.
  4. Alvi, A.Z.
  5. Chau, D.
  6. Coles, J.E.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN);Alberta Univ, Edmonton ALTA (CAN) Faculty of Pharmacy and Pharmaceutical Sciences
Abstract
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 IgG sub 1 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 antibody, 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.
Keywords
ELISA (Enzyme Linked Immunosorben);Sequencing;Recombinant antibody technology;Genetic analysis;VEE virus
Report Number
DRDC-SUFFIELD-SL-2001-171 — Reprint
Date of publication
14 May 2003
Number of Pages
9
Reprinted from
J. Biochem, vol 133, 2003, p 59-66
DSTKIM No
CA022366
CANDIS No
519232
Format(s):
Hardcopy;Document Image stored on Optical Disk

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