title: 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.
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 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 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.
authors.affiliation: Chemical Biological Defence Section, Defence Research & Development Canada-Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada T1A 8K6.
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Hu
authors.forename: Azhar Z
authors.initials: AZ
authors.lastname: Alvi
authors.forename: Damon
authors.initials: D
authors.lastname: Chau
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Coles
authors.forename: R Elaine
authors.initials: RE
authors.lastname: Fulton
authors.forename: Mavanur R
authors.initials: MR
authors.lastname: Suresh
authors.forename: Les P
authors.initials: LP
authors.lastname: Nagata
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology; D015922:Complement C1q / Q000378:metabolism; D004666:Encephalitis Virus, Venezuelan Equine / Q000276:immunology*; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; C029152:G-substrate; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C064956:glycoprotein E2, equine encephalitis virus; D015922:Complement C1q