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.

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis

“…The synthetic APA antibody was directed against P. aeruginosa (strain O6) and consists of a single‐chain variable fragment (Hemachandra et al. , 2001) fused to the human IgG1 heavy chain constant region (CH1–CH3), which adds effector functions to the antibody (Hu et al. , 2003).…”

“…The construct used to express human IL-10 in plants (IL10-ELP) contains a translational fusion with an elastin-like polypeptide (ELP) tag and was described in detail by Conley et al (2009a). The synthetic APA antibody was directed against P. aeruginosa (strain O6) and consists of a single-chain variable fragment (Hemachandra et al, 2001) fused to the human IgG1 heavy chain constant region (CH1-CH3), which adds effector functions to the antibody (Hu et al, 2003). A (His) 6 tag was included for detection of the APA protein.…”

Generation and characterization of a single‐gene mouse–human chimeric antibody against hepatitis B surface antigen

“…An scFv‐Fc is a single‐gene product, which can dimerize through hinge region cysteine residues, thus providing the avidity effect. Several systems have been developed for the expression of such antibody‐like molecules 35–37 . Such fusion molecules have a prolonged serum half‐life in vivo 38 .…”

“…Such glycosylation is considered to be important for induction of effector functions 42 . The scFv‐Fc fusion molecules bind to C1q 37 and can trigger superoxide production through FcγRI 41 . It has been observed that scFv‐Fc fusion proteins can activate complement cascade and induce antibody‐dependent cell‐mediated cytotoxicity 43,44 .…”

“…Several systems have been developed for the expression of such antibody-like molecules. [35][36][37] Such fusion molecules have a prolonged serum half-life in vivo. 38 Powers et al have shown that the large size of a dimeric scFv-Fc results in a prolonged serum half-life in vivo, with the half-life of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc.…”