Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1

Abstract

SUMMARYWe have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 10 9 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment.

Phage Displayed scFv: pIII Scaffold May Fine Tune Binding Specificity

“…However, soluble scFv molecules have previously been shown to have negligible binding to target as compared to parent phage antibody. (17) This altered specificity of soluble scFv molecules is proposed to be due to their poor stability, smaller size, and lack of structural support provided by phage minor coat protein pIII, to which scFv are often fused. Similar phenomena of soluble scFv showing …”

Protective and Therapeutic Capacity of Human Single-Chain Fv-Fc Fusion Proteins against West Nile Virus

“…A bivalent molecule and/or addition of the Fc region is typically required for effective neutralization and in vivo efficacy by scFvs (16,22). The small size (26 kDa) of the monovalent scFvs results in their rapid clearance, with a half-life as short as only 3.5 h (13), and is a limiting factor for their use in passive immunization.…”

Construction of Human Fab Library and Isolation of Monoclonal Fabs with Rabies Virus‐Neutralizing Ability

“…The isolation of Fab with high specificity and affinity for the antigen is possible even if the size of the library is not enormous in the case of an "immunelibrary," described in the current study, because peripheral lymphocytes with antibody repertoires roughly oriented to an antigen-specific antibody group were used to construct the Fab library. Ray et al described two RV-neutralizing scFv-Fc fusion proteins isolated from a human synthetic scFv phage display library (19). The molecules revealed neutralizing activity against the RV PV strain after fusing to the human Fc protein, in a standard in vivo neutralization assay where the virus was incubated with the molecules before inoculation in mice.…”