Novel bifunctional single-chain variable antibody fragments to enhance virolysis by complement: generation and proof-of-concept.

Georg Huber,Renate Kunert,Heribert Stoiber,Zoltán Bánki

doi:10.1155/2014/971345

Abstract

When bound to the envelope of viruses, factor H (FH), a soluble regulator of complement activation, contributes to the protection against a potent immune defense mechanism, the complement-mediated lysis (CML). Thus, removing FH from the surface renders viruses, such as HIV, susceptible to CML. For a proof of concept, we developed a construct consisting of recombinant bifunctional single-chain variable fragment (scFv) based on a monoclonal antibody against Friend murine leukemia virus (F-MuLV) envelope protein gp70, which was coupled to specific binding domains (short consensus repeats 19-20; SCR1920) of FH. We used Pichia pastoris as expression system in common shake flasks and optimized expression in high density bench top fermentation. Specific binding of recombinant scFv was proven by flow cytometry. The recombinant scFv-SCR significantly enhanced CML of F-MuLV in vitro implying that FH binding to the viral surface was impaired by the scFv-SCR. This novel concept to enhance virolysis may provide a new approach for antiviral treatment.

Highlights

Monoclonal antibodies are undoubtedly indispensable for many regiments in cancer and antiviral therapies
For a proof of concept, we developed a construct consisting of recombinant bifunctional single-chain variable fragment based on a monoclonal antibody against Friend murine leukemia virus (F-MuLV) envelope protein gp70, which was coupled to specific binding domains of factor H (FH)
First we determined the sequence of HC and LC of monoclonal Ab clone 48 (mAb48) [17], which targets a specific conformational epitope on F-MuLV envelope protein gp70

Summary

Introduction

Monoclonal antibodies (mAbs) are undoubtedly indispensable for many regiments in cancer and antiviral therapies. In applications in which Fc-dependent effector functions are not essential, smaller fragments such as single-chain variable fragments (scFv) have several advantages over their parental antibodies. They offer rapid blood clearance and enhanced tumor penetration, which makes them a good choice for oncologic imaging [1]. A scFv is composed of variable fragments of the light chain (vL) and heavy chain (vH) connected by a short hydrophilic polypeptide linker providing flexibility to join the two fragments They are expressed as a single polypeptide and are easier to produce compared to full-length antibodies. Expression by the yeast Pichia pastoris provides a straightforward and cost-effective microbial culturing system

Methods

Results

Conclusion