High-Affinity Target Binding Engineered via Fusion of a Single-Domain Antibody Fragment with a Ligand-Tailored SH3 Domain

Annika Järviluoma,Tomas Strandin,Anna R Mäkelä,Matthias Geyer,Jérôme Bouchet,Sebastian Lülf,Serge Benichou,Kalle Saksela,Stefan Dübel

doi:10.1371/journal.pone.0040331

Abstract

Monoclonal and recombinant antibodies are ubiquitous tools in diagnostics, therapeutics, and biotechnology. However, their biochemical properties lack optimal robustness, their bacterial production is not easy, and possibilities to create multifunctional fusion proteins based on them are limited. Moreover, the binding affinities of antibodies towards their antigens are suboptimal for many applications where they are commonly used. To address these issues we have made use of the concept of creating high binding affinity based on multivalent target recognition via exploiting some of the best features of immunoglobulins (Ig) and non-Ig-derived ligand-binding domains. We have constructed a small protein, named Neffin, comprised of a 118 aa llama Ig heavy chain variable domain fragment (VHH) fused to a ligand-tailored 57 aa SH3 domain. Neffin could be readily produced in large amounts (>18 mg/L) in the cytoplasm of E. coli, and bound with a subpicomolar affinity (Kd 0.54 pM) to its target, the HIV-1 Nef protein. When expressed in human cells Neffin could potently inhibit Nef function. Similar VHH-SH3 fusion proteins could be targeted against many other proteins of interest and could have widespread use in diverse medical and biotechnology applications where biochemical robustness and strong binding affinity are required.

Highlights

Specific recognition and strong binding to chosen target molecules is the cornerstone of modern therapeutic and diagnostic practices
We have generated a fusion protein comprised of an single-domain antibodies (sdAb) fragment derived from a llama immunized against the HIV-1 pathogenicity factor Nef with a synthetic library-derived SH3 domain optimized for binding to Nef
Construction of Neffin Bivalent target recognition is an attractive concept for generating high affinity binding polypeptides for therapeutic and diagnostic applications

Summary

Introduction

Specific recognition and strong binding to chosen target molecules is the cornerstone of modern therapeutic and diagnostic practices. Recombinant antibodies containing only the Fab fragment and single-chain antibodies (scFv) comprised only of the variable domains of heavy and light chains joined by a flexible linker peptide represent simpler and smaller alternatives to complete immunoglobulins. The possibility to select recombinant antibodies from synthetic libraries and to optimize their properties by random and targeted mutagenesis combined with powerful in vitro affinity selection schemes have been fruitfully exploited in various biotechnology applications. These approaches enable rational targeting of antibody binding, including target epitopes that might be poorly immunogenic, as well as overcoming the affinity ceiling of monoclonal antibodies. While most natural antibodies have Kd values in the range of 1028 to 10211 M [6,7], orders of magnitude tighter binding has been reported for optimized recombinant antibodies [8]

Methods

Results

Conclusion