Plasma Half-life Extension of Small Recombinant Antibodies by Fusion to Immunoglobulin-binding Domains

Felix Unverdorben,Roland E. Kontermann,Aline Färber-Schwarz,Meike Hutt,Fabian Richter

doi:10.1074/jbc.m111.311522

Abstract

Many therapeutic proteins possessing a small size are rapidly cleared from circulation. Half-life extension strategies have therefore become increasingly important to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Here, we performed a comparative analysis of the half-life extension properties of various bacterial immunoglobulin-binding domains (IgBDs) derived from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), and Finegoldia (formerly Peptostreptococcus) protein L (PpL). These domains, composed of 50-60 amino acid residues, were fused to the C terminus of a single-chain Fv and a bispecific single-chain diabody, respectively. All fusion proteins were produced in mammalian cells and retained their antigen-binding properties. The half-lives of the antibody molecules were prolonged to varying extents for the different IgBDs. The strongest effects in mice were observed for domain C3 of SpG (SpG(C3)) followed by domains B and D of SpA, suggesting that SpG(C3) is particularly useful to extend the plasma half-life of small proteins.

Highlights

Half-life extension has become increasingly important for therapeutic proteins
ScDb-immunoglobulin-binding domains (IgBDs) and single-chain Fv fragment (scFv)-IgBD fusion proteins were generated by fusing IgBDs from Staphylococcus protein A (SpA), Streptococcus protein G (SpG), or peptostreptococcal protein L (PpL) to the C terminus of a bispecific single-chain diabody directed against Carcinoembryonic antigen (CEA) and CD3
We have generated a panel of novel IgBD fusion proteins using IgBDs from various bacterial immunoglobulin receptors

Summary

Background

Half-life extension has become increasingly important for therapeutic proteins. Results: Fusion of different bacterial immunoglobulin-binding domains to small recombinant antibodies prolonged their half-life to varying extents. With a growing number of small protein therapeutics being developed, half-life extension strategies have become increasingly important to improve their pharmacokinetic and pharmacodynamic properties [1, 2] These strategies include approaches to increase the hydrodynamic radius of the protein, e.g. by hyperglycosylation, coupling to polyethylene glycol (PEG) chains or other hydrophilic polymers, and fusion to PEGmimetic polypeptide chains or long circulation plasma protein and fragments thereof. We performed a comparative analysis of the capacity of various IgBDs derived from SpA, SpG, and PpL to extend the half-life of small recombinant antibodies, a scDb possessing a molecular mass of 55 kDa and a single-chain Fv fragment (scFv) with a molecular mass of 28 kDa. We show that half-lives in mice are prolonged to various extents depending on the applied IgBDs. The longest terminal half-life was seen for the fusion proteins containing domain C3 of SpG opening new applications as half-life extension module

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION