Abstract
We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) Science 287∶1279–1283]. IgGBP fusion increases the in vivo half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the in vitro and in vivo properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in Escherichia coli and bind specifically to human IgG with an affinity of ∼40 nM and ∼20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn in vitro. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.
Highlights
Protein-based therapeutic sales in the biotechnology industry continue to soar reaching,$64 billion in 2012, a remarkable,18% increase compared to 2011 [1]
We determined the ability of immunoglobulin G (IgG) from various species to bind the target proteins using a competition based enzyme-linked immunosorption assay (ELISA) in which either mKate or mKate-IgGFc domain binding peptide (IgGBP) are absorbed to a 96-well plate and donkey IgG-HRP is used as the detection reagent (Figure S1a in File S1)
We describe a simple approach to substantially improve protein half-life without the necessity to increase molecular weight by engineering serum IgG binding using a low molecular weight IgGFc binding peptide fused to the C-terminus of a model protein
Summary
Protein-based therapeutic sales in the biotechnology industry continue to soar reaching ,$64 billion in 2012, a remarkable ,18% increase compared to 2011 [1]. Unlike mAbs, these classes of human proteins are rapidly eliminated from circulation due to their small size, which necessitates their frequent injection or continuous infusion to maintain therapeutic blood concentrations. To overcome these limitations a number of protein half-life extension strategies have been devised [2] including chemical conjugation [3] or genetic fusion to high molecular weight polymers [4], genetic fusion to the Fc-domain of immunoglobulin G (IgG) [5] or monomeric IgG domains [6,7,8], and genetic fusion to albumin [9]. Engineering proteins to interact with serum IgG or albumin has the potential to significantly increase half-life by reducing both renal clearance and intracellular catabolism
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