Rational Design and Engineering of Interferon-β 1a for Improving Biophysical and Pharmacokinetic Properties

Abstract

Recombinant human IFN-β 1a (rhIFN-β 1a) is a single glycosylated protein (at N80, 1N) with anti-viral, anti-proliferation, and immunomodulatory activities. rhIFN-β 1a has been approved as a drug for the treatment of multiple sclerosis (MS). rhIFN-β is a highly hydrophobic protein that has a strong propensity for aggregation. It was already well known that aggregation reduces the activity of rhIFN-β and can also contribute to low production yield in mammalian culture systems, resulting in the high price of therapeutics high cost of production. As with other protein drugs, rhIFN-βs also have relatively short serum half-lives that may necessitate frequent parenteral administration to achieve efficacy. The frequent injections and related local skin reactions are the most common inconveniences associated with treatment. Modification of protein drugs by the attachment of an oligosaccharide moiety or polyethylene glycol (PEG) can improve patient compliance through sustained clinical response with less frequent dosing. This is due to the fact that glycoengineered or PEGylated protein could exhibit improved thermal stability and solubility, thus prolonging the circulating half-life. Moreover, it could reduce immunogenicity of the protein drug. To address these unmet needs by designing a biobetter version of rhIFN-β, an oligosaccharide moiety and PEG were sequentially introduced on rhIFN-β 1a. Site-specific hyperglycosylation was introduced into rhIFN-β 1a via site-directed mutagenesis. Glycoengineered rhIFN-β 1a was characterized by western blotting, isoelectric focusing, enzyme immunoassay, and glycosylation analysis. Glycoengineering of rhIFN-β 1a resulted in the production of a new molecular entity, R27T, with which we could obtain valuable competitive intellectual property rights. Glycoengineering successfully resulted in a product that exhibited unaltered ligand-receptor binding, with no observed change in the specific activity. R27T displayed improved stability and solubility, reduced aggregation, and increased half-life in pharmacokinetic (PK) studies, suggesting that hyperglycosylated rhIFN-β could be a biobetter version rhIFN-β for the treatment of MS. The addition of PEG is well known as an effective strategy to alter the PK profiles of a variety of drugs, thereby improving therapeutic potential. To obtain a more dramatic improvement in PK property, PEG was conjugated to R27T using several methods. PEG-R27T exhibited improved in vivo circulation half-lives over their corresponding native forms, although there was little activity loss. Taken together, rational design and engineering of rhIFN-β 1a using glycoengineering and PEGylation resulted in improved biophysical and PK properties, suggesting that these modification products could serve as new improved therapeutics for MS.