Novel long-lasting interferon alpha derivatives designed by glycoengineering

Human alpha interferons (hIFN-alpha) comprise a family of closely related proteins that block viral infection, inhibit cell proliferation and modulate cell differentiation. Recombinant hIFN-alpha2 has proved useful for the treatment of a variety of human viral diseases and cancers. However, the clinical use of this cytokine has been restricted due to its short circulating half-life, which makes frequent dosing over an extended period necessary. To circumvent this problem, a glycoengineering strategy was carried out using site-directed mutagenesis. Fourteen mutants were constructed by the insertion of one N-glycosylation consensus sequence into different positions of the cytokine. Mutations were focused on amino acid positions that were believed not to be critical for the protein's structure or function. Taking into account the retained specific in vitro bioactivity and the higher carbohydrate content, five N-glycosylation positions were selected to be introduced into the molecule. Successive increases in molecular weight were observed after each addition of a functional consensus sequence, resulting in analogs with 4 and 5 N-linked carbohydrates (4N- and 5N-IFN) with increased size and charge, factors that reduce renal clearance of proteins. Pharmacokinetic experiments showed a similar behavior of 4N- and 5N-IFN variants, with a 25-fold increase in the elimination half-life and a 20-fold decrease in the systemic clearance rate compared with the non-glycosylated rhIFN-alpha2 following subcutaneous administration to rats. Besides, both distribution and elimination half-lives of the 4N analog were longer in comparison with the non-glycosylated cytokine, determining a 10-fold increase in the area under the curve after intravenous inoculation. Thus, herein we describe for the first time heavily glycosylated IFN analogs with a remarkable improvement in pharmacokinetic properties, which allow us to project drugs that combine less frequency of administration with enhanced therapeutic efficacy.