Prolonging hIFN-alpha2 half-life through glycoengineering.

NATALIA CEAGLIO; MARCOS OGGERO; MARINA ETCHEVERRIGARAY; RICARDO KRATJE

Praga. República Checa

Congreso; Protein Expression Europe; 2007

Cambridge Healthtech Institute

Human alpha interferons comprise a family of closely related proteins that exhibit antiviral, growth inhibitory and immune modulator functions. The recombinant cytokine has proved efficacy for the treatment of a diversity of human viral diseases and cancers. However, due to its rapid clearance, patients have to be treated by daily or thrice weekly injections. To address this problem, a long-acting IFN-alpha2 mutein was designed by introduction of N-linked glycosylation consensus sequences by site directed mutagenesis. Fourteen mutants were prepared focusing on amino acid substitutions that were believed not to be critical for the protein´s structure and function. Each IFN analog was expressed in CHO cells and, taking into account the retained specific biological activity and the higher carbohydrate content, five N-glycosylation positions were selected to be sequentially introduced into the cytokine. Analogs with increased size and charge were concomitantly obtained with each addition of functional consensus sequence, resulting in highly glycosylated molecules with four and five N-glycosylation sites (4N-IFN and 5N-IFN). 4N-IFN and 5N-IFN muteins were found to have a similar 20-fold decrease in systemic clearance rate and a 25-fold increase in elimination half-life after subcutaneous inoculation in rats, compared to the non-glycosylated rhIFN-alpha2b. Additionally, despite the lower in vitro activity, the 4N-IFN had a noteworthy increased in vivo potency as shown by its anti-proliferative effect on human prostate carcinoma implanted in nude mice. Therefore, the 4N-IFN derivative turns out to be a potential drug for clinical use, combining less frequency of administration with enhanced therapeutic efficacy.