A novel hGM-CSF-derived peptide to generate highly O-glycosylated hIFN-alfa2b variants

VERÓNICA FERRANDO; MARCOS OGGERO; MARINA ETCHEVERRIGARAY; RICARDO KRATJE; NATALIA CEAGLIO

Villa General Belgrano

Simposio; GlycoAR 2016; 2016

CONICET-UNC-INSTITUTO LELOIR-IBYME-SOC LATIN GLICOBIOLOGÍA

Currently, the clinical use of therapeutic recombinant proteins entails certain limitations due to their low stability and short half-life. In this work, a strategy for the production of highly O-glycosylated rhIFN-a2b variants was carried out by fusion to a novel peptide (GMOPm). This tag has 6 potential O-glycosylation sites and derives from the human granulocyte and macrophage-colony stimulating factor (hGM-CSF). Our goal is to improve the properties of IFN by increasing its glycosylation content and, consequently, decreasing its plasma clearance leading to a better in vivo biological activity. Five variants were designed by adding the GMOPm tag to the N- and/or C-terminal ends of the IFN sequence in different proportions, to obtain molecules with 7 to 29 potential O-glycosylation sites. The amino acid sequences of the chimeras were analyzed using different online predictors to study the probability of O-glycosylation. The results were consistent with the molecular masses of the variants assessed by SDS-PAGE followed by western blot, indicating the successful incorporation of O-linked glycans. Thus, the applied strategy allowed the generation of novel GMOPm/rhIFN-a2b chimeras, which presented higher molecular masses (31-60 kDa) than the CHO-K1-derived wild type rhIFN-a2b (21.5 kDa) and retained both in vitro antiviral (20-100%) and antiproliferative (2-47%) bioactivity relative to the unmodified rhIFN-a2b.