GMOPm: An hGM-CSF-derived peptide as a novel O-glycoengineering tool

ITURRASPE, FRANCISCO; GUGLIOTTA, AGUSTINA; KRATJE, RICARDO; OGGERO, MARCOS; CEAGLIO, NATALIA

Copenhague

Congreso; 26th Meeting of the European Society for Animal Cell Technology (ESACT); 2019

European Society for Animal Cell Technology (ESACT)

One of the major concerns regarding administration of protein biotherapeutics for disease treatment lies in their low stability and short plasma half-life. To circumvent this problem, we have developed a 15-mer peptide tag named GMOPm. This sequence comprises the first 7 amino acids of the N-terminal region of human granulocyte-macrophage colony stimulating factor (hGM-CSF) together with 8 more residues that have been added aiming to generate 6 potential O-glycosylation sites. The goal of this work was to study the ability of GMOPm to improve the pharmacokinetic of a widely used biotherapeutic, human interferon-α2b (hIFN-α2b), as a mean to increase its efficacy.  Five chimeras were constructed by adding GMOPm to the N- and/or C-terminal ends of hIFN-α2b in different proportions to obtain variants with 7 to 29 potential O-glycosylation sites as predicted in silico: GMOPm-IFN; (GMOPm)2-IFN; (GMOPm)3-IFN; (GMOPm)2-IFN-GMOPm and (GMOPm)3-IFN-GMOPm. The variants were purified, in vitro characterized, and analyzed regarding their pharmacokinetic parameters.  CHO-K1 recombinant cell lines were cultured for IFN variants production and the chimeras were purified from culture supernatant by affinity chromatography, with yields ranging from 40 to 100%. SDS-PAGE and IEF analysis demonstrated that the more the number of GMOPm tags added, the higher the molecular mass and the lower the isoelectric point of the fusion protein. Interestingly, all IFN variants retained in vitro antiviral activity, although it decreased concomitantly with the number of fused tags. Pharmacokinetic experiments in rats demonstrated that the highest glycosylated variants, GMOPm2-IFN-GMOPm and GMOPm3-IFN-GMOPm, exhibited a plasma half-life 2 and 4-fold higher and a clearance rate 3 and 5 fold-lower than the variant with a single GMOPm tag, respectively, demonstrating the success of this O-glycoengineering tool for improving IFN properties.