Improvement of in vitro stability and pharmacokinetics of hIFN-alfa by fusing the carboxyl-terminal peptide of hCG beta subunit

CEAGLIO, NATALIA; GUGLIOTTA, AGUSTINA; TARDIVO, MARÍA BELÉN ; CRAVERO, DIANELA; ETCHEVERRIGARAY, MARINA; KRATJE, RICARDO; OGGERO EBERHARDT, MARCOS

JOURNAL OF BIOTECHNOLOGY

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2016 vol. 221 p. 13 - 24

0168-1656

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for thebiopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-2b), whichbelongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, showsa poor stability in solution and an extremely short plasma half-life which determines a strict therapeu-tic regimen comprising high and repeated doses. In this work, we have used a strategy based on thefusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) -subunit, bear-ing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-2b.Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were effi-ciently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferativebioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through thisapproach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasmaprotease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by theCTP moiety. These results constitute the first report that postulates CTP as a tag for improving both thein vitro and in vivo stability of rhIFN-2b which, in turn, would positively influence its in vivo bioactivity.