title: Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG β-subunit.
abstract: Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) β-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-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 plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity.
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo", C.C. 242, S3000ZAA Santa Fe, Argentina. Electronic address: nceaglio@fbcb.unl.edu.ar.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ceaglio
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo", C.C. 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Agustina
authors.initials: A
authors.lastname: Gugliotta
authors.affiliation: Zelltek S.A., PTLC RN 168, S3000ZAA Santa Fe, Argentina.
authors.forename: María Belén
authors.initials: MB
authors.lastname: Tardivo
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo", C.C. 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Dianela
authors.initials: D
authors.lastname: Cravero
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo", C.C. 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Marina
authors.initials: M
authors.lastname: Etcheverrigaray
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo", C.C. 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Kratje
authors.affiliation: Cell Culture Laboratory, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje "El Pozo", C.C. 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Marcos
authors.initials: M
authors.lastname: Oggero
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000493:pharmacokinetics; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry* / Q000235:genetics; D003412:Cricetulus; D054697:Cytostatic Agents / Q000378:metabolism / Q000493:pharmacokinetics; D004355:Drug Stability; D057809:HEK293 Cells; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism* / Q000493:pharmacokinetics*
chemical_list: D000998:Antiviral Agents; D018997:Chorionic Gonadotropin, beta Subunit, Human; D054697:Cytostatic Agents; D016898:Interferon-alpha; D010446:Peptide Fragments; D011994:Recombinant Proteins
keywords: Carboxyl-terminal peptide; O-Glycosylation; Pharmacokinetics; Recombinant human interferon alpha; hCG β-subunit; in vitro protein stability