title: Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway.
abstract: Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.
authors.affiliation: Schering-Plough Research Institute, Biotechnology Development, Bioanalytical and Process Development, Union, New Jersey 07083 and Aventis Pharmaceuticals, Medical Affairs-Oncology, Bridgewater, New Jersey 08807, USA. michael.grace@spcorp.com
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Grace
authors.forename: Seoju
authors.initials: S
authors.lastname: Lee
authors.forename: Sheri
authors.initials: S
authors.lastname: Bradshaw
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Chapman
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Spond
authors.forename: Stuart
authors.initials: S
authors.lastname: Cox
authors.forename: Marc
authors.initials: M
authors.lastname: Delorenzo
authors.forename: Diana
authors.initials: D
authors.lastname: Brassard
authors.forename: David
authors.initials: D
authors.lastname: Wylie
authors.forename: Susan
authors.initials: S
authors.lastname: Cannon-Carlson
authors.forename: Constance
authors.initials: C
authors.lastname: Cullen
authors.forename: Stephen
authors.initials: S
authors.lastname: Indelicato
authors.forename: Marcio
authors.initials: M
authors.lastname: Voloch
authors.forename: Ronald
authors.initials: R
authors.lastname: Bordens
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004337:Drug Carriers / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000494:pharmacology; D053613:Janus Kinase 1; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D000998:Antiviral Agents; D004268:DNA-Binding Proteins; D004337:Drug Carriers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D011092:Polyethylene Glycols; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; D053613:Janus Kinase 1; C417083:peginterferon alfa-2b