Site of Pegylation and Polyethylene Glycol Molecule Size Attenuate Interferon-α Antiviral and Antiproliferative Activities through the JAK/STAT Signaling Pathway

Therapeutic pegylated interferon-αs (IFN-α) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-α molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-α 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-α2b that were monopegylated using 5–30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys1, His34, Lys31, Lys83, Lys121, Lys131, and Lys134. The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-α2b control. The highest residual activity was observed with the His34 positional isomers, and the lowest was observed with the Cys1 positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys134 > Lys83 ∼ Lys131 ∼ Lys121 > Lys31. The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-α to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His34 positional isomer suggests that this site may be favorable for pegylating IFN-α2b molecules.