A Long-Acting, Highly Potent Interferon α-2 Conjugate Created Using Site-Specific PEGylation

Recombinant interferon α-2 (IFN-α2) is used clinically to treat a variety of viral diseases and cancers. IFN-α2 has a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of IFN-α2 by modifying lysine residues of the protein with amine-reactive poly(ethylene glycol) (PEG) reagents. However, amine-PEGylated IFN-α2 comprises a heterogeneous product mixture with low specific activity due to the large number and critical locations of lysine residues in IFN-α2. In an effort to overcome these problems we determined the feasibility of creating site-specific, mono-PEGylated IFN-α2 analogues by introducing a free (unpaired) cysteine residue into the protein, followed by modification of the added cysteine residue with a maleimide-PEG reagent. IFN-α2 cysteine analogues were expressed in Escherichia coli and purified, and their in vitro bioactivities were measured in the human Daudi cell line growth inhibit...