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 inhibition assay. Several cysteine analogues were identified that do not significantly affect in vitro biological activity of IFN-α2. Certain of the cysteine analogues, but not wild-type IFN-α2, reacted with maleimide-PEG to produce mono-PEGylated proteins. The PEG−Q5C analogue retained high in vitro bioactivity (within 3- to 4-fold of wild-type IFN-α2) even when modified with 20- and 40-kDa PEGs. Pharmacokinetic experiments indicated that the 20-kDa PEG−Q5C and 40-kDa PEG−Q5C proteins have 20-fold and 40-fold longer half-lives, respectively, than IFN-α2 following subcutaneous administration to rats. These studies demonstrate the feasibility of using site-specific PEGylation technology to create a long-acting, mono-PEGylated IFN-α2 protein with high specific activity.