Site-specific PEGylation enhances the pharmacokinetic properties and antitumor activity of interferon beta-1b

Abstract

Interferon beta (IFN-β) is widely used to ameliorate disease progression in patients with Multiple Sclerosis. IFN-β has a short half-life in humans, necessitating frequent administration for optimum effectiveness. Covalent modification of IFN-β with polyethylene glycol (PEG) improves the pharmacokinetic properties of the protein, but can adversely affect the protein's in vitro bioactivity. Random modification of lysine residues in IFN-β with amine-reactive PEGs decreased the in vitro bioactivity of the protein 50-fold, presumably due to modification of lysine residues near critical receptor binding sites. PEGylated IFN-β proteins that retained high in vitro bioactivity could be obtained by selective modification of the N-terminus of the protein with PEG. Here we use site-specific PEGylation technology (targeted attachment of a cysteine-reactive-PEG to an engineered cysteine residue in IFN-β) to identify several additional amino acid positions where PEG can be attached to IFN-β without appreciable loss of in vitro bioactivity. Unexpectedly, we found that most of the PEG-IFN-β analogs showed 11- to 78-fold improved in vitro bioactivities relative to their unPEGylated parent proteins and to IFN-β-1b. In vivo studies showed that a lead PEG-IFN-β protein had improved pharmacokinetic properties compared to IFN-β and was significantly more effective than IFN-β at inhibiting growth of a human tumor xenograft in athymic mice.

FIG. 1.

Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Interferon beta (IFN-β)-1b and 12 IFN-β-1b cysteine analogs. Molecular weight markers (MW) are indicated to the left.

FIG. 2.

Dose response curves for IFN-β-1b (Betaseron), IFN-β-1b prepared by us (BBT IFN-β-1b), and representative IFN-β-1b cysteine analogs and PEGylated cysteine analogs for inhibiting in vitro proliferation of human Daudi B cells. Daudi cells were incubated with serial dilutions of the indicated test proteins and incubated for 4 days at 37°C in a tissue culture incubator. On day 4, cell number was quantified using Cell-Titer AqueousOne solution. Absorbance on the Y-axis is proportional to cell number. (A) Compares in vitro bioactivities of IFN-β-1b (Betaseron), BBT IFN-β-1b, and the T112C protein. (B) Compares IFN-β-1b (Betaseron), N25C, and 5 kDa-PEG-N25C. (C) Compares F111C and F111C modified with 20 kDa-, 30 kDa-, and 40 kDa-PEGs. Data are means±SD of triplicate wells for each protein dilution. SD generally were <10% of the means.

FIG. 3.

Nonreducing SDS-PAGE analysis of purified F111C and PEGylated F111C proteins. Lane 1, molecular weight markers; Lane 2, F111C, Lane 3, 20 kDa-PEG-F111C; Lane 4, 30 kDa-PEG-F111C; Lane 5, 40 kDa-PEG-F111C.

FIG. 4.

Clearance of IFN-β-1b (Betaseron) and 40 kDa-PEG F111C following intravenous (A) and subcutaneous (B) administration to rats. Proteins were dosed at 100 μg/kg and were quantitated in plasma samples by ELISA. Data are means±SD for 3 rats/group.

FIG. 5.

Dose response curves for IFN-β-1b (Betaseron) and PEG-F111C proteins for inhibiting in vitro proliferation of human NIH: OVCAR-3 cells. NIH: OVCAR-3 cells were incubated with serial dilutions of the indicated test proteins for 4 days at 37°C in a tissue culture incubator. On day 4, cell number was quantified as described in the legend to Fig. 2. Data are means±SD of triplicate wells for each protein dilution. SD generally were <10% of the means.

FIG. 6.

Inhibition of human tumor xenograft growth in athymic mice by IFN-β-1b (Betaseron) and 40 kDa-PEG-F111C. Athymic mice received an injection of 5×10⁶ NIH: OVCAR-3 cells on day 0. Mice received 3X/week subcutaneous injections of vehicle solution or 15 μg protein/injection of IFN-β-1b (Betaseron) or 40 kDa-PEG-F111C and tumor volumes were measured over time. Data are means±SE for 10 to 11 mice per group.