N-Terminally PEGylated Human Interferon-β-1a with Improved

Dec 3, 2005 - To prepare a PEGylated form of human interferon-β-1a (IFN-β-1a) suitable for ... 106 units of PEGylated IFN-β-1a resulted in a 58% re...
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Bioconjugate Chem. 2006, 17, 179−188

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N-Terminally PEGylated Human Interferon-β-1a with Improved Pharmacokinetic Properties and in Vivo Efficacy in a Melanoma Angiogenesis Model§ Darren P. Baker,*,†,§ Edward Y. Lin,† KoChung Lin,† Maria Pellegrini,† Russell C. Petter,† Ling Ling Chen,† Robert M. Arduini,† Margot Brickelmaier,† Dingyi Wen,† Donna M. Hess,† Liqing Chen,† Donna Grant,† Adrian Whitty,† Alan Gill,† Daniel J. Lindner,‡ and R. Blake Pepinsky† BiogenIdec Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, and Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195. Received August 4, 2005; Revised Manuscript Received October 14, 2005

PEGylation of IFN-R has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-β-1a (IFN-β-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal R-amino group of the cytokine. The PEGylated protein retained ∼50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced ∼30-fold and ∼4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was ∼13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SKMEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 × 106 units of unmodified IFN-β-1a resulted in a 29% reduction in vessel number, while 1 × 106 units of PEGylated IFN-β-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFNβ-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-β-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 × 106 units of unmodified IFN-β-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 × 106 units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFNβ-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-β-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-β is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehydemodified IFN-β-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-β-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.

INTRODUCTION (IFNs)1

Interferons are a family of cytokines that mediate antiviral, antiproliferative, and immunomodulatory effects in response to biological and chemical stimuli (1-3). Two types of IFN are recognized on the basis of their physical and biological properties: Type I IFNs, members of which include the IFN-R subtypes, IFN-β, IFN-τ, and IFN-ω, and Type II, the only member of which is IFN-γ. The Type I IFN’s signal through a common cell surface receptor composed of the IFNAR1 and IFNAR2 chains (4-7). The intracellular portions § This paper is dedicated to the memory of my friend, mentor, and colleague Professor Charles A. Fewson, OBE (Sept 8, 1937 to Aug 28, 2005), whose personal and intellectual contributions to the scientific community will be sorely missed. * Corresponding author. e-mail: [email protected]; telephone: 617-679 2165; fax: 617-679 2304. † BiogenIdec Inc. ‡ Taussig Cancer Center. 1 Abbreviations. IFN, interferon; PEG, poly(ethylene glycol); mPEG, methoxy poly(ethylene glycol); PEGylated, poly(ethylene glycol)modified; EMC, encephalomyocarditis; PBS, phosphate-buffered saline; AUC, area under the curve; Cmax, maximal concentration; CL, systemic clearance; t1/2, elimination half-life; Vss, volume of distribution at steady state.

of IFNAR1 and IFNAR2 associate with the Janus tyrosine kinases Tyk2 and JAK1, respectively (8-11), and, following ligand binding, the kinases phosphorylate each other and the receptor chains, thereby creating binding sites for STAT proteins (12, 13). The STAT proteins are subsequently phosphorylated, dimerize, migrate to the nucleus, and drive the expression of IFN-stimulated genes (14). Type I IFNs have been approved for the treatment of a number of diseases for which their antiviral, antiproliferative, and immunomodulatory activities underlie their mechanism of action. Recombinant IFN-R-2b (Intron-A, Schering-Plough) has been approved for the treatment of chronic hepatitis B and hepatitis C virus (HCV) infection, genital warts, AIDS-related Kaposi’s sarcoma, follicular non-Hodgkin’s lymphoma, malignant melanoma, and hairy cell leukemia, while recombinant IFN-R-2a (Roferon-A, Hoffman La-Roche) is approved for the treatment of HCV, hairy cell leukemia, and chronic phase Philadelphia chromosome-positive chronic myelogenous leukemia. In addition, IFN-R-2b has been shown to be effective for the treatment of giant cell angioblastoma, a destructive pediatric tumor (15), and both IFN-R-2b and IFN-R-2a have been used for the treatment of life-threatening pediatric hemangiomas (16, 17). Recombinant forms of IFN-β-1a (Avonex, BiogenIdec; Rebif, Serono) and IFN-β-1b (Betaferon, Schering

10.1021/bc050237q CCC: $33.50 © 2006 American Chemical Society Published on Web 12/03/2005

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AG) are approved for the treatment of multiple sclerosis, while nonrecombinant forms of IFN-β (e.g. Feron, Toray) are approved in Japan for the treatment of HCV. As with other small protein drugs, Type I IFNs have relatively short circulating half-lives that may necessitate frequent parenteral administration to achieve efficacy. To reduce the dosing frequency, and to improve the pharmacokinetics and efficacy, PEGylated forms of type I IFN’s have been developed (1820). PEGylation of IFN-R-2b with a linear 12 kDa PEG (PEGIntron, Schering-Plough) increased the half-life following subcutaneous administration from 4-7 to 30-40 h (21) and improved the sustained virological response (SVR) against HCV from 12 to ∼24% (22). When given in combination with the antiviral drug ribavirin, the SVR with PEG-Intron increased to 54% (23). Similarly, PEGylation of IFN-R-2a with a branched 40 kDa PEG (Pegasys, Hoffman La-Roche) increased the halflife following intravenous administration from 3-8 to 65 h (24) and improved the SVR from 19 to 39% in HCV patients (25), and from 8 to 30% in HCV patients with concomitant cirrhosis of the liver (26). When administered in combination with ribavirin, the SVR of PEGylated IFN-R-2a improved to 56% (27). In addition to a significant improvement in the SVR, the frequency of dosing for the PEGylated proteins was reduced from thrice weekly to only once per week, a factor that increases patient compliance and therefore the effectiveness of the overall treatment regimen. A similar reduction in dosing frequency was observed upon modification of recombinant granulocyte colonystimulating factor with a linear 20 kDa PEG (Neulasta, Amgen), administered for the treatment of neutropenia in cancer patients undergoing chemotherapy (28). While the PEGylation of IFN-R-2b and IFN-R-2a serves as a clear example of the benefits that can be achieved by administering a longer half-life molecule, it is perhaps surprising that a PEGylated form of IFN-β has not been tested in clinical trials of either multiple sclerosis or HCV, especially when considering that the unmodified proteins used to treat these diseases often require frequent dosing to achieve efficacy. For multiple sclerosis, two of the three approved IFN-β products are administered multiple times per week: IFN-β-1a (Rebif) is injected subcutaneously thrice weekly (29), while IFN-β-1b (Betaferon) is injected subcutaneously every other day (30). For HCV, efficacy of recombinant IFN-β in European patients has been established following thrice weekly subcutaneous administration (31), and following daily dosing for 6 days a week for twelve weeks by intravenous administration (32). In Japan, where nonrecombinant forms of IFN-β have been used widely for the treatment of HCV, the unmodified protein is also administered frequently, typically every day for 2 weeks followed by thrice weekly for the remainder of the treatment regimen, although twice-daily regimens have also been tested (33-35). Therefore, development of a PEGylated form of IFN-β may be useful for the treatment of diseases such as multiple sclerosis or HCV where some of the current therapies require frequent dosing. Moreover, the use of a PEGylated form of IFN-β for the treatment of HCV may, in addition to reducing the dosing frequency, also offer the potential of a better safety profile compared to PEGylated IFN-R-2b and IFN-R-2a. While many patients must reduce the dose of PEGylated IFN-R or stop treatment due to adverse reactions, data from clinical trials of IFN-β in multiple sclerosis and HCV indicate that treatment with this cytokine is associated with a good safety and tolerability profile (36). With the aim of developing a PEGylated form of IFN-β-1a suitable for testing in preclinical studies and potentially in human clinical trials, we have synthesized 20 kDa mPEG-O-2methylpropionaldehyde for targeting the N-terminal R-amino group of the protein. Since the N-terminus of human IFN-β-1a

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Figure 1. Schematic representation of the steps used in the synthesis of 20 kDa mPEG-O-2-methylpropionaldehyde.

is not critical for activity (alanine scanning mutagenesis of the N-terminal region (37), and deletion of the N-terminal methionine (38) have no effect on the antiviral activity), it was likely that modification of the protein with 20 kDa mPEG-O-2methylpropionaldehyde would result in a protein that retained significant activity and that had improved pharmacokinetic properties. Indeed, Pepinsky et al. (39) have shown that modification of the N-terminal R-amino group of IFN-β-1a with 20 kDa mPEG-propionaldehyde resulted in a protein that retained full in vitro antiviral activity, had improved pharmacokinetic properties in mice, rats, and rhesus monkeys, and was pharmacologically active in the latter species as judged by the induction of the IFN-responsive markers neopterin and β2microglobulin. However, while this report described the effects of PEGylation on the pharmacokinetic and pharmacodynamic properties of the protein, the unmodified and PEGylated proteins were not tested in an animal model of disease. Therefore, it remained an open question as to whether the improved pharmacokinetic properties would translate into improved efficacy. Here we report on the synthesis and characterization of 20 kDa mPEG-O-2-methylpropionaldehyde, and the preparation and characterization of IFN-β-1a modified at the N-terminus with this PEG group. We further report on the pharmacokinetics of the unmodified and PEGylated proteins in rats, and the efficacy of the two proteins in a murine dermis model of angiogenesis inhibition. The improved efficacy of the PEGylated protein is shown to correlate with the improvement in its pharmacokinetic properties.

MATERIALS AND METHODS Materials. SP-Sepharose, a Superose 6 HR 10/30 column, and 3H-thymidine were obtained from GE Healthcare (Piscatawy, NJ); 20 kDa mPEG-OH was from Sunbio (Anyang City, South Korea); recombinant human interferon-β-1a was from BiogenIdec (Cambridge, MA); sodium cyanoborohydride was from Sigma-Aldrich (St. Louis, MO); human serum albumin was from Instituto Grifols (Barcelona, Spain); endoproteinase Lys-C was from Wako Pure Chemical Industries (Osaka, Japan); sodium hydride (60% dispersion in mineral oil), 3-bromo-2methylpropene, borane tetrahydrofuran complex (1 M in THF), and 30% hydrogen peroxide were from Aldrich (Milwaukee, WI); potassium iodide, sodium hydroxide, sodium bicarbonate, and sodium thiosulfate were from Fisher (Fair Lawn, NJ); and Dess-Martin periodinane was from Lancaster (Windham, NH). Synthesis of 20 kDa mPEG-O-2-methylpropionaldehyde. The synthesis of 20 kDa mPEG-O-2-methylpropionaldehyde is given in Figure 1. Linear mPEG-OH with a molecular weight of 20000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1 mmol) was treated with 60% NaH (32 mg, 0.8 mmol) in THF (35 mL). 3-Bromo2-methylpropene (0.67 g, 5 mmol) and a catalytic amount of potassium iodide were added, and the mixture was heated to reflux for 17 h. The solvent was then removed under vacuum. CH2Cl2 (20 mL) was added to the residue, and the organic layer was separated and dried over anhydrous Na2SO4, and the volume was reduced to approximately 6 mL. The CH2Cl2 solution was added to ether (150 mL) dropwise. The resulting white precipitate was collected and dried under vacuum, yielding 1.8

N-Terminally PEGylated Human Interferon-β-1a

g (∼90% recovery) of compound 1. 1H NMR (CDCl3, 400 MHz) and 1H-13C HSQC 2D NMR (CDCl3, 400 MHz) showed 1H δ 4.92 (s, 1H), 4.85 (s, 1H), 3.89 (s), 3.35 (s), 1.70 (s, 3H); 13C δ 112, 76, 59, 20. To compound 1 (0.77 g, 0.04 mmol) in THF (7 mL) and CH2Cl2 (0.7 mL) at 0 °C was added BH3‚ THF (1.0 M, 1.4 mL). The mixture was stirred in an ice bath for 1 h. NaOH (2 M, 1.0 mL) was added slowly, followed by 30% H2O2 (0.3 mL). The reaction was warmed to ambient temperature and stirred for 16 h. The above workup procedure was followed (CH2Cl2 extraction, followed by precipitation from ether and drying), to yield 0.60 g (∼78% recovery) of compound 2 as a white solid. 1H NMR (CDCl3, 400 MHz), 1H-1H TOCSY 2D NMR (CDCl3, 400 MHz), and 1H-13C HSQC 2D NMR (CDCl3, 400 MHz) showed 1H δ 3.35 (s), 2.01 (m, 1H), 0.84 (d, 3H); 13C δ 59, 36, 13. Compound 2 (250 mg, 0.013 mmol) was dissolved in CH2Cl2 (2.5 mL) and Dess-Martin periodinane (DMP; 25 mg, 0.059 mmol) was added with stirring. The reaction was stirred for 30 min at ambient temperature. Saturated aqueous NaHCO3 and Na2S2O3 (2 mL each) were added, and the mixture was stirred at ambient temperature for 1 h. The above workup procedure was followed (CH2Cl2 extraction, followed by precipitation from ether and drying) to yield 194 mg (∼77% recovery) of compound 3 (20 kDa mPEG-O-2methylpropionaldehyde) as a white solid. 1H NMR (CDCl3, 400 MHz), 1H-1H TOCSY 2D NMR (CDCl3, 400 MHz), and 1H13C HSQC 2D NMR (CDCl , 400 MHz) showed 1H δ 9.70 (s, 3 1H), 3.35, 2.63 (m), 1.09 (d, 3H); 13C δ 59, 47, 11. The synthesis of 20 kDa mPEG-O-2-methylpropionaldehyde as described was repeated numerous times, up to a 20 g scale. Preparation of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a. Recombinant human IFN-β-1a (5.6 mg at 1.1 mg/mL in 50 mM Na2HPO4 pH 6.0, 270 mM NaCl) was treated with 20 kDa mPEG-O-2-methylpropionaldehyde (5 mg/mL) in the presence of sodium cyanoborohydride (5 mM), in the dark for 19.25 h at ambient temperature. The reaction mixture was diluted with 4 volumes of 20 mM MES pH 5.0 and loaded under gravity onto a 4.5 mL (1 cm internal diameter × 5.7 cm) column of SP-Sepharose Fast Flow resin that had been equilibrated in 5 mM Na2HPO4 pH 5.5. The column was washed with 45 mL of 5 mM Na2HPO4 pH 5.5 and then bound protein eluted with 25 mM MES pH 6.4, 400 mM NaCl. Fractions containing protein were pooled and then concentrated with Centricon YM 3 concentrators (Millipore). The unmodified and PEGylated forms of IFN-β-1a were separated on a Superose 6 HR 10/30 column run at 0.4 mL/min in 5 mM Na2HPO4 pH 5.5, 150 mM NaCl as the mobile phase. Fractions were collected and analyzed by SDS-PAGE, and those containing only the singly modified protein were pooled. The PEGylated protein was filter-sterilized (0.2 µm), and the concentration of the protein determined as described below. The final yield of the PEGylated IFN-β-1a (excluding the mass of the attached PEG) was 27%. Sufficient protein was removed for analytical characterization, and the remainder was formulated at 100 µg/ mL in 5 mM Na2HPO4 pH 5.5, 150 mM NaCl containing 45.6 mg/mL human serum albumin and stored at -70 °C. Determination of Protein Concentration. The concentration of unmodified and PEGylated IFN-β-1a was determined by absorbance measurements at 280 nm using the predicted extinction coefficient of 29990 L mol-1 cm-1 based on the tryptophan, tyrosine, and cystine (disulfide) content (40). The same value for the extinction coefficient was used for the PEGylated protein as for the unmodified protein since the PEG group does not contribute to the absorbance at 280 nm (a 1 mg/mL solution of 20 kDa mPEG-O-2-methylpropionaldehyde was found to have an absorbance of 0.002 as compared to 1.50 for 1 mg/mL IFNβ-1a). Therefore, an identical absorbance at 280 nm equates to the same mass of IFN-β-1a irrespective of the presence of the

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attached PEG. When referring to the mass of the PEGylated protein, the contribution of the PEG moiety is not included. Peptide Mapping. Peptides were generated by digestion of ∼13 µg of unmodified or PEGylated IFN-β-1a with 20% (w/w) endoproteinase Lys-C in 100 µL of PBS pH 7.6, 1 mM EDTA, 5 mM DTT for 24 h at ambient temperature. 4 µL of 1 M DTT and 100 µL of 8 M urea were then added, and the digest was loaded onto a YMC (Milford, MA) C18 (1.0 mm internal diameter × 250 mm) reverse-phase HPLC column (AA12S052501WT). The column was developed with the following gradient at 0.07 mL/min where A ) water, 0.03% TFA and B ) acetonitrile, 0.024% TFA: 0-50% B in 50 min, 50-70% B in 10 min, 70% B in 10 min. Peptide peaks were detected on-line with a 2690/ZMD LC-MS system (Waters, Milford, MA). The digests were also loaded onto a Vydac (Hesperia, CA) C4 (1.0 mm internal diameter × 250 mm) reverse-phase HPLC column (214TP51). The column was developed with the following gradient where A ) water, 0.1% TFA and B ) acetonitrile, 0.08% TFA: 0-63% B in 70 min, 63-80% B in 10 min. Peptide peaks were detected on-line with a Waters (Milford, MA) Model 996 photodiode array detector. Determination of Antiviral Activity. The specific antiviral activity of IFN-β-1a was determined using a cytopathic effect (CPE) bioassay that measures the ability of the protein to protect human lung carcinoma A549 cells (grown at 37 °C/5% CO2) challenged with encephalomyocarditis (EMC) virus. For the assay, 3 × 104 A549 cells in 100 µL of Dulbecco’s modified eagles medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin, were added to wells of 96-well microtiter plates and incubated for 5-6 h. Serial dilutions of duplicate IFN-β-1a standards and test samples were then added, and the cells were incubated for a further 24 h. The medium was removed, and EMC virus was added. The cells were incubated for 48 h, and 50 µL of 5 mg/ mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) in PBS was then added. After a 1 h incubation, the medium was removed, the wells were washed with 100 µL of PBS, and the cells and dye were solubilized with 100 µL of 1.2 N HCl in 2-propanol. The plates were then read at 450 nm, and the data were analyzed using four-parameter curve fitting with Softmax Pro software (Molecular Devices, Sunnyvale, CA). Determination of Antiproliferative Activity. The specific antiproliferative activity of IFN-β-1a was determined using a bioassay that measures the ability of the protein to inhibit the growth of Daudi human B cells (grown at 37 °C/5% CO2). For the assay, 1 × 104 Daudi cells in 150 µL of RPMI-1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine were added to wells of 96-well microtiter plates and incubated for 48 h with serial dilutions of quadruplet IFN-β-1a samples. 3HThymidine (1 µCi in 50 µL medium/well) was added, the cells incubated for a further 16 h, and the cultures were harvested using a Tomtec (Hamden, CT) 96 Mach III harvester. Thymidine incorporation was measured using a Perkin-Elmer (Wellesley, MA) 1450 MicroBeta JET scintillation counter. The data were analyzed using four-parameter curve fitting with DeltaGraph software version 4.5 (SPSS Inc., Chicago, IL). Determination of Pharmacokinetic Parameters. Pharmacokinetic parameters following intravenous administration of unmodified and PEGylated IFN-β-1a were determined using precannulated (jugular vein) female Lewis rats (weight 200250 g) obtained from Charles River Laboratories (Research Triangle Park, Raleigh-Durham, NC). Three rats were used for the unmodified IFN-β-1a and three for the PEGylated protein. Animals were acclimated in the BiogenIdec animal facility prior to the experiment. Food was withheld overnight prior to beginning the study. Water was available ad libitum. Experimental procedures were carried out according to an approved

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Institutional Animal Care and Use Committee (IACUC) protocol. Unmodified and PEGylated IFN-β-1a were formulated in 15 mg/mL human serum albumin in PBS, and a single dose of unmodified IFN-β-1a (80 µg/kg) or PEGylated IFN-β-1a (24 µg/kg) was administered. Blood samples (200 µL) were drawn at the following times: unmodified IFN-β-1a; 0, 0.083, 0.25, 0.5, 1.25, 3, and 5 h; PEGylated IFN-β-1a; 0, 0.083, 0.25, 0.5, 1.25, 3, 24, 48, and 72 h. To minimize blood volume depletion, each blood sample removed was replaced with an equivalent volume of sterile normal saline. Blood samples were allowed to clot for 60 min at ambient temperature and centrifuged, and serum was separated and stored at -70 °C. The amount of unmodified and PEGylated IFN-β-1a present in the serum samples was determined using the A549 cell/EMC virus CPE bioassay described above, using the unmodified or PEGylated protein as the standard, respectively. Pharmacokinetic parameters were calculated using WinNonLin software version 3 (Pharsight, Mountain View, CA) using noncompartmental analysis. Determination of in Vivo Antiangiogenic Activity. Unmodified and PEGylated IFN-β-1a were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors grown in athymic nude homozygous (nu/nu) mice. SK-MEL-1 cells (2 × 106) were inoculated on day 0 into the dermis in the flanks of the mid axillary line of 3-week-old nu/nu NCR mice. 24 h later (day 1), groups of four mice each received the following subcutaneous doses of human serum albumin solution (HSA, vehicle control), or HSA containing either unmodified or PEGylated IFN-β-1a: Group 1, 0.1 mL of 45.6 mg/mL HSA once on day 1 only; Group 2, 0.1 mL of 45.6 mg/mL HSA containing 1 × 106 units of unmodified IFN-β-1a once on day 1 only; and Group 3, 0.1 mL of 45.6 mg/mL HSA containing 1 × 106 units of PEGylated IFN-β-1a once on day 1 only. Mice were sacrificed on day 10 and the tumor inoculation site assessed for neovascularization, measured by an observer blinded as to the treatment (41). Every radially oriented vessel entering the periphery of the tumor was scored as a single vessel. In an independent study, groups of three mice each received the following subcutaneous doses of HSA, or HSA containing either unmodified or PEGylated IFN-β-1a: Group 1: 0.1 mL of 45.6 mg/mL HSA once on day 1 only; Group 2, 0.1 mL of 45.6 mg/mL HSA containing 1 × 106 units of PEGylated IFN-β-1a once on day 1 only; Group 3, 0.1 mL of 45.6 mg/mL HSA on days 1-9 inclusive; Group 4, 0.1 mL of 45.6 mg/mL HSA containing 1 × 106 units of unmodified IFN-β-1a on days 1-9 inclusive. Mice were sacrificed on day 10 and neovascularization of the tumors determined as described above. Experimental procedures were carried out according to an approved IACUC protocol. Statistical analysis of the data was carried out using GraphPad Prism software version 4 (GraphPad Software, San Diego, CA).

RESULTS PEGylation of IFN-β-1a. IFN-β-1a was modified with 20 kDa mPEG-O-2-methylpropionaldehyde, and the pharmacokinetic and efficacy profiles of the unmodified and PEGylated proteins were compared. Reaction of IFN-β-1a with 20 kDa mPEG-O-2-methylpropionaldehyde resulted in the formation of predominantly singly modified protein, with lesser amounts of more highly modified forms present (data not shown). The unmodified and PEGylated forms were separated from one another by size exclusion chromatography. Figure 2, lane C shows that the singly modified protein was purified to homogeneity, with no unmodified or multiply modified forms detected. Incorporation of a single PEG resulted in an increase in the apparent Mr of the protein by reducing SDS-PAGE from ∼25 kDa (Figure 2, lane B) to ∼55 kDa (Figure 2, lane C). By

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Figure 2. SDS-PAGE of unmodified and PEGylated human IFN-β1a. Purified proteins (4 µg) were electrophoresed on a reducing 1020% gradient gel and stained with Coomassie brilliant blue. Lane A, molecular weight markers; lane B, unmodified IFN-β-1a; lane C, PEGylated IFN-β-1a.

size exclusion chromatography, the singly modified protein had an apparent Mr of 320000 Da. The ∼10-fold increase in the apparent mass compared to the unmodified protein results from a large increase in the hydrodynamic volume of the PEGylated protein, a typical consequence of this type of modification. The specificity of the PEGylation reaction was determined by peptide mapping. Peptide maps of unmodified and PEGylated IFN-β-1a were generated with endoproteinase Lys-C (Figure 3). For the PEGylated protein, 93% of the PEG was attached to the N-terminal peptides consisting of residues 1-19 (MSYNLLGFLQRSSNFQCQK) and 2-19 (SYNLLGFLQRSSNFQCQK) as evident by their disappearance from the map (Figure 3, compare panels A and B). The presence of two N-terminal peptides stems from the fact that the preparation of IFN-β-1a used contained a small amount (11%) of a truncated species in which the N-terminal methionine was missing, and in which the R-amino group of the second residue, serine, was modified. The mapping data therefore show that the PEG is primarily attached to the N-terminal peptide(s). Furthermore, the observation that the peaks corresponding to the adjacent peptides (residues 20-30 and 20-33) do not decrease in size (Figure 3, compare panels A and B) suggests that the modification is at the N-terminal R-amino group rather than at the -amino group of Lys-19. Had Lys-19 been modified, endoproteinase Lys-C would not be expected to cleave the peptide bond between Lys19 and Leu-20, and the amount of peptide 20-30 and 20-33 would have subsequently been reduced. However, while the PEGylated protein consists primarily of a species in which the N-terminal R-amino group is modified, the observation that some (∼7%) of the N-terminal peptide corresponding to residues 1-19 remains (Figure 3, panel B) indicates that the preparation must also contain singly modified form(s) in which the PEG is attached at site(s) other than at the N-terminus. However, these are not readily assigned based on a comparison of the maps for unmodified and PEGylated protein, since they represent only a small fraction of the total population and the modification(s) may occur at more than one site. To determine the effect of PEGylation on the specific antiviral activity of IFN-β-1a, the unmodified and PEGylated proteins were assayed in the A549 cell/EMC virus CPE bioassay. Figure 4A shows that incorporation of a single 20 kDa mPEG-O-2methylpropionaldehyde moiety at the N-terminus of IFN-β-1a produces an approximately 2-fold loss of specific activity as compared to the unmodified protein. For the unmodified protein, the mean EC50 value was 5.7 ( 0.76 pg/mL (n ) 4 replicates), while for the PEGylated protein, the mean EC50 value was 11.9 ( 0.85 pg/mL (n ) 4 replicates). Therefore, the specific antiviral activity of the PEGylated IFN-β-1a is ∼1 × 108 units/mg. The unmodified and PEGylated proteins were also tested for their

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Figure 3. Endoproteinase Lys-C peptide maps of unmodified (A) and PEGylated (B) IFN-β-1a. Peptide peaks were detected on-line with a 2690/ ZMD LC-MS system. Peptides are denoted by residue number and asterisks denote peptides from endoproteinase Lys-C generated by protease autodigestion.

Figure 4. (A) Antiviral activity of unmodified and PEGylated IFNβ-1a assayed on human lung carcinoma A549 cells challenged with EMC virus. Assays were performed in duplicate and error bars show the spread between the determinations. The data were analyzed using four-parameter curve fitting. Unmodified IFN-β-1a (O), PEGylated IFNβ-1a (b). (B) Antiproliferative activity of unmodified and PEGylated IFN-β-1a assayed on Daudi human B cells. Assays were performed in quadruplet and error bars show the standard deviation about the mean. The data were analyzed using four-parameter curve fitting. Unmodified IFN-β-1a (O), PEGylated IFN-β-1a (b).

ability to inhibit the growth of Daudi human B cells. Figure 4B shows that the PEGylated protein was 2.4-fold less potent than the unmodified IFN-β-1a. For the unmodified protein, the IC50 value was 30 pg/mL, while for the PEGylated protein, the IC50 value was 73 pg/mL. Therefore, the PEGylated protein showed the same ∼2-fold loss of potency as compared to the unmodified protein in assays measuring antiviral and antiproliferative activities. Pharmacokinetics of Unmodified and PEGylated IFN-β1a. Pharmacokinetic parameters were determined for unmodified and PEGylated IFN-β-1a following intravenous administration in female Lewis rats. The mean blood serum curves for unmodified and PEGylated IFN-β-1a are shown in Figure 5

panels A and B, respectively. Systemic clearance, elimination half-life (t1/2), and the volume of distribution at steady state (Vss) were calculated and are shown in Table 1. Incorporation of 20 kDa mPEG-O-2-methylpropionaldehyde at the N-terminus of IFN-β-1a reduced the clearance by ∼30-fold and increased t1/2 by ∼13-fold as compared to the unmodified protein (Table 1). In addition, the volume of distribution decreased ∼4-fold for the PEGylated protein, suggesting that the PEG moiety reduces the ability of the protein to distribute outside of the central vasculature. The maximal observed concentration (Cmax) for the unmodified protein was ∼2-fold greater than for the PEGylated protein although this was not unexpected since the unmodified protein was dosed at 80 µg/kg as compared to 24 µg/kg for the PEGylated protein, i.e., ∼3-fold greater amount in terms of IFNβ-1a equivalents. The unmodified protein was dosed at a higher level to ensure that sufficient protein remained in circulation to be detected in the CPE bioassay and thus enable calculation of its pharmacokinetic parameters. The AUC for the PEGylated protein was ∼10-fold greater than for the unmodified protein, despite being dosed at a level ∼3-fold lower than the unmodified protein. Therefore, PEGylation of the N-terminus of IFN-β-1a with 20 kDa mPEG-O-2-methylpropionaldehyde results in a significant increase in the systemic exposure as compared to the unmodified protein. In Vivo Efficacy of Unmodified and PEGylated IFN-β1a. To determine whether the improved pharmacokinetic properties of the PEGylated IFN-β-1a translated into an improvement in efficacy, the unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SKMEL-1 melanoma tumors grown in athymic nude homozygous (nu/nu) mice. This murine dermis model, described by Bauer et al. (42), has been used previously to evaluate the antiangiogenic effects of unmodified and PEGylated IFN-R-2b and was therefore a useful system for comparing the efficacy of unmodified and PEGylated IFN-β-1a. Two studies were performed to compare the efficacy of the unmodified and PEGylated proteins. In one study, groups of four mice carrying SK-MEL-1 melanoma tumors received either human serum albumin solution (HSA, vehicle control) once on day 1 only (Group 1), HSA containing 1 × 106 units of unmodified IFN-β-1a once on day 1 only (Group 2), or HSA containing 1 × 106 units of PEGylated IFN-β-1a once on day

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Figure 5. Pharmacokinetic profiles of unmodified and PEGylated IFN-β-1a in rats following intravenous administration. Female Lewis rats were injected with unmodified (A) or PEGylated (B) IFN-β-1a. Blood was collected at the time points indicated and assayed for serum levels of interferon by activity in the A549 cell/EMC virus CPE bioassay. Data points are means of values obtained from three rats. Pharmacokinetic parameters were calculated using noncompartmental analysis. For visual presentation, the data were fitted using a two-compartment model. Unmodified IFN-β-1a (O), PEGylated IFN-β-1a (b). Table 1. Pharmacokinetic Parameters for Unmodified and PEGylated Human IFN-β-1a in Female Lewis Rats Following Intravenous Administration protein

dose (µg/kg)

Cmax (pg/mL)

AUC (pg/h/mL)

t1/2 (h)

CL (mL/h/kg)

Vss (mL/kg)

unmodified PEGylated

80 24

1400000 720000

510000 4800000

0.98 13

160 5.0

160 39

1 only (Group 3) (Figure 6A). Since the PEGylated protein was shown to have a specific activity 2-fold lower than that of the unmodified protein (see above), the mice were administered with 5 µg of unmodified IFN-β-1a or 10 µg of PEGylated IFN-β-1a thus ensuring equal dosing on a unit (antiviral/antiproliferative activity) basis. As shown in Figure 6A, treatment of the mice on day 1 only with a single dose of 1 × 106 units of unmodified IFN-β-1a resulted in a mean vessel number of 36.0 ( 4.2 as compared to 50.8 ( 5.3 for the vehicle-treated mice (Figure 6A, compare Groups 2 and 1, respectively). By contrast, treatment with a single dose of 1 × 106 units of PEGylated IFN-β-1a resulted in a mean vessel number of 21.3 ( 3.9 (Figure 6A, compare Groups 3 and 1, respectively). Therefore, a single dose of the PEGylated protein resulted in a 58% reduction in vessel number as compared to a 29% reduction for the unmodified protein. Analysis of variance followed by a Newmann-Keuls multiple comparison test indicated that both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice (p < 0.01 for the unmodified IFN-β-1a-treated mice and p < 0.001 for the PEGylated IFN-β-1a-treated mice). Furthermore, the mean vessel number of the PEGylated IFN-β-1a-treated mice was statistically significantly different from that of the unmodified IFN-β-1a-treated mice (p < 0.01). To determine whether the efficacy of the PEGylated IFN-β1a could be matched by increasing the dose and frequency of the unmodified protein, an independent study was carried out in which groups of three mice carrying SK-MEL-1 melanoma tumors received either HSA once on day 1 only (Group 1), HSA containing 1 × 106 units of PEGylated IFN-β-1a once on day 1 only (Group 2), HSA daily on days 1-9 inclusive (Group 3), or HSA containing 1 × 106 units of unmodified IFN-β-1a daily on days 1-9 inclusive (Group 4) (Figure 6B). Results from Group 2 (single dose of PEGylated IFN-β-1a) were compared to Group 1 (single dose of vehicle control), while results from Group 4 (unmodified IFN-β-1a administered daily) were compared to Group 3 (vehicle control administered daily) to control for any effects resulting from the multiple dosing regimen. As shown in Figure 6B, treatment of the mice on day

1 only with a single dose of 1 × 106 units of PEGylated IFNβ-1a resulted in a mean vessel number of 17.3 ( 2.5 as compared to 51.0 ( 5.6 for the mice treated with vehicle control once on day 1 only (Figure 6B, compare Groups 2 and 1, respectively). For the multiply dosed mice, treatment with 1 × 106 units of unmodified IFN-β-1a daily resulted in a mean vessel number of 21.7 ( 4.0 as compared to 44.0 ( 4.6 for the mice treated daily with vehicle control (Figure 6B, compare Groups 4 and 3, respectively). Hence, a single dose of 1 × 106 units of the PEGylated protein resulted in a 66% reduction in vessel number compared to a 51% reduction following daily administration of 1 × 106 units of the unmodified protein. Analysis of the data using an unpaired Student’s t test indicated that both treatments resulted in statistically significant reductions in mean vessel number as compared to the respective vehicle control (p ) 0.0032 for the unmodified IFN-β-1a-treated mice, and p ) 0.0007 for the PEGylated IFN-β-1a-treated mice). Further analysis was carried out to determine whether the difference in mean vessel number between the unmodified IFN-β-1a-treated mice and the PEGylated IFN-β-1a-treated mice was statistically significant. Mean vessel numbers for the test and the corresponding control group were ranked. Differences were calculated between each test and control value at the same rank level. The decrease in mean vessel number was 22.33 ( 0.33 for the unmodified IFN-β-1a-treated mice and 33.67 ( 1.86 for the PEGylated IFN-β-1a-treated mice. The mean decreases were then tested using an unpaired t-test that gave a P value of 0.0039, i.e., the resulting mean vessel number following a single dose of the PEGylated protein on day 1 only was significantly different from that when the unmodified protein was given on days 1-9 inclusive.

DISCUSSION PEGylation of therapeutic proteins is an established method to improve pharmacokinetic properties, to reduce dosing frequency, and to potentially improve pharmacodynamic responses and clinical efficacy. The benefit of this technology is clearly seen in the case of IFN-R-2b and IFN-R-2a where dosing frequency was reduced and efficacy against HCV infection improved, following PEGylation (22, 23, 25-27). To prepare a PEGylated form of IFN-β-1a that could be tested and compared to the unmodified protein in preclinical studies and potentially in human clinical trials, we modified the protein at the N-terminal R-amino group with 20 kDa mPEG-O-2methylpropionaldehyde. N-Terminal PEGylation of IFN-β-1a with 20 kDa mPEGO-2-methylpropionaldehyde significantly increased the systemic

N-Terminally PEGylated Human Interferon-β-1a

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Figure 6. In vivo antiangiogenic activity of unmodified and PEGylated IFN-β-1a. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors grown in athymic nude homozygous (nu/nu) mice. Mice were injected with SK-MEL-1 cells (2 × 106) on day 0, followed by subcutaneous administration of either human serum albumin solution (HSA, vehicle control), HSA containing 1 × 106 units of unmodified IFN-β-1a, or HSA containing 1 × 106 units of PEGylated IFN-β-1a. For (A), groups of four mice received either HSA once on day 1 only (Group 1), HSA containing 1 × 106 units of unmodified IFN-β-1a once on day 1 only (Group 2), or HSA containing 1 × 106 units of PEGylated IFN-β-1a once on day 1 only (Group 3). For (B), groups of three mice received either HSA once on day 1 only (Group 1), HSA containing 1 × 106 units of PEGylated IFN-β-1a once on day 1 only (Group 2), HSA on days 1-9 inclusive (Group 3), or HSA containing 1 × 106 units of unmodified IFN-β-1a on days 1-9 inclusive (Group 4). Mice were sacrificed on day 10, and the tumor inoculation site was assessed for neovascularization, measured by an observer blinded as to the treatment. The mean vessel number ( the standard error of the mean is shown. Asterisks denote statistically significant differences between the unmodified or PEGylated IFN-β-1a-treated mice and the corresponding vehicle-treated mice.

exposure in rats following intravenous administration. The increase in t1/2 and AUC for the PEGylated protein was ∼13fold and ∼10-fold, respectively, compared to the unmodified protein, resulting from decreases in the clearance and volume of distribution at steady state (Table 1). While a direct sideby-side comparison of the pharmacokinetic parameters of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a and 20 kDa mPEG-propionaldehyde-modified IFN-β-1a was not carried out, a comparison of the t1/2 values obtained for unmodified IFN-β-1a and the PEGylated forms in this and in the previously published study (39) indicates that they were comparable with respect to half-life. For the unmodified and 20 kDa mPEG-propionaldehyde-modified IFN-β-1a, the t1/2 values were 1.47 and 10.13 h, respectively (39), while for the unmodified and 20 kDa mPEG-O-2-methylpropionaldehydemodfied IFN-β-1a (this study), the t1/2 values were 0.98 and 13 h, respectively (Table 1). While we did not determine the pharmacokinetic properties of 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a following subcutaneous or intraperitoneal administration in rats, we anticipate that it will also show improved pharmacokinetic parameters compared to the unmodified protein since human IFN-β-1a and rat IFN-β modified at the N-terminus with 20 kDa mPEG-propionaldehyde both show improved pharmacokinetic parameters as compared to their respective unmodified forms following these routes of administration (39, 43). Similar improvements in pharmacokinetic properties were observed for IFN-β-1a modified on Cys17 with a 40 kDa branched PEG following subcutaneous administration to cynamologous monkeys (44). The unmodified and 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a were tested in a murine dermis model to determine whether the improved pharmacokinetics of the modified protein would translate into an improvement in efficacy. When a single dose of 1 × 106 units of unmodified IFN-β-1a was given once on day 1 only, the number of radially oriented blood vessels entering the periphery of the SK-MEL-1 tumors at day 10 was reduced 29% as compared to the vehicle control. By contrast, a single dose of 1 × 106 units of PEGylated IFN-β-1a given once on day 1 only resulted in a 58% reduction (Figure 6A). Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicletreated mice, with the PEGylated IFN-β-1a-treated mice showing a statistically significant reduction in mean vessel number as compared to the unmodified IFN-β-1a-treated mice. Therefore,

the attached 20 kDa PEG moiety significantly improved the efficacy of the protein, indicating that the pharmacokinetic properties and efficacy are correlated. To address the question as to whether the efficacy of the unmodified protein could be improved to a level commensurate with that of the modified protein, a single dose of 1 × 106 units of PEGylated IFN-β-1a was administered once on day 1 only, and compared to administration of 1 × 106 units of unmodified IFN-β-1a given daily on days 1-9 inclusive. Treatment of the mice with a single dose of 1 × 106 units of PEGylated IFN-β-1a once on day 1 only resulted in a mean vessel number of 17.3 ( 2.5 as compared to 51.0 ( 5.6 for the mice treated with vehicle control once on day 1 only (66% reduction, Figure 6B). This result was very similar to that obtained for the same samples in the single dose comparison study, where a single dose of the PEGylated protein resulted in a mean vessel number of 21.3 ( 3.9 as compared to 50.8 ( 5.3 for the vehicle control (Figure 6A). For the multiply dosed mice, treatment with 1 × 106 units of unmodified IFN-β-1a daily on days 1-9 inclusive resulted in a mean vessel number of 21.7 ( 4.0 as compared to 44.0 ( 4.6 for the mice treated daily with vehicle control (51% reduction, Figure 6B). Both treatments resulted in statistically significant reductions in mean vessel number as compared to the respective vehicle-treated mice, with the PEGylated IFNβ-1a-treated mice showing a statistically significant reduction in mean vessel number as compared to the unmodified IFN-β1a-treated mice. Therefore, even though the cumulative total dose of the unmodified protein was 9-fold greater than that of the PEGylated protein on a unit (activity) basis (4.5-fold greater on an IFN-β-1a mass basis), the PEGylated protein was still more efficacious than the unmodified protein. A similar study using the same murine dermis model was carried out to compare the efficacy of unmodified IFN-R-2b (Intron-A) and PEGylated IFN-R-2b (PEG-Intron) (42). In this study, mice received either 0, 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 units of unmodified IFN-R-2b on days 1-10 inclusive, and the mean number of vessels compared to mice that received the same amount (units) of PEGylated IFN-R-2b (PEG-Intron) as a single dose. While at 1 × 103 units there was no effect with either protein, at 1 × 104 units and above there was a significant reduction in the vessel number as compared to the control-treated mice. However, there was no significant difference observed between mice receiving either a single dose of PEGylated IFNR-2b or daily administration of the unmodified protein. Fol-

186 Bioconjugate Chem., Vol. 17, No. 1, 2006

lowing a single dose of 1 × 106 units of PEGylated IFN-R-2b, the reduction in vessel number was ∼70% (42), similar to the values obtained in this study for 1 × 106 units of PEGylated IFN-β-1a (58% and 66%, Figure 6). PEGylation of IFN-β-1a at the N-terminus with 20 kDa mPEG-O-2-methylpropionaldehyde, in addition to improving pharmacokinetic properties and in vivo efficacy, also has the benefit of yielding a product with essentially a single site of modification; thereby simplifying product characterization. By contrast, PEGylated IFN-R-2b (PEG-Intron), which is modified with a linear 12 kDa PEG using a succinimidyl carbonate chemistry, is a mixture of 14 different monoPEGylated positional isomers in which the PEG chain is attached to either lysine, tyrosine, histidine, serine, or cysteine residues (45). Similarly, for PEGylated IFN-R-2a (Pegasys), modification with a branched 40 kDa PEG using an N-hydroxysuccinamide ester derivative results in a mixture of 6 different monoPEGylated positional isomers, all of which are attached to lysine residues (46). A consequence of using these nonspecific amine-coupling chemistries is that the product is a heterogeneous mixture that requires extensive analytical characterization following production to ensure batch-to-batch consistency. Furthermore, PEG chains can become attached at site(s) that interfere with the binding of the protein to the IFNAR1/IFNAR2 receptor. Indeed, many of the residues that are modified in these proteins have been shown by site-specific mutagenesis to be important for activity (4), and it is therefore not surprising that both PEGylated IFNs-R show a significant reduction in their in vitro antiviral activity as compared to their respective unmodified form. For PEG-Intron, the antiviral activity of the mixture is 28% of that of the unmodified protein and ranges from 6 to 37% for the individual species (45), while for Pegasys, the mixture has an antiviral activity of only 7% of the unmodified IFN-R-2a (46). The data for 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-β-1a (50% retention of activity), and for 20 kDa mPEG-propionaldehyde-modified IFN-β-1a (39) and 20 kDa mPEG-propionaldehyde-modified rat IFN-β (43), both of which retain full in vitro antiviral activity, highlights the importance of modifying sites distal to receptor binding site(s) if retention of activity is a desired outcome. The N-terminus of IFN-β-1a therefore serves as an attractive site for modification since alanine scanning mutagenesis of the N-terminal region (37) and deletion of the N-terminal methionine (38) have no affect on activity, and analysis of the preliminary X-ray crystal structure of IFN-β-1a complexed with the extracellular portion of the human IFNAR2 chain shows that the N-terminus does not participate in the binding site (Ann Boriack-Sjodin, personal communication). In addition to IFN-β-1a, the N-terminus of IFN-R-2 also appears to be suitable for PEGylation. Using sitespecific mutagenesis to replace glutamine at position 5 with a cysteine, Rosendahl et al. (47) have recently shown that the cysteine can be modified with linear 5 kDa, 10 kDa, or 20 kDa maleimide-PEGs, or with a branched 40 kDa maleimide PEG, resulting in the production of site-specific monoPEGylated proteins. The 5 kDa PEG-maleimide-modified protein retained ∼50% of the antiproliferative activity of the unmodified wild type or Q5C mutant protein, while the activities of the 10 kDa, 20 kDa, and 40 kDa PEG-maleimide-modified proteins were within 3-4-fold of the unmodified wild type or Q5C mutant (47). As expected, the pharmacokinetic properties of the PEGylated proteins were superior to that of the unmodified protein. Following intravenous administration in rats, the t1/2 value for the unmodified wild-type protein was 0.5 h, while for the 10 kDa, 20 kDa, and 40 kDa PEG-maleimide-modified proteins, the t1/2 values were 22, 24, and 32 h, respectively (47). The superior retention of activity of the 40 kDa PEG-maleimidemodified IFN-R-2 (3-4-fold lower than that of the unmodified

Baker et al.

protein) as compared to Pegasys (14-fold lower) presumably reflects the fact that the N-terminal region of IFN-R-2, like that of IFN-β-1a, is not critical for receptor binding. Indeed, sitedirected mutagenesis and modeling studies indicate that the N-terminus of the cytokine does not interact with the IFNAR2 chain, rather the binding interface between IFN-R-2 and IFNAR2 is formed by a primarily hydrophobic patch composed of several residues, the most N-terminal of which is leucine-26 (48-50). PEGylation of the N-terminus of IFN-β-1a, with the retention of significant activity, is also desirable since a lower dose may be required to achieve efficacy as compared to the unmodified protein, or as compared to a protein that has lost substantial activity following modification. This not only affects factors such as the cost of making the drug, but may also play an important role in reducing immunogenicity and the formation of neutralizing antibodies. For IFN-β-1a, for example, administration of the two different products currently available for the treatment of multiple sclerosis results in the formation of different levels of neutralizing antibodies. In a side-by-side comparison of the efficacious dose of Rebif (Serono), given as a 44 µg subcutaneous injection thrice weekly, and Avonex (BiogenIdec), given as a 30 µg intramuscular injection once per week, 25% of the multiple sclerosis patients receiving Rebif were positive for neutralizing antibodies (defined as titers of g20 neutralizing units/mL), as compared to only 2% of patients receiving Avonex (51). Therefore, a higher dose given more frequently correlates with a higher incidence of neutralizing antibody formation. The ability to produce a PEGylated form of IFN-β-1a with high potency may enable a lower protein mass to be administered and possibly reduce the incidence and level of neutralizing antibodies. However, immunogenicity data from clinical trial(s) would be required to fully address this issue. In summary, we have prepared a modified form of IFN-β-1a in which 20 kDa mPEG-O-2-methylpropionaldehyde is covalently attached to the N-terminal R-amino group of the protein. The modified protein retains ∼50% of the in vitro antiviral and antiproliferative activity of the unmodified form, has improved pharmacokinetic properties in rats, and has improved efficacy with respect to its ability to inhibit the formation of tumorinduced blood vessels entering the periphery of human SKMEL-1 melanoma tumors grown in athymic nude homozygous (nu/nu) mice. With the known efficacy of unmodified IFN-β in multiple sclerosis and HCV, this modified form of the protein may serve as a useful adjunct for the treatment of these diseases, not only in terms of potentially increasing efficacy but also by reducing dosing frequency, and thereby patient convenience and compliance. N-Terminally PEGylated IFN-β-1a may also be useful for the treatment of certain cancers, either as sole therapy or in combination with other drugs such as tamoxifen for example, that has been shown to have additive/synergistic antitumor effects when administered with IFN-β (41, 52), or thalidomide, that has been shown to have synergistic antitumor effect when administered with IFN-R-2b (42).

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